tretinoin and Leukemia--Myeloid--Acute

to review the current diagnostic and therapeutic landscape of AML in Latin America as a reflection of other low- and middle-income countries and regions of the world. Encompassing both acute promyelocytic and non-promyelocytic disease types.. We reviewed the literature and study registries concerning epidemiological features of patients with AML/APL treated in Latin America, as well as evaluated diagnostic and genetic stratification and patient fitness assessment challenges, the importance of early mortality and supportive care capacity, intensive and non-intensive chemotherapy alternatives, consolidation, and maintenance strategies including novel agents and hematopoietic stem cell transplantation.. Although most of the current technologies and treatment options are available in the region, a significant fraction of patients have only limited access to them. In addition, mortality in the first weeks from diagnosis is higher in the region compared to developed countries.. Disparities in access to technologies, supportive care capacity, and availability of novel agents and HSCT hinder results in our region, reflecting barriers common to other LMICs. Recent developments in the diagnosis and treatment of this disease must be implemented through education, collaborative clinical research, and advocacy to improve outcomes.

Topics: Antineoplastic Combined Chemotherapy Protocols; Hematopoietic Stem Cell Transplantation; Humans; Latin America; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin

Loads of new therapeutic regimes have been turned up to manage Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), particularly in elderly patients who are unfit for intensive chemotherapy. Despite accumulating research, the best MDS and AML management approach is indeterminate. Myelodysplastic syndrome implies a group of various hematopoietic stem cell disorders that may progress to acute myeloid leukemia. These disorders are more frequent in older adults. To the high rate of morbidity and abundant toxicities related to the therapeutic approaches, also, the treatment would be challenging. The clinical effectiveness of valproic acid, a histone deacetylase inhibitor, in MDS and AML patients is unknown, even though it has demonstrated positive activities to promote differentiation and apoptosis in cancer cells. We investigated the clinical research on the effects of valproic acid in conjunction with various drugs, including low-dose cytarabine, all-trans retinoic acid, DNA-hypomethylating agents, hydrazine, and theophylline. We conclude that VPA is a safe and effective treatment option for MDS and AML patients, particularly when used in conjunction with all-trans retinoic acid, DNA-hypomethylating drugs, and hydralazine. However, more randomized clinical studies are required to identify an ideal regimen.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Tretinoin; Valproic Acid

Because there are few evidence-based guidelines and an extremely low incidence rate, managing and treating patients who have transitioned from acute promyelocytic leukemia (APL), which was diagnosed during pregnancy, to acute myeloid leukemia (AML), can be difficult.. In this case, a 34-year-old pregnant patient was diagnosed with APL in medium-risk group in June 2017. After the all-trans retinoic acid and arsenic trioxide-based full-course treatment, the patients achieved complete remission (CR) and were well-tolerated. After 5 years, the patient complained of fatigue for 3 months.. Bone marrow examination revealed hypercellularity with approximately 50% immunophenotypic abnormal myeloblasts with MLL-AF9 fusion gene. Based on the AML diagnosis criteria of the World Health Organization, the patient was eventually diagnosed with a rare transformation from APL to AML.. The patient was treated with two cycles of induction chemotherapy and an allogeneic hematopoietic stem cell transplantation (allo-HSCT).. Until now, the patient is in continuous remission with no signs of APL and AML.. Despite the rarity of APL to AML transformation, it is crucial to track the disease's progress and administer treatment on time. It remains uncertain whether the risk stratification and clinical outcomes of secondary AML with MLL-AF9 are equivalent to those of de novo AML with MLL-AF9. The management and treatment of these patients should be personalized and require further observation.

Topics: Adult; Female; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Myeloid-Lymphoid Leukemia Protein; Oncogene Proteins, Fusion; Pregnancy; Remission Induction; Tretinoin

Differentiation therapy using all-trans retinoic acid (ATRA) is well established for the treatment of acute promyelocytic leukemia (APL). Several attempts have been made to treat non-APL acute myeloid leukemia (AML) patients by employing differentiation inducers, such as hypomethylating agents and low-dose cytarabine, with encouraging results. In the present review, I focus on other possible differentiation inducers: kinase inhibitors and interferons (IFNs). A number of kinase inhibitors have been reported to induce differentiation, including CDK inhibitors, GSK3 inhibitors, Akt inhibitors, p38 MAPK inhibitors, Src family kinase inhibitors, Syk inhibitors, mTOR inhibitors, and HSP90 inhibitors. Other powerful inducers are IFNs, which were reported to enhance differentiation with ATRA. Although clinical trials for these kinase modulators remain scarce, their mechanisms of action have been, at least partly, clarified. The Raf/MEK/ERK MAPK pathway and the RARα downstream are affected by many of the kinase inhibitors and IFNs and seem to play a pivotal role for the induction of myeloid differentiation. Further clarification of the mechanisms, as well as the establishment of efficient combination therapies with the kinase inhibitors or IFNs, may lead to the development of effective therapeutic strategies for AML.

Topics: Antineoplastic Agents; Cell Differentiation; Glycogen Synthase Kinase 3; Humans; Interferons; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Protein Kinase Inhibitors; Tretinoin

Acute myeloid leukemia (AML) is an aggressive and often fatal hematopoietic malignancy. A very attractive way to treat myeloid leukemia, called "differentiation therapy", was proposed when in vitro studies showed that some compounds are capable of inducing differentiation of AML cell lines. One of the differentiation-inducing agents, all-

Topics: Antineoplastic Agents; Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Vitamin D; Vitamins

Acute myeloid leukemia is characterized by arrested differentiation, and agents that overcome this block are therapeutically useful, as shown by the efficacy of all-trans retinoic acid in acute promyelocytic leukemia. However, the early promise of differentiation therapy did not translate into clinical benefit for other subtypes of acute myeloid leukemia, in which cytotoxic chemotherapeutic regimens remained the standard of care. Recent advances, including insights from sequencing of acute myeloid leukemia genomes, have led to the development of targeted therapies, comprising agents that induce differentiation of leukemic cells in preclinical models and clinical trials, thus rejuvenating interest in differentiation therapy. These agents act on various cellular processes including dysregulated metabolic programs, signaling pathways, epigenetic machinery and the cell cycle. In particular, inhibitors of mutant IDH1/2 and FLT3 have shown clinical benefit, leading to approval by regulatory bodies of their use. Besides the focus on recently approved differentiation therapies, this review also provides an overview of differentiation- inducing agents being tested in clinical trials or investigated in preclinical research. Combinatorial strategies are currently being tested for several agents (inhibitors of KDM1A, DOT1L, BET proteins, histone deacetylases), which were not effective in clinical studies as single agents, despite encouraging anti-leukemic activity observed in preclinical models. Overall, recently approved drugs and new investigational agents being developed highlight the merits of differentiation therapy; and ongoing studies promise further advances in the treatment of acute myeloid leukemia in the near future.

Topics: Antineoplastic Agents; Cell Differentiation; Histone Demethylases; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin

Differentiation Syndrome (DS) has been identified in a subset of patients undergoing treatment with novel classes of differentiating therapies for acute myeloid leukemia (AML) such as IDH and FLT3 inhibitors. While DS is a well-known treatment-related complication in acute promyelocytic leukemia (APL), efforts are still ongoing to standardize diagnostic and treatment parameters for DS in AML. Though the rates of incidence vary, many of the signs and symptoms of DS are common between APL and AML. So, DS can lead to fatal complications in AML, but prompt management is usually effective and rarely necessitates interruption or discontinuation of AML therapy.

Topics: Acute Kidney Injury; Adrenal Cortex Hormones; Antineoplastic Agents; Arsenic Trioxide; Cell Differentiation; Clinical Trials as Topic; Cytokine Release Syndrome; Edema; Enzyme Inhibitors; Epigenesis, Genetic; Fever; fms-Like Tyrosine Kinase 3; Humans; Hypotension; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Myelopoiesis; Neoplasm Proteins; Pleural Effusion; Respiration Disorders; Tretinoin

The unraveling of the pathophysiology of acute myeloid leukemia (AML) has resulted in rapid translation of the information into clinical practice. After more than 40 years of slow progress in AML research, the US Food and Drug Administration has approved nine agents for different AML treatment indications since 2017. In this review, we detail the progress that has been made in the research and treatment of AML, citing key publications related to AML research and therapy in the English literature since 2000. The notable subsets of AML include acute promyelocytic leukemia (APL), core-binding factor AML (CBF-AML), AML in younger patients fit for intensive chemotherapy, and AML in older/unfit patients (usually at the age cutoff of 60-70 years). We also consider within each subset whether the AML is primary or secondary (therapy-related, evolving from untreated or treated myelodysplastic syndrome or myeloproliferative neoplasm). In APL, therapy with all-trans retinoic acid and arsenic trioxide results in estimated 10-year survival rates of ≥80%. Treatment of CBF-AML with fludarabine, high-dose cytarabine, and gemtuzumab ozogamicin (GO) results in estimated 10-year survival rates of ≥75%. In younger/fit patients, the "3+7" regimen (3 days of daunorubicin + 7 days of cytarabine) produces less favorable results (estimated 5-year survival rates of 35%; worse in real-world experience); regimens that incorporate high-dose cytarabine, adenosine nucleoside analogs, and GO are producing better results. Adding venetoclax, FLT3, and IDH inhibitors into these regimens has resulted in encouraging preliminary data. In older/unfit patients, low-intensity therapy with hypomethylating agents (HMAs) and venetoclax is now the new standard of care. Better low-intensity regimens incorporating cladribine, low-dose cytarabine, and other targeted therapies (FLT3 and IDH inhibitors) are emerging. Maintenance therapy now has a definite role in the treatment of AML, and oral HMAs with potential treatment benefits are also available. In conclusion, AML therapy is evolving rapidly and treatment results are improving in all AML subsets as novel agents and strategies are incorporated into traditional AML chemotherapy. LAY SUMMARY: Ongoing research in acute myeloid leukemia (AML) is progressing rapidly. Since 2017, the US Food and Drug Administration has approved 10 drugs for different AML indications. This review updates the research and treatment pathways for AML.

Topics: Age Factors; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Bridged Bicyclo Compounds, Heterocyclic; Cladribine; Core Binding Factors; Cytarabine; Daunorubicin; Gemtuzumab; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Maintenance Chemotherapy; Mutation; Myelodysplastic Syndromes; Myeloproliferative Disorders; Neoplasm, Residual; Sulfonamides; Survival Rate; Translational Research, Biomedical; Tretinoin; Vidarabine

Among elderly patients with acute myeloid leukemia (AML), especially those who are unfit for intensive chemotherapy, a policy of reduced-intensity chemotherapy or conservative observation has been chosen, resulting in unmet medical needs. Clinical trials using anticancer drugs including antimetabolites or drugs targeted to cell cycle-related molecules failed to show superiority over conventional treatments. Recently, drugs targeted to Bcl-2, SMO, FLT3, and IDH1/2 have been shown to prolong overall survival alone or in combination with reduced-intensity chemotherapy. These treatments are likely to reshape the therapeutic landscape of AML, which will be personalized for individual patients based on leukemia genetics.

Topics: Aged; Aged, 80 and over; Aminopyridines; Aniline Compounds; Antineoplastic Agents; Arsenic Trioxide; Azacitidine; Benzimidazoles; Bridged Bicyclo Compounds, Heterocyclic; Decitabine; fms-Like Tyrosine Kinase 3; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Phenylurea Compounds; Precision Medicine; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Smoothened Receptor; Staurosporine; Sulfonamides; Survival Rate; Tretinoin; Triazines

Management of acute myeloid leukemia during pregnancy (P-AML) is a challenging endeavor with limited evidence-based information available. To truly achieve the goal of improving P-AML patients, additional evidence-based research is necessary. We retrospectively reviewed cases of 17 patients diagnosed with P-AML, including seven for acute promyelocytic leukemia (APL) from January 2012 to June 2019. Among the non-APL, 90% patients (9/10) ended pregnancy prior to induction chemotherapy. The median intervals between diagnosis and start of chemotherapy were 5 days (range 1-14 days). Four patients elected to delay chemotherapy by more than one week. Of the seven APL patients, six received all-trans retinoic acid (ATRA) before the diagnostic molecular results. Five patients underwent cesarean sections (CS) and all newborns were alive (four preterm and one full-term deliveries). Overall, approximately 94% of the patients (16/17) are currently alive in remission. To treat P-AML patients in a safer manner, balancing the risk of progressing to advanced disease and proceeding with pregnancy is required. We consider a slight delay (less than 14 days) in the termination of pregnancy may not differ the prognosis to patients with non-APL. For APL, patients will benefit from prompt administration of ATRA for highly suspected cases.

Topics: Adult; Cesarean Section; Evidence-Based Practice; Female; Humans; Leukemia, Myeloid, Acute; Pregnancy; Pregnancy Complications, Hematologic; Pregnancy Outcome; Prognosis; Remission Induction; Retrospective Studies; Risk Assessment; Time Factors; Tretinoin; Young Adult

Acute myeloid leukemia (AML) is an aggressive, often fatal hematopoietic malignancy.

Topics: Animals; Apoptosis; Hematopoiesis; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Neoplastic Stem Cells; Tretinoin

After being in the therapeutic wilderness for several decades, acute myeloid leukemia has been recently thrust into the limelight with a series of drug approvals. Technical refinements in production, genetic manipulation and chemical modification of monoclonal antibodies led to growing interest in antibodies-based treatment strategies. Much of the focus of these efforts in acute myeloid leukemia has been on CD33 as a target. On September 2, 2017, the U.S. Food and Drug Administration approved gemtuzumab ozogamicin for treatment of relapsed or refractory CD33

Topics: Aged; Animals; Antineoplastic Agents; Antineoplastic Agents, Immunological; Antineoplastic Combined Chemotherapy Protocols; Arsenicals; Calicheamicins; Chlorides; Clinical Trials as Topic; Cytarabine; Daunorubicin; Drug Approval; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Gemtuzumab; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mice; Middle Aged; Multicenter Studies as Topic; Recurrence; Sialic Acid Binding Ig-like Lectin 3; Tretinoin

Acute myeloid leukaemia (AML) is the most common acute leukaemia affecting adults. Most patients diagnosed with AML are at advanced age and present with co-morbidities, so that intensive therapy such as stem cell transplantation (SCT) is impossible to provide or is accompanied by high risks for serious adverse events and treatment-related mortality. Especially for these patients, it is necessary to find out whether all-trans retinoic acid (ATRA), an intermediate of vitamin A inducing terminal differentiation of leukaemic cell lines, added to chemotherapy confers increased benefit or harm when compared with the same chemotherapy alone.. This review aims to determine benefits and harms of ATRA in addition to chemotherapy compared to chemotherapy alone for adults with AML (not those with acute promyelocytic leukaemia (non-APL)).. We searched the Central Register of Controlled Trials (CENTRAL), MEDLINE, study registries and relevant conference proceedings up to July 2018 for randomised controlled trials (RCTs). We also contacted experts for unpublished data.. We included RCTs comparing chemotherapy alone with chemotherapy plus ATRA in patients with all stages of AML. We excluded trials if less than 80% of participants were adults or participants with AML, and if no subgroup data were available. Patients with myelodysplastic syndrome (MDS) were included, if they had a refractory anaemia and more than 20% of blasts.. Two review authors independently extracted data and assessed the quality of trials. We contacted study authors to obtain missing information. We used hazard ratios (HR) for overall survival (OS) and disease-free survival (DFS; instead of the pre-planned event-free survival, as this outcome was not reported), and we calculated risk ratios (RR) for the other outcomes quality of life, on-study mortality and adverse events. We presented all measures with 95% confidence intervals (CIs). We assessed the certainty of evidence using GRADE methods.. Our search resulted in 2192 potentially relevant references, of which we included eight trials with 28 publications assessing 3998 patients. Overall, we judged the potential risk of bias of the eight included trials as moderate. Two of eight trials were published as abstracts only. All the included trials used different chemotherapy schedules and one trial only evaluated the effect of the hypomethylating agent decitabine, a drug know to affect epigenetics, in combination with ATRA.The addition of ATRA to chemotherapy resulted in probably little or no difference in OS compared to chemotherapy only (2985 participants; HR 0.94 (95% confidence interval (CI) 0.87 to 1.02); moderate-certainty evidence). Based on a mortality rate at 24 months of 70% with chemotherapy alone, the mortality rate with chemotherapy plus ATRA was 68% (95% CI 65% to 71%).For DFS, complete response rate (CRR) and on-study mortality there was probably little or no difference between treatment groups (DFS: 1258 participants, HR 0.99, 95% CI 0.87 to 1.12; CRR: 3081 participants, RR 1.02, 95% CI 0.96 to 1.09; on-study mortality: 2839 participants, RR 1.02, 95% CI 0.81 to 1.30, all moderate-certainty evidence).Three trials with 1428 participants reported the adverse events 'infection' and 'cardiac toxicity': There was probably no, or little difference in terms of infection rate between participants receiving ATRA or not (RR 1.05, 95% CI 0.96 to 1.15; moderate-certainty evidence). We are uncertain whether ATRA decreases cardiac toxicity (RR 0.46, 95% CI 0.24 to 0.90; P = 0.02, very low certainty-evidence, however, cardiac toxicity was low).Rates and severity of diarrhoea and nausea/vomiting were assessed in two trials with 337 patients and we are uncertain whether there is a difference between treatment arms (diarrhoea: RR 2.19, 95% CI 1.07 to 4.47; nausea/vomiting: RR 1.46, 95% CI 0.75 to 2.85; both very low-certainty evidence).Quality of life was not reported by any of the included trials.. We found no evidence for a difference between participants receiving ATRA in addition to chemotherapy or chemotherapy only for the outcome OS. Regarding DFS, CRR and on-study mortality, there is probably no evidence for a difference between treatment groups. Currently, it seems the risk of adverse events are comparable to chemotherapy only.As quality of life has not been evaluated in any of the included trials, further research is needed to clarify the effect of ATRA on quality of life.

Topics: Adult; Antineoplastic Agents; Decitabine; Diarrhea; Disease Progression; Heart; Humans; Infections; Leukemia, Myeloid, Acute; Nausea; Recurrence; Tretinoin; Vomiting

Recent reports of recurrent mutations in childhood acute myeloid leukemia (AML) have identified potential targets for new therapeutic strategies. Acute promyelocytic leukemia (APL) is characterized commonly by a fusion between the PML gene and the RARA gene, genes targetable by arsenic (ATO) and retinoic acid (ATRA), respectively. A mutation in GATA1, common in AML of Down syndrome (ML-DS), renders cells more susceptible to cytarabine and anthracyclines, thus permitting targeted dose reductions to preserve high survival rates while reducing toxicity. In all other patients, Ras pathway mutations, KMT2A and other methyltransferase mutations, FLT3 mutations, and KIT mutations are all relatively common in childhood AML and all are potentially "druggable". The focus of this review is on those therapies likely to be clinically available in the near future. The preclinical and clinical data providing a rationale for testing in children of specific agents in children is discussed. Whether the expression of a potential target is sufficient to predict response to a targeted therapy is an open question in childhood AML. Development of clinical trials to evaluate targeted therapies in small molecularly defined subsets of AML will be the next great challenge for all cooperative groups in North America and Europe.

Topics: Adolescent; Adult; Arsenic Trioxide; Arsenicals; Child; Child, Preschool; Clinical Trials as Topic; Down Syndrome; Epigenesis, Genetic; GATA1 Transcription Factor; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Medical Oncology; Molecular Medicine; Mutation; Oxides; ras Proteins; Receptor Protein-Tyrosine Kinases; Treatment Outcome; Tretinoin

Topics: Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Consolidation Chemotherapy; Cytarabine; Daunorubicin; Drug Discovery; Etoposide; Hematopoietic Stem Cell Transplantation; Humans; Idarubicin; Induction Chemotherapy; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Maintenance Chemotherapy; Mercaptopurine; Methotrexate; Molecular Targeted Therapy; Mutation; Recurrence; Tretinoin; Vincristine

The important role of cell cycle regulatory molecules in all trans-retinoic acid (ATRA)- and vitamin D3-induced growth inhibition and differentiation induction has been intensively studied in both acute myeloid leukaemia primary cells and a variety of leukaemia cell lines. Cyclin-dependent kinases (CDK)-activating kinase has been demonstrated to interact with retinoic acid receptor (RAR)α in acute promyelocytic leukaemia cells, and inhibition of CDK-activating kinase by ATRA causes hypophosphorylation of PML-RARα, leading to myeloid differentiation. In many cases, downregulation of CDK activity by ATRA and vitamin D3 is a result of elevated p21- and p27-bound CDKs. Activation of p21 is regulated at the transcriptional level, whereas elevated p27 results from both (indirectly) transcriptional activation and post-translational modifications. CDK inhibitors (CKIs) of the INK family, such as p15, p16 and p18, are mainly involved in inhibition of cell proliferation, whereas CIP/KIP members, such as p21, regulate both growth arrest and induction of differentiation. ATRA and vitamin D3 can also downregulate expression of G1 CDKs, especially CDK2 and CDK6. Inhibition of cyclin E expression has only been observed in ATRA- but not in vitamin D3-treated leukaemic cells. In vitro, not only dephosphorylation of pRb but also elevation of total pRb is required for ATRA and vitamin D3 to suppress growth and trigger their differentiation. Finally, sharp reduction in c-Myc has been observed in several leukaemia cell lines treated with ATRA, which may regulate expression of CDKs and CKIs.

Topics: Cell Differentiation; Cholecalciferol; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinases; Humans; Leukemia, Myeloid, Acute; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

MicroRNA MicroRNA s (miRNAs) are small noncoding RNAs acting as endogenous regulators of gene expression. Their discovery is one of the major recent breakthroughs in molecular biology. miRNAs establish a multiplicity of relationships with target mRNAs and exert pleiotropic biological effects in many cell physiological pathways during development and adult life. The dynamic nature of gene expression regulation by Retinoic Acid Retinoic acid (RA) is consistent with an extensive functional interplay with miRNA activities. In fact, RA regulates the expression of many different miRNAs, thus suggesting a relevant function of miRNAs in RA-controlled gene expression programmes. miRNAs have been extensively studied as targets and mediators of the biological activity of RA during embryonic development as well as in normal and neoplastic cells. However, relatively few studies have experimentally explored the direct contribution of miRNA function to the RA signalling pathway. Here, we provide an overview of the mechanistic aspects that allow miRNA biogenesis, functional activation and regulation, focusing on recent evidence that highlights a functional interplay between miRNAs and RA-regulated molecular networks. We report examples of tissue-specific roles of miRNAs modulated by RA in stem cell pluripotency maintenance and regeneration, embryonic development, hematopoietic and neural differentiation, and other biological model systems, underlining their role in disease pathogenesis. We also address novel areas of research linking the RA signalling pathway to the nuclear activity of miRNAs.

Topics: Cell Differentiation; Embryo, Mammalian; Embryonic Development; Gene Expression Regulation; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Pluripotent Stem Cells; Protein Binding; Protein Multimerization; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Signal Transduction; Tretinoin

Acute myeloblastic leukemia (AML) is a disease which may be completely cured by intensive chemotherapy or stem cell transplantation. However, the prognoses are poor in elderly, refractory or recurrence cases. Molecular targeted drugs have been expected to improve the prognoses of patients with various cancers, but there are few kinds of molecular target drugs for AML. On the other hand, excellent drug exists such as tretinoin for acute promyelocytic leukemia. Molecular mechanisms have been elucidated in AML cells, and the molecules which can be the good target of the treatment have been identified. Novel molecular target drugs are also expected.

Topics: Aminoglycosides; Antibodies, Monoclonal, Humanized; Benzoates; Gemtuzumab; Humans; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Tetrahydronaphthalenes; Tretinoin

All-trans-retinoic acid (ATRA) is a physiologically active metabolite of vitamin A. Its antitumour activities have been extensively studied in a variety of model systems and clinical trials; however, to date the only malignancy responsive to ATRA treatment is acute promyelocytic leukaemia (APL) where it induces complete remission in the majority of cases when administered in combination with light chemotherapy and/or arsenic trioxide. After decades of studies, the efficacy of ATRA to treat other acute myeloid leukaemia (AML) subtypes and solid tumours remains poor. Recent studies directed to improve ATRA responsiveness in non-APL AML seem to indicate that the lack of effective ATRA response in these tumours may be primarily due to aberrant epigenetics, which negatively affect ATRA-regulated gene expression and its antileukaemic activity. Epigenetic reprogramming could potentially restore therapeutic effects of ATRA in all AML subtypes. This review discusses the current progresses in the understanding how ATRA can be utilised in the therapy of non-APL AML and other cancers.

Topics: Animals; Antineoplastic Agents; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Tretinoin

Acute myeloid leukemia (AML) is a hematologic malignancy with great variability in the pathogenesis, clinical features and treatment outcomes. Advances in molecular research have greatly improved our understanding of the leukemogenesis in AML. In addition to the conventional risk factors molecular genetic alterations, such as mutations of NPM1, CEBPA, c-KIT, AML1/RUNX1, WT1, FLT3 and others, are also important prognostic factors in AML patients. Risk-adapted treatment may not only improve the prognosis, but also reduce the toxicity from the chemotherapy in patients with AML.

Topics: Antineoplastic Agents; CCAAT-Enhancer-Binding Proteins; Core Binding Factor Alpha 2 Subunit; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Humans; Imatinib Mesylate; Leukemia, Myeloid, Acute; Mutation; Neoplasm Proteins; Nuclear Proteins; Nucleophosmin; Proto-Oncogene Proteins c-kit; Treatment Outcome; Tretinoin; WT1 Proteins

Despite its dual role in determining cell fate in a wide array of solid cancer cell lines, autophagy has been robustly shown to suppress or kill acute myeloid leukemia cells via degradation of the oncogenic fusion protein that drives leukemogenesis. However, autophagy also induces the demise of acute leukemia cells that do not express the known fusion protein, though the molecular mechanism remains elusive. Nevertheless, since it can induce cooperation with apoptosis and differentiation in response to autophagic signals, autophagy can be manipulated for a better therapy on acute myeloid leukemia.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Molecular Targeted Therapy; Oncogene Proteins, Fusion; Tretinoin

Recent cytogenetical findings and novel molecular biology results of acute myeloid leukaemia have shed new lights of our understanding in the diagnosis and treatment of the disease. Acute myeloid leukaemia is not only represented by the wide variety of morphological and immunophenotypic diversity but also demonstrates cytogenetical and molecular biological heterogeneity of its own. It has an unfavorable prognosis, especially in the elderly. Overall survival of younger patients (<50-60 years) has increased in the past years due to high dose chemotherapy (daunorubicine, cytarabine). But in case of unfavorable prognostic factors (not only cytogenetical but also molecular biological characters of the disease), allogeneic stem cell transplantation is needed for successful overall outcome. Better understanding the biology of acute myeloid leukaemia could establish novel targeted therapies and help us eventually to cure the disease.

Topics: Age Factors; Aminoglycosides; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Boronic Acids; Bortezomib; Cytarabine; Disease-Free Survival; Drug Resistance, Neoplasm; fms-Like Tyrosine Kinase 3; Gemtuzumab; Hematopoietic Stem Cell Transplantation; Humans; Idarubicin; Leukemia, Myeloid, Acute; Mitoxantrone; Molecular Targeted Therapy; Oxides; Prognosis; Pyrazines; Recurrence; Transplantation, Homologous; Treatment Outcome; Tretinoin

Since the 1970s, the concept of differentiation therapy has been viewed as a promising and revolutionary approach for the treatment of acute myeloid leukemia (AML) and other cancers. However, the successful clinical application of differentiation therapy has only been realized since the late 1980s and only in one subtype of AML, acute promyelocytic leukemia (APL). The use of all-trans-retinoic acid (ATRA) and arsenic trioxide, both of which induce degradation of the progressive multifocal leukoencephalopathy/retinoic acid receptor alpha oncoprotein, in combination with chemotherapy is currently the accepted treatment of APL, presenting a potential paradigm for differentiation therapy in clinical oncology.. We have begun to understand why ATRA fails to induce differentiation in AML. The underlying reasons identified thus far are associated with an inability to target the removal of leukemogenic fusion proteins, aberrant epigenetic regulation of genes involved in the ATRA signaling pathway and the presence of factors that interfere with proper retinoic acid receptor alpha function.. Here, we examine the reasons why the exquisite sensitivity of APL to ATRA-based differentiation therapy has not been extended to other of AML subtypes. Current differentiation-based combinatorial approaches to target AML will also be analyzed. Finally, we will evaluate the potential of novel strategies, high-throughput screening, and functional genomics to uncover new differentiation-based therapies for AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Humans; Leukemia, Myeloid, Acute; Oxides; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Sensitivity and Specificity; Tretinoin

Therapy-related myelodysplastic syndrome (t-MDS) and therapy-related acute myelogenous leukemia (t-AML) in patients with acute promyelocytic leukemia (APL) are rare events. The cumulative exposure to chemotherapy with alkylating agents and topoisomerase II inhibitors is associated with t-AML that may develop any time after the completion of the treatment. We report the case of an acquired AML who previously received therapy for APL, after two years of being diagnosed. The diagnosis was established by morphologic findings, membrane markers, cytogenetic studies, and fluorescence in situ hybridization (FISH). To our knowledge this is the first documented case in Puerto Rico of a patient with APL that developed a t-AML without the characteristic chromosomal and morphologic findings of APL.

Topics: Adult; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 7; Cytarabine; Daunorubicin; Drug Synergism; Fatal Outcome; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Mercaptopurine; Methotrexate; Mitoxantrone; Monosomy; Neoplasms, Second Primary; Translocation, Genetic; Tretinoin

The fundamental principle of the treatment of AML (acute myeloid leukemia) is "total cell kill. " For remission induction, "response-oriented individualized therapy" was developed in Japan. However, the similar response rate was obtained by "set therapy," which became the present standard regimen. Regarding the post-remission therapy, consolidation therapy is conducted without further long-term maintenance/intensification therapy. For poor-risk patients, hematopoietic stem cell transplantation should be considered. To improve the therapeutic efficacy, the development of molecular targeted therapy will be indispensable. As for CML (chronic myeloid leukemia), the development of imatinib has completely changed the treatment strategy. The eradication of CML stem cells is the next challenging issue.

Topics: Antineoplastic Agents; Benzamides; Cytarabine; Drug Design; Drug Therapy, Combination; Gene Targeting; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Piperazines; Precision Medicine; Pyrimidines; Remission Induction; Risk; Tretinoin

Great progress on insight into genetic aberrations of myeloid leukemia via gene expression profiling has led to better understanding of the pathobiology of this heterogeneous disorder. It enabled the development of specific treatment modalities targeted to underlying oncogenic abnormalities, with well established examples of all-trans retinoic acid for the treatment of acute promyelocytic leukemia and imatinib for chronic myeloid leukemia. However, these strategies have not been completely developed yet in that most of brand new targeted therapies have been somewhat far from achieving cure of leukemia and that many problems with regards to drug resistance and recurrence from minimal residual disease remain to be solved. On the other hand, concept of cancer(leukemic) stem cell and its niche has been shedding new light on oncological field these days. This review summarizes the current clinical trials using new targeted therapies and research trends on myeloid leukemia.

Topics: Animals; Antineoplastic Agents; Benzamides; Chromosome Aberrations; Clinical Trials as Topic; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Neoplastic Stem Cells; Piperazines; Pyrimidines; Translational Research, Biomedical; Tretinoin

The heterogeneity of acute myeloid leukemia (AML) results from a complex network of cytogenetic aberrations and molecular mutations. These genetic markers are the basis for the categorization of cases within distinct subgroups and are highly relevant for the prediction of prognosis and for therapeutic decisions in AML. Clinical variances within distinct genetically defined subgroups could in part be linked to the interaction of diverse mutation classes, and the subdivision of normal karyotype AML on the basis of recurrent molecular mutations gains increasing relevance for therapeutic decisions. In parallel to these important insights in the complexity of the genetic networks in AML, a variety of diverse new compounds is being investigated in preclinical and clinical studies. These approaches aim to develop targeted treatment concepts that are based on interference with molecular genetic or epigenetic mechanisms. This review provides an overview on the most relevant genetic markers, which serve as basis for targeted therapy approaches now or might represent options for such approaches in the future, and summarizes recent results of targeted therapy studies.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Cycle; Cell Division; Chromosome Aberrations; Genetic Markers; Humans; Karyotyping; Leukemia, Myeloid, Acute; Models, Genetic; Mutation; Oxides; Proto-Oncogene Proteins c-kit; Transcription, Genetic; Tretinoin

Currently, no standard treatment is available for elderly patients with de novo/secondary acute myeloid leukemia (AML) who are not eligible for intensive chemotherapy. New, less aggressive therapies are therefore needed. Histone deacetylase inhibitors (HDACi) are known to reduce proliferation and induce differentiation in hematological malignancies. With all-trans retinoic acid (ATRA) these effects have been reported to be even enhanced. Valproic acid (VPA) is an HDACi and has been known as anti-epileptic agent for many years. We treated 21 patients with de novo/secondary AML and 1 patient with myelodysplastic syndrome with ATRA (45 mg/m(2)/day in 2 doses, 14 days, q29 days) and VPA (150 mg/day 1 week, then 300 mg/day, continuously). Treatment was tolerated well with moderate side effects. 4 patients revealed hematological improvement and another 4 patients experienced a reduction in transfusion dependency. The overall response rate was 27%. Our study is presented together with an overview of the literature on the topic.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Enzyme Inhibitors; Humans; Leukemia, Myeloid, Acute; Middle Aged; Myelodysplastic Syndromes; Palliative Care; Prevalence; Retrospective Studies; Treatment Outcome; Tretinoin; Valproic Acid

The same progress in the recent therapeutic strategy for older adults with hematological malignancies has also been seen in younger adults. The standard initial therapy for elderly acute promylocytic leukemia is the combination with all-trans retinoic acid and anthracyclines. For other acute myeloid leukemias (AML), many trials of combination chemotherapy have not improved the outcome of elderly patients. Gemtuzumab ozogamicin,which is an immunoconjugate binding to CD 33 on the surface of AML blasts, has produced good results for elderly patients in either monotherapy or in combination with conventional chemotherapeutic drugs. One of the BCR-ABL tyrosine kinase inhibitors, imatinib mesylate, is active for elderly Philadelphia-positive leukemia including acute lymphoblastic leukemia and chronic myeloid leukemia. In the treatment of elderly diffuse large B cell lymphoma, combination of rituximab and cyclophosphamide+doxorubicin+vincristine+prednisone (CHOP) has become the therapy of choice based upon a Groupe d'Etude des Lymphomes de l'Adulte (GELA) trial even though there are some other trials for elderly patients such as dose-dense CHOP therapy. For follicular lymphoma, combination therapies of rituximab and cytotoxic drugs such as R-CHOP and R-CVP are also considered as promising therapies. For the management of multiple myeloma, high-dose chemotherapy, mainly melphalan with autologous stem cell transplantation, has become the standard treatment even for elderly patients less than 65 years of age.

Topics: Aged; Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Cyclophosphamide; Doxorubicin; Drug Administration Schedule; Gemtuzumab; Hematologic Neoplasms; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Multiple Myeloma; Prednisone; Rituximab; Survival Rate; Tretinoin; Vincristine

Topics: Adult; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Blood Component Transfusion; Child; Child, Preschool; Female; Hemorrhage; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tretinoin

Topics: Evidence-Based Medicine; Hematopoietic Stem Cell Transplantation; Histocompatibility; HLA Antigens; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Remission Induction; Transplantation, Autologous; Transplantation, Homologous; Treatment Outcome; Tretinoin

In eukaryotic organisms cellular fate and tissue specific gene expression are regulated by the activity of proteins known as transcription factors that by interacting with specific DNA sequences direct the activation or repression of target genes. The post genomic era has shown that transcription factors are not the unique key regulators of gene expression. Epigenetic mechanisms such as DNA methylation, post-translational modifications of histone proteins, remodeling of nucleosomes and expression of small regulatory RNAs also contribute to regulation of gene expression, determination of cell and tissue specificity and assurance of inheritance of gene expression levels. The relevant contribution of epigenetic mechanisms to a proper cellular function is highlighted by the effects of their deregulation that cooperate with genetic alterations to the development of various diseases and to the establishment and progression of tumors.

Topics: Chromatin; DNA; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation; Gene Silencing; Genetic Diseases, Inborn; Heterochromatin; Histones; Humans; Leukemia, Myeloid, Acute; Models, Biological; Neoplasms; Nucleosomes; Oligodeoxyribonucleotides; Protein Processing, Post-Translational; Protein Structure, Tertiary; RNA; RNA Interference; Sequence Analysis, DNA; Transcription, Genetic; Tretinoin

Topics: Animals; Antineoplastic Agents; Benzamides; fms-Like Tyrosine Kinase 3; Genetic Therapy; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Piperazines; Proto-Oncogene Proteins; Pyrimidines; Receptor Protein-Tyrosine Kinases; Tretinoin

Acute leukemia is classified broadly as either acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL). The main treatments remain remission induction therapy and postremission chemotherapy. The advances in chemotherapy for pediatric patients with ALL have been dramatic, with some 95% achieving complete remission, and long-term survival is 60-80%. Among adults, high long-term survival rates due to improvements in chemotherapy for B-cell type ALL and core binding factor leukemias have been reported. For adult leukemias overall, however, long-term survival rates have stalled at 15-40% despite the high remission rate attained. In most cases this is due to a recurrence. Among the prognostic factors reported for acute leukemia, chromosome type may be cited as the currently most reliable. Acute leukemia patients are classified based on chromosome type, and the postremission therapeutic strategy is considered in terms of an appropriate combination of chemotherapy and hematopoietic stem cell transplant. This accounts for an important part of the treatment given today. Target-based therapies such as all-trans-retinoic acid (ATRA) for AML have brought dramatic improvements in treatment results. The effect of imanitib against Philadelphia chromosome positive ALL, for which the prognosis is poor, is also attracting attention. Moreover, promising new treatment strategies that have been developed, including cord blood transplant, mini-transplant, antibody therapy, and immunotherapy, Clinical studies of PCR and other means to reveal small residual lesions and estimate prognosis are also making progress. In the future it will be possible to identify prognostic factors in genetic tests such as DNA microarrays and single nucleotide polymorphisms, so that the optimum treatment can be selected for individual patients.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Chromosome Aberrations; Combined Modality Therapy; Female; Humans; Leukemia, Myeloid, Acute; Male; Philadelphia Chromosome; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Stem Cell Transplantation; Survival Rate; Survivors; Tretinoin

The WHO classification of adultory acute myeloid leukemia puts the main emphasis on prognosis, based mainly upon cytogenetic findings and gene-expression profiles. The complex prognostic assessment provides a more solid basis for early therapeutic stratification. This review focuses mainly on medical therapy. Induction phase is quite uniform, it consists of antracyclin and cytosin arabinosid. High-dose cytosin arabinosid is the predominant tool of postinduction therapy, especially in the favorable cytogenetic pattern cases. All-trans retinoic acid resulted in extremely good results in the promyelocytic cases, and this therapy seems to be advantageous in respect of acute DIC, too. Arsenic trioxide could be the drug of choice in relapsed promyelocytic leukemia. Some new agents are promising in refractory or relapsed cases, i.e. antibodies (Mylotarg) or farnesyltransferase inhibitors. The near future may bring about new therapeutic approaches, involving immunotherapy (dendritic cell vaccines) or gene-therapy as well.

Topics: Adult; Anthracyclines; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Cytarabine; Drug Resistance, Neoplasm; Gene Expression Profiling; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Oligonucleotide Array Sequence Analysis; Oxides; Palliative Care; Prognosis; Remission Induction; Tretinoin

Despite major recent advances in the understanding of the molecular biology of adult acute myeloblastic leukemia(AML), the treatment of the disease remains challenging. This review summarizes literature in the field of curative chemotherapy for adult acute myeloblastic leukemia(AML). In particular, detailed analysis of curing leukemia in patients treated with risk-oriented chemotherapy and in patients with APL was presented. In addition, results using our curative chemotherapy approach for adult AML were reported. Lastly, future directions for curing adult AML were discussed.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Chromosome Aberrations; Clinical Trials as Topic; Humans; Leukemia, Myeloid, Acute; Prognosis; Risk; Tretinoin

Topics: Alkyl and Aryl Transferases; Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Benzoates; Cyclosporine; Cytarabine; Daunorubicin; Drug Design; Farnesyltranstransferase; Gemtuzumab; Humans; Leukemia, Myeloid, Acute; Neoplasm Recurrence, Local; Nucleosides; Oxides; Prognosis; Radioisotopes; Remission Induction; Tetrahydronaphthalenes; Topoisomerase Inhibitors; Tretinoin; Verapamil

Malignancies in childhood occur with an incidence of 13-14 per 100,000 children under the age of 15 years. Acute lymphoblastic leukaemia with an incidence of 29% is the most common paediatric malignancy, whereas acute myeloid leukaemias account for about 5%. The treatment of acute leukaemias consists of sequential therapy cycles (induction, consolidation, intensification, maintenance therapy) with different cytostatic drugs over a time period of up to 1.5-3 years. Over the last 25 years of clinical trials, a significant rise in the rate of complete remissions as well as an increase in long-term survival has been achieved. Therefore, growing attention is now focused on the long-term effects of antileukaemic treatment. Several cytostatic drugs administered in the treatment of acute leukaemia in childhood are known to cause long-term adverse effects. Anthracyclines may induce chronic cardiotoxicity, alkylating agents are likely to cause gonadal damage and secondary malignancies and the use of glucocorticoids may cause osteonecrosis. Most of the long-term adverse effects have not been analysed systematically. Approaches to minimising long-term adverse effects without jeopardising outcome have included: the design of new drugs such as a liposomal formulation of anthracyclines, the development of anthracycline-derivates with lower toxicity, the development of cardioprotective agents or, more recently, the use of targeted therapy;alternative administration schedules like continuous infusion or timed sequential therapy; and risk group stratification by the monitoring of minimal residual disease. Several attempts have been made to minimise the cardiotoxicity of anthracyclines: decreasing concentrations delivered to the myocardium by either prolonging infusion time or using liposomal formulated anthracyclines or less cardiotoxic analogues, or the additional administration of cardioprotective agents. The advantage of these approaches is still controversial, but there are ongoing clinical trials to evaluate the long-term effects. The use of new diagnostic methods, such as diagnosis of minimal residual disease, which allow reduction or optimisation of dose, offer potential advantages compared with conventional treatment in terms of reducing the risk of severe long-term adverse effects. Most options for minimising long-term adverse effects have resulted from theoretical models and in vitro studies, but only some of the modalities such as the use of dexrazoxane, the con

Topics: Alkylating Agents; Anthracyclines; Antimetabolites; Antineoplastic Agents; Asparaginase; Asparagine; Cardiovascular Diseases; Child; Clinical Trials as Topic; Glucocorticoids; Gonadal Disorders; Humans; Leukemia; Leukemia, Myeloid, Acute; Osteonecrosis; Podophyllotoxin; Polyethylene Glycols; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tretinoin; Vinca Alkaloids

Topics: DNA Methylation; Gene Expression Profiling; Histone Deacetylase Inhibitors; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Translocation, Genetic; Tretinoin

In vitro studies that showed RA could cause growth arrest and differentiation of myelogenous leukemia and neuroblastoma led to clinical trials of retinoids in APL and neuroblastoma that increased survival for both of those diseases. In the case of APL, ATRA has been the drug of choice, and preclinical and clinical data support direct combinations of ATRA with cytotoxic chemotherapy. For neuroblastoma, a phase I study defined a dose of 13-cis-RA, which was tolerable in patients after myeloablative therapy, and a phase III trial that showed postconsolidation therapy with 13-cis-RA improved EFS for patients with high-risk neuroblastoma. Preclinical studies in neuroblastoma indicate that ATRA or 13-cis-RA can antagonize cytotoxic chemotherapy and radiation, so use of 13-cis-RA in neuroblastoma is limited to maintenance after completion of cytotoxic chemotherapy and radiation. A limitation on the antitumor benefit of ATRA in APL is the marked decrease in drug levels that occurs during therapy as a result of induction of drug metabolism, resulting in a shorter drug half-life and decreased plasma levels. Although early studies sought to overcome the pharmacologic limitations of ATRA therapy in APL, the demonstration that ATO is active against APL in RA-refractory patients has led to a focus on studies employing ATO. Use of 13-cis-RA in neuroblastoma has avoided the decreased plasma levels seen with ATRA. It is likely that recurrent disease seen during or after 13-cis-RA therapy in neuroblastoma is due to tumor cell resistance to retinoid-mediated differentiation induction. Studies in neuroblastoma cell lines resistant to 13-cis-RA and ATRA have shown that they can be sensitive, and in some cases collaterally hypersensitive, to the cytotoxic retinoid fenretinide. Fenretinide induces tumor cell cytotoxicity rather than differentiation, acts independently from RA receptors, and in initial phase I trials has been well tolerated. Clinical trials of fenretinide, alone and in combination with ceramide modulators, are in development.

Topics: Antineoplastic Agents; Child; Clinical Trials as Topic; Fenretinide; Humans; Isotretinoin; Leukemia, Myeloid, Acute; Neoplasms; Neuroblastoma; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Retinoids; Tretinoin

Topics: Antineoplastic Agents; Clinical Trials as Topic; Cytarabine; Humans; Karyotyping; Leukemia, Myeloid, Acute; Multicenter Studies as Topic; Prognosis; Risk Factors; Survival Analysis; Tretinoin

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Animals; Antigens, CD; Antigens, Differentiation; Apoptosis; Gene Expression Regulation; Hematopoiesis; Humans; Immunotoxins; Leukemia, Myeloid, Acute; Membrane Glycoproteins; Multienzyme Complexes; NAD+ Nucleosidase; Tretinoin

A differentiation block with accumulation of immature myeloid cells characterizes acute myelogenous leukemia (AML). However, native AML cells often show some morphological signs of differentiation that allow a classification into different subsets, and further differentiation may be induced by exposure to various soluble mediators, e.g., all trans-retinoic acid (ATRA) and several cytokines. Combination therapy with ATRA and chemotherapy should now be regarded as the standard treatment for the acute promyelocytic leukemia variant of AML. Several agents can induce leukemic cell differentiation for other AML subtypes, although these effects differ between patients. Differentiation may then be associated with induction of apoptosis, and differentiation-inducing therapy may therefore become useful in combination with intensive chemotherapy to increase the susceptibility of AML blasts to drug-induced apoptosis. However, it should be emphasized that differentiation and apoptosis can occur as separate events with different regulation in AML cells, and future studies in AML should therefore focus on: A) the identification of new agents with more predictable effects on differentiation and apoptosis; B) the use of clinical and laboratory parameters to define new subsets of AML patients in which differentiation/apoptosis induction has a predictable and beneficial effect, and C) further characterization of how AML blast sensitivity to drug-induced apoptosis is modulated by differentiation induction.

Topics: Adult; Apoptosis; Cell Differentiation; Genes, Tumor Suppressor; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Prognosis; Proto-Oncogene Proteins c-bcl-2; Tretinoin

The current situation and perspectives for the treatment of acute myelogenous leukemia in adults were summarized. Due to the recent progress of chemotherapy, the complete remission (CR) rate is close to 80% in adults with newly diagnosed acute myelogenous leukemia in Japan. On the other hand, the long-term disease-free survival rate is 30-40% in CR cases. The allogeneic bone marrow transplantation (allo BMT) during the first CR has a low relapse rate compared with that of chemo therapy. However, the therapy-related mortality is not inconsiderable, so, the time of allo BMT is controversial. Idarubicin has a superior CR rate as well as long-term disease-free survival in comparison with daunorubicin, and it is becoming the first line treatment for acute myelogenous leukemia in adults.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Clinical Trials as Topic; Combined Modality Therapy; Disease-Free Survival; Drug Administration Schedule; Humans; Idarubicin; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Remission Induction; Survival Rate; Tretinoin

Due to advances in chemotherapy, differentiation therapy and bone marrow transplantation (BMT), adult acute myeloid leukemia (AML) has become a curable disease, and we are making further efforts to heighten the cure rate. The JALSG AML 89 study resulted in a 77% complete remission (CR) rate in 326 adults with AML, and a 38% 4.5-year disease-free survival (DFS) in CR cases. The JALSG AML92 study for APL with all-trans retinoic acid resulted in a 89% CR rate in 196 and 64% 4-year DFS in CR cases.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Cytarabine; Daunorubicin; Drug Resistance, Neoplasm; Humans; Infusions, Intravenous; Leukemia, Myeloid, Acute; Mercaptopurine; Middle Aged; Prednisolone; Tretinoin

The standard treatment of patients with acute myeloid leukemia (AML) has depended on the elimination of the leukemic clone with cytotoxic myeloablation. Differentiation therapy is receiving increasing attention due to the remarkable activity of a vitamin A derivative, all-trans retinoic acid (ATRA), in patients with acute promyelocytic leukemia (APL). The majority of patients treated have achieved complete remission and with rapid resolution of the life-threatening bleeding diathesis. Phase II studies with ATRA in APL indicate certain limitations in the therapeutic use of retinoic acid. Patients achieving complete remission with ATRA alone require postremission chemotherapy, once remission is achieved, to extend remission. The results of differentiating therapy in APL patients has encouraged the use of differentiating therapy in patients with non-M3 AML. A number of areas await research, such as the need to develop ways to overcome acquired retinoic resistance and the development of cytochrome P450 inhibitors. Other novel retinoids, such as 9-cis-retinoic acid, may be even more effective than ATRA in APL. Additional important areas of research involve studying combinations of retinoids and other putative differentiating agents, and identifying different selected treatments targeted at specific molecular defects.

Topics: Antineoplastic Agents; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Randomized Controlled Trials as Topic; Survival Rate; Tretinoin

Treatment failure in drug sensitive malignancies is a complex phenomenon resulting from both drug resistance and from the rapid regrowth of malignant cells ('regrowth resistance'). Attempts to overcome regrowth resistance during the treatment of the aggressive lymphomas by increasing the frequency of cytotoxic therapy appears to have failed. An alternative approach of significant potential would be to administer biologically active agents to directly slow the regrowth of neoplastic cells between courses of full dose cytotoxic therapy. To facilitate this approach a new system for classifying treatment failure in the leukemias and lymphomas is needed so that the extent of regrowth resistance and the effects of treatment on regrowth resistance can be directly assessed. Accordingly, a new classification system is proposed.

Topics: Antineoplastic Combined Chemotherapy Protocols; Drug Resistance; Genes, myc; Genes, p53; Humans; Interferons; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lymphoma; Treatment Failure; Tretinoin

Topics: Carrier Proteins; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Myelodysplastic Syndromes; Neoplasms; Receptors, Retinoic Acid; Retinoids; Teratogens; Tretinoin

Numerous clinical trials in adult patients with newly diagnosed acute myelogenous leukemia indicate that the survival rate is approximately 20%. A limited number of favorable prognostic features have been identified; patients who present with specific cytogenetic abnormalities such as t(15;17), t(8;21) and inv (16) tend to have survival rates approaching 40%. However, such patients constitute only a small proportion of the total denominator and it has therefore been a major focus of scientific endeavor to develop new drugs for the treatment of this disease. A wide variety of agents have undergone preliminary testing in this regard and include both natural substances such as homoherringtonine, a drug that has demonstrated modest complete remission rates in this disease and all-trans retinoic acid, a compound that has revolutionized the treatment of acute promyelocytic leukemia. This review will put in perspective some of the newer drugs for the treatment of acute myelogenous leukemia and allow for some conclusions to be drawn as to their impact in the treatment of this disease.

Topics: Amsacrine; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Cytarabine; Daunorubicin; Drug Evaluation; Humans; Idarubicin; Isomerism; Leukemia, Myeloid, Acute; Mitoxantrone; Remission Induction; Survival Rate; Tretinoin

The aim of differentiation therapy is to induce a maturation of the leukemic clone. Various approaches have been used: suppression of proliferation by low dose Ara-C in acute myeloid leukemia (AML); enhancement of differentiation by retinoic acid derivatives in acute promyelocytic leukemia (APL) or by differentiation-inducing factors; modulation of cell metabolism by breaking an autocrine loop in hairy cell leukemia (HCL). In all cases the treatment is given continuously at small doses over a long period of time. The very significant clinical results which have been obtained mainly in APL with all-trans retinoic acid and in HCL with alpha-interferon are briefly discussed.

Topics: Cell Differentiation; Cytarabine; Humans; Interferon Type I; Leukemia; Leukemia, Hairy Cell; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Myelodysplastic Syndromes; Tretinoin

Differentiation therapies try to change the malignant cell in order to acquire a more mature or normal phenotype. Various ways were tested in leukemia: suppression the proliferative pressure by low dose Ara-C, enhancement of the differentiation by retinoic acid derivatives or by differentiation factors, and modulation of the cell metabolism interrupting an autocrine loop (a growth factor and its receptor). The treatment is given continuously at small doses, during a long period of time. In all these cases it seems necessary to tailor the differentiation therapy to each category of leukemia.

Topics: Cell Differentiation; Cell Division; Cytarabine; Humans; Interferon Type I; Leukemia; Leukemia, Hairy Cell; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Myelodysplastic Syndromes; Tretinoin

In this review we have discussed the data which indicates that differentiation can indeed occur to a limited degree in the neoplastic cells in acute leukemia and myelodysplastic syndromes. Attempts to increase the degree of differentiation as an approach to the treatment of these disorders have utilised a variety of chemical agents which have been reported to exert such an effect in vitro. Analysis of the results, however, indicates that beneficial effects of these agents in patients is due to cytotoxic action which selectively reduces the neoplastic cell population. Leukemic cells can also undergo incomplete differentiation in vitro when exposed to appropriate growth factors, which raises the question of whether leukemia and myelodysplasia can be treated by the induction of differentiation in the malignant cells following the administration of these agents. This question, and questions about the nature of complete remission, will be answered in the coming years by clinical trials of growth factors, and further studies of the DNA in mature cells to analyse whether these cells are derived from leukemic progenitors.

Topics: Cell Differentiation; Cytarabine; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Tretinoin

Topics: Arachidonic Acid; Arachidonic Acids; Cell Differentiation; Cell Line; Cells, Cultured; Cyclic AMP; Humans; Leukemia; Leukemia, Myeloid, Acute; Lymphokines; Monocytes; Oncogenes; Receptors, Formyl Peptide; Receptors, Immunologic; T-Lymphocytes; Tretinoin

Human myeloid leukemia cells have a growth advantage over normal cells because they do not differentiate into functional end-cells but remain in the proliferative pool. Several agents have been identified as inducers of differentiation of leukemia cells; among them are the retinoids, 1 alpha,25-dihydroxyvitamin D3 and human tumor necrosis factor alpha, which will be discussed in the following chapter.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Calcitriol; Cell Differentiation; Clinical Trials as Topic; Humans; Leukemia, Experimental; Leukemia, Myeloid, Acute; Mice; Middle Aged; Myelodysplastic Syndromes; Recombinant Proteins; Retinoids; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Adolescent; Adult; Age Factors; Bone Marrow Transplantation; Cell Differentiation; Child, Preschool; Cytarabine; Drug Therapy, Combination; Female; Hematopoiesis; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Tretinoin; Vitamin D

A prominent phenotypic abnormality of human acute myelogenous leukemia cells is the inability of the cells to differentiate to functional mature cells; instead, the cells are blocked at an early stage of development and remain in the proliferative pool and rapidly accumulate. Investigation of the induction of myeloid leukemic cell differentiation has made recent advances with the development of several human myelogenous leukemia cell lines. The lines provide models to study the biology of myeloid differentiation and to identify inducers of differentiation of myeloid leukemic blood cells. This review critically examines the inducers of leukemic cell differentiation and their potential therapeutic importance.

Topics: 3-Deazauridine; Acetamides; Alkaloids; Animals; Calcitriol; Cell Differentiation; Cell Line; Cells, Cultured; Dimethyl Sulfoxide; Glycoproteins; Granulocytes; Growth Inhibitors; Humans; Hypoxanthines; Interferons; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Myeloid, Acute; Lymphokines; Lyngbya Toxins; Macrophages; Mice; Phorbol Esters; Piperidones; Polyethylene Glycols; Tretinoin

Topics: Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Decitabine; Humans; Leukemia, Myeloid, Acute; Tretinoin

Acute myeloid leukaemia with mutated NPM1 is associated with high CD33 expression and intermediate-risk cytogenetics. The aim of this study was to evaluate intensive chemotherapy with or without the anti-CD33 antibody-drug conjugate gemtuzumab ozogamicin in participants with newly diagnosed, NPM1-mutated acute myeloid leukaemia.. This open-label, phase 3 trial was conducted at 56 hospitals in Germany and Austria. Eligible participants were 18 years or older and had newly diagnosed NPM1-mutated acute myeloid leukaemia and an Eastern Cooperative Oncology Group performance status of 0-2. Participants were randomly assigned, using age as a stratification factor (18-60 years vs >60 years), 1:1 to the two treatment groups using allocation concealment; there was no masking of participants and investigators to treatment groups. Participants received two cycles of induction therapy (idarubicin, cytarabine, and etoposide) plus all-trans retinoic acid (ATRA) followed by three consolidation cycles of high-dose cytarabine (or an intermediate dose for those older than 60 years) and ATRA, without or with gemtuzumab ozogamicin (3 mg/m. Between May 12, 2010, and Sept 1, 2017, 600 participants were enrolled, of which 588 (315 women and 273 men) were randomly assigned (296 to the standard group and 292 to the gemtuzumab ozogamicin group). No difference was found in short-term event-free survival (short-term event-free survival at 6-month follow-up, 53% [95% CI 47-59] in the standard group and 58% [53-64] in the gemtuzumab ozogamicin group; hazard ratio [HR] 0·83; 95% CI 0·65-1·04; p=0·10) and overall survival between treatment groups (2-year overall survival, 69% [63-74] in the standard group and 73% [68-78] in the gemtuzumab ozogamicin group; 0·90; 0·70-1·16; p=0·43). There was no difference in complete remission or CRi rates (n=267 [90%] in the standard group vs n=251 [86%] in the gemtuzumab ozogamicin group; odds ratio [OR] 0·67; 95% CI 0·40-1·11; p=0·15) and complete remission or CRh rates (n=214 [72%] vs n=195 [67%]; OR 0·77; 0·54-1·10; p=0·18), whereas the complete remission rate was lower with gemtuzumab ozogamicin (n=172 [58%] vs n=136 [47%]; OR 0·63; 0·45-0·80; p=0·0068). Cumulative incidence of relapse was significantly reduced by gemtuzumab ozogamicin (2-year cumulative incidence of relapse, 37% [95% CI 31-43] in the standard group and 25% [20-30] in the gemtuzumab ozogamicin group; cause-specific HR 0·65; 0·49-0·86; p=0·0028), and there was no difference in the cumulative incidence of death (2-year cumulative incidence of death 6% [4-10] in the standard group and 7% [5-11] in the gemtuzumab ozogamicin group; HR 1·03; 0·59-1·81; p=0·91). There were no differences in the number of days in hospital across all cycles between treatment groups. The most common treatment-related grade 3-4 adverse events were febrile neutropenia (n=135 [47%] in the gemtuzumab ozogamicin group vs n=122 [41%] in the standard group), thrombocytopenia (n=261 [90%] vs n=265 [90%]), pneumonia (n=71 [25%] vs n=64 [22%]), sepsis (n=85 [29%] vs n=73 [25%]). Treatment-related deaths were documented in 25 participants (4%; n=8 [3%] in the standard group and n=17 [6%] in the gemtuzumab ozogamicin group), mostly due to sepsis and infections.. The primary endpoints of the trial of event-free survival and overall survival were not met. However, an anti-leukaemic efficacy of gemtuzumab ozogamicin in participants with NPM1-mutated acute myeloid leukaemia is shown by a significantly lower cumulative incidence of relapse rate, suggesting that the addition of gemtuzumab ozogamicin might reduce the need for salvage therapy in these participants. The results from this study provide further evidence that gemtuzumab ozogamicin should be added in the standard of care treatment in adults with NPM1-mutated acute myeloid leukaemia.. Pfizer and Amgen.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Female; Gemtuzumab; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplasm Recurrence, Local; Nuclear Proteins; Treatment Outcome; Tretinoin; Young Adult

The prognosis of AML patients with adverse genetics, such as a complex, monosomal karyotype and TP53 lesions, is still dismal even with standard chemotherapy. DNA-hypomethylating agent monotherapy induces an encouraging response rate in these patients. When combined with decitabine (DAC), all-trans retinoic acid (ATRA) resulted in an improved response rate and longer overall survival in a randomized phase II trial (DECIDER; NCT00867672). The molecular mechanisms governing this in vivo synergism are unclear. We now demonstrate cooperative antileukemic effects of DAC and ATRA on AML cell lines U937 and MOLM-13. By RNA-sequencing, derepression of >1200 commonly regulated transcripts following the dual treatment was observed. Overall chromatin accessibility (interrogated by ATAC-seq) and, in particular, at motifs of retinoic acid response elements were affected by both single-agent DAC and ATRA, and enhanced by the dual treatment. Cooperativity regarding transcriptional induction and chromatin remodeling was demonstrated by interrogating the HIC1, CYP26A1, GBP4, and LYZ genes, in vivo gene derepression by expression studies on peripheral blood blasts from AML patients receiving DAC + ATRA. The two drugs also cooperated in derepression of transposable elements, more effectively in U937 (mutated TP53) than MOLM-13 (intact TP53), resulting in a "viral mimicry" response. In conclusion, we demonstrate that in vitro and in vivo, the antileukemic and gene-derepressive epigenetic activity of DAC is enhanced by ATRA.

Topics: Decitabine; Humans; Karyotype; Karyotyping; Leukemia, Myeloid, Acute; Tretinoin

All-trans-retinoic acid (ATRA) is highly active in acute promyelocytic leukemia but not in other types of acute myeloid leukemia (AML). Previously, we showed that ATRA in combination with Lysine-specific demethylase 1 (LSD1) inhibition by tranylcypromine (TCP) can induce myeloid differentiation in AML blasts. This phase I/II clinical trial investigated the safety and efficacy of TCP/ATRA treatment as salvage therapy for relapsed/refractory (r/r) AML. The combination was evaluated in 18 patients, ineligible for intensive treatment. The overall response rate was 20%, including two complete remissions without hematological recovery and one partial response. We also observed myeloid differentiation upon TCP/ATRA treatment in patients who did not reach clinical remission. Median overall survival (OS) was 3.3 months, and one-year OS 22%. One patient developed an ATRA-induced differentiation syndrome. The most frequently reported adverse events were vertigo and hypotension. TCP plasma levels correlated with intracellular TCP concentration. Increased H3K4me1 and H3k4me2 levels were observed in AML blasts and white blood cells from some TCP/ATRA treated patients. Combined TCP/ATRA treatment can induce differentiation of AML blasts and lead to clinical response in heavily pretreated patients with r/r AML with acceptable toxicity. These findings emphasize the potential of LSD1 inhibition combined with ATRA for AML treatment.

Topics: Adult; Aged; Antidepressive Agents; Antineoplastic Agents; Arabidopsis Proteins; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Therapy, Combination; Female; Follow-Up Studies; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Proof of Concept Study; Prospective Studies; Salvage Therapy; Survival Rate; Transcription Factors; Tranylcypromine; Tretinoin; Young Adult

In preclinical studies, the lysine-specific histone demethylase 1A (LSD1) inhibitor tranylcypromine (TCP) combined with all-trans retinoic acid (ATRA) induces differentiation and impairs survival of myeloid blasts in non-acute promyelocytic leukemia acute myeloid leukemia (AML). We conducted a phase I clinical trial (NCT02273102) to evaluate the safety and activity of ATRA plus TCP in patients with relapsed/refractory AML and myelodysplasia (MDS).. Seventeen patients were treated with ATRA and TCP (three dose levels: 10 mg twice daily, 20 mg twice daily, and 30 mg twice daily).. ATRA-TCP had an acceptable safety profile. The MTD of TCP was 20 mg twice daily. Best responses included one morphologic leukemia-free state, one marrow complete remission with hematologic improvement, two stable disease with hematologic improvement, and two stable disease. By intention to treat, the overall response rate was 23.5% and clinical benefit rate was 35.3%. Gene expression profiling of patient blasts showed that responding patients had a more quiescent CD34. These data indicate that LSD1 inhibition sensitizes AML cells to ATRA and may restore ATRA responsiveness in subsets of patients with MDS and AML.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Female; Gene Expression Regulation, Leukemic; Histone Demethylases; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Transcriptome; Tranylcypromine; Tretinoin

DNA-hypomethylating agents are studied in combination with other epigenetic drugs, such as histone deacetylase inhibitors or differentiation inducers (eg, retinoids), in myeloid neoplasias. A randomized, phase II trial with a 2 × 2 factorial design was conducted to investigate the effects of the histone deacetylase inhibitor valproate and all-. Two hundred patients (median age, 76 years; range, 61-92 years) ineligible for induction chemotherapy received decitabine (20 mg/m. The addition of ATRA resulted in a higher remission rate (21.9% with ATRA. The addition of ATRA to decitabine resulted in a higher remission rate and a clinically meaningful survival extension in these patients with difficult-to-treat disease, without added toxicity.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Decitabine; Disease-Free Survival; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin; Valproic Acid

High CD33 expression in acute myeloid leukemia (AML) with mutated. Between May 2010 and September 2017, patients ≥ 18 years old and considered eligible for intensive therapy were randomly assigned up front for induction therapy with idarubicin, cytarabine, etoposide, and all-. Five hundred eighty-eight patients were randomly assigned (standard arm, n = 296; GO arm, n = 292). EFS in the GO arm was not significantly different compared with that in the standard arm (hazard ratio, 0.83; 95% CI, 0.65 to 1.04;. The trial did not meet its early primary end point of EFS, mainly as a result of a higher early death rate in the GO arm. However, in patients achieving CR/CRi after induction therapy, significantly fewer relapses occurred in the GO compared with the standard arm.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Immunological; Antineoplastic Combined Chemotherapy Protocols; Consolidation Chemotherapy; Cytarabine; Etoposide; Female; Gemtuzumab; Humans; Idarubicin; Induction Chemotherapy; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Nuclear Proteins; Nucleophosmin; Progression-Free Survival; Prospective Studies; Tretinoin; Young Adult

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD-Scid-IL2rγ(null)mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies.

Topics: Amino Acid Substitution; Animals; Blast Crisis; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Survival; Female; Granulocytes; HL-60 Cells; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Male; Mice; Mice, Nude; Mutation, Missense; Neoplasm Proteins; Tretinoin; Xenograft Model Antitumor Assays

Outcome of patients with primary refractory acute myeloid leukemia remains unsatisfactory. We conducted a prospective phase II clinical trial with gemtuzumab ozogamicin (3 mg/m(2) intravenously on day 1), all-trans retinoic acid (45 mg/m(2) orally on days 4-6 and 15 mg/m(2) orally on days 7-28), high-dose cytarabine (3 g/m(2)/12 h intravenously on days 1-3) and mitoxantrone (12 mg/m(2) intravenously on days 2-3) in 93 patients aged 18-60 years refractory to one cycle of induction therapy. Primary end point of the study was response to therapy; secondary end points included evaluation of toxicities, in particular, rate of sinusoidal obstruction syndrome after allogeneic hematopoietic cell transplantation. Complete remission or complete remission with incomplete blood count recovery was achieved in 47 (51%) and partial remission in 10 (11%) patients resulting in an overall response rate of 61.5%; 33 (35.5%) patients had refractory disease and 3 patients (3%) died. Allogeneic hematopoietic cell transplantation was performed in 71 (76%) patients; 6 of the 71 (8.5%) patients developed moderate or severe sinusoidal obstruction syndrome after transplantation. Four-year overall survival rate was 32% (95% confidence interval 24%-43%). Patients responding to salvage therapy and undergoing allogeneic hematopoietic cell transplantation (n=51) had a 4-year survival rate of 49% (95% confidence intervaI 37%-64%). Patients with fms-like tyrosine kinase internal tandem duplication positive acute myeloid leukemia had a poor outcome despite transplantation. In conclusion, the described regimen is an effective and tolerable salvage therapy for patients who are primary refractory to one cycle of conventional intensive induction therapy. (clinicaltrials.gov identifier: 00143975).

Topics: Adult; Aminoglycosides; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Comorbidity; Consolidation Chemotherapy; Cytarabine; Female; Gemtuzumab; Hematopoietic Stem Cell Transplantation; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Remission Induction; Salvage Therapy; Treatment Outcome; Tretinoin; Young Adult

Acute myeloid leukemia (AML) is predominantly a disease of older patients with a poor long-term survival. Approval of decitabine (DAC) in the European Union (EU) in 2012 for the treatment of patients with AML ≥65 years marks the potential for hypomethylating agents in elderly AML. Nevertheless the situation is dissatisfactory and the quest for novel treatment approaches, including combination epigenetic therapy is actively ongoing. The given randomized trial should be helpful in investigating the question whether combinations of DAC with the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and/or all-trans retinoic acid (ATRA), which in vitro show a very promising synergism, are superior to the DAC monotherapy. The accompanying translational research project will contribute to find surrogate molecular end points for drug efficacy and better tailor epigenetic therapy. An additional aim of the study is to investigate the prognostic value of geriatric assessments for elderly AML patients treated non-intensively.. DECIDER is a prospective, randomized, observer blind, parallel group, multicenter, Phase II study with a 2x2 factorial design. The primary endpoint is objective best overall response (complete remission (CR) and partial remission (PR)). The target population is AML patients aged 60 years or older and unfit for standard induction chemotherapy. Patients are randomized to one of the four treatment groups: DAC alone or in combination with VPA or ATRA or with both add-on drugs. One interim safety analysis was planned and carried out with the objective to stop early one or more of the treatment arms in case of an unacceptable death rate. This analysis showed that in all treatment arms the critical stopping rule was not reached. No important safety issues were observed. The Data Monitoring Committee (DMC) recommended continuing the study as planned. The first patient was included in December 2011. A total of 189 out of 200 planned patients were randomized since then (status 31.12.2014).. ClinicalTrials.gov identifier: NCT00867672 (registration date 23.03.2009); German clinical trials registry number: DRKS00000733 (registration date 19.04.2011).

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Clinical Protocols; Decitabine; Female; Humans; Induction Chemotherapy; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin; Valproic Acid

We conducted a phase I study using midostaurin (25 or 50 mg orally twice daily), all-trans retinoic acid (ATRA) and CLAG chemotherapy to target multiple pathways in relapsed/refractory AML. 10 patients received the combination and no dose-limiting toxicities were observed. Two patients (22 %) achieved complete remission and 1 patient (11 %) achieved complete remission with incomplete count recovery. Pharmacokinetic data showed that the 25 mg dosing of midostaurin achieved therapeutic levels with no significant interaction between midostaurin and ATRA. With evidence of activity of ATRA in NPM1-mutated AML and midostaurin in FLT3-ITD AML, this combination warrants further investigation.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cladribine; Cytarabine; Female; fms-Like Tyrosine Kinase 3; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Nuclear Proteins; Nucleophosmin; Recurrence; Remission Induction; Staurosporine; Treatment Outcome; Tretinoin

The outcome of patients with acute myeloid leukemia who are older than 60 years has remained poor because of unfavorable disease characteristics and patient-related factors. The randomized German-Austrian AML Study Group 06-04 protocol was designed on the basis of in vitro synergistic effects of valproic acid (VPA) and all-trans retinoic acid with chemotherapy. Between 2004 and 2006, 186 patients were randomly assigned to receive 2 induction cycles with idarubicin, cytarabine, and all-trans retinoic acid either with VPA or without (STANDARD). In all patients, consolidation therapy was intended. Complete remission rates after induction tended to be lower in VPA compared with STANDARD (40% vs 52%; P = .14) as a result of a higher early death rate (26% vs 14%; P = .06). The main toxicities attributed to VPA were delayed hematologic recovery and grade 3/4 infections, observed predominantly during the second induction cycle. After restricting VPA to the first induction cycle and reducing the dose of idarubicin, these toxicities dropped to rates observed in STANDARD. After a median follow-up time of 84 months, event-free and overall survival were not different between the 2 groups (P = .95 and P = .57, respectively). However, relapse-free-survival was significantly superior in VPA compared with STANDARD (24.4% vs 6.4% at 5 years; P = .02). Explorative subset analyses revealed that AML with mutated Nucleophosmin 1 (NPM1) may particularly benefit from VPA. This trial was registered at www.clinicaltrials.gov as #NCT00151255.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Critical Care; Cytarabine; Disease-Free Survival; Drug Synergism; Enzyme Inhibitors; Female; Follow-Up Studies; Humans; Idarubicin; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Nuclear Proteins; Nucleophosmin; Survival Rate; Tretinoin; Valproic Acid

Heat shock protein (HSP) 70 and HSP90 are released by primary human acute myeloid leukemia (AML) cells during stress-induced spontaneous in vitro apoptosis. The AML cells also show constitutive release of several cytokines and the systemic serum levels of several soluble mediators are altered in patients with untreated AML. In the present study, we have investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving AML-stabilizing palliative treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Patients with untreated AML showed increased HSP90 levels and a distinct serum cytokine profile when compared with healthy controls, and low pre-therapy HSP90 levels were associated with a prolonged survival during treatment with ATRA + valproic acid + theophyllin. Hierarchical cluster analysis showed a close association between HSP70, HSP90, IL-1 receptor antagonist (IL-1ra), and hepatocyte growth factor (HGF) levels. Furthermore, disease-stabilizing therapy altered the serum-cytokine profile, but the correlations between HSP70/HSP90/IL-1ra/HGF were maintained only when ATRA + valproic acid were combined with theophyllin but not when combined with cytarabine. We conclude that both HSP levels and serum cytokine profiles are altered and may represent possible therapeutic targets or prognostic markers in human AML.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Cytokines; Female; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Male; Middle Aged; Neoplasm Proteins; Palliative Care; Prognosis; Theophylline; Tretinoin; Tumor Cells, Cultured; Valproic Acid

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Cohort Studies; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; MicroRNAs; Myelodysplastic Syndromes; Treatment Outcome; Tretinoin; Valproic Acid

To optimize treatment for younger patients with acute myeloid leukemia and high-risk myelodysplastic syndrome by comparing induction options and the number of consolidation courses and whether consolidation should include transplantation.. We randomly assigned 1,658 patients younger than age 60 years to receive mitoxantrone/cytarabine/etoposide versus cytarabine/daunorubicin/etoposide and subsequently 1,193 patients to daunorubicin/cytarabine/thioguanine (DAT) where the cytarabine dose was standard (S-DAT) versus double the standard dose (H-DAT). Patients in this randomization were randomly assigned to all-trans-retinoic acid or not. In consolidation, 992 patients were randomly assigned between a total of four courses versus five courses, and 324 patients who were not good risk were randomly assigned to transplantation or chemotherapy as the final course.. Complete remission (CR) was achieved in 74% of patients and CR without recovery was achieved in an additional 11%; overall survival (OS) at 8 years was 38%. No differences in CR, relapse-free survival, relapse, or OS were seen between any of the induction randomizations except for a reduction in relapse risk (RR) on the mitoxantrone arm, which was offset by increased myelosuppression and deaths in CR. The addition of a fifth course did not improve OS and may be detrimental in older patients. Although transplantation reduced RR, it did not improve OS for the intermediate-risk group but was probably of benefit in high-risk patients.. Several chemotherapy schedules achieved similar remission rates and OS. Four courses of chemotherapy are adequate, but the addition of transplantation as a final course does not improve OS. New agents are required to enhance conventional chemotherapy.

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Cytarabine; Daunorubicin; Etoposide; Female; Humans; Infant; Infant, Newborn; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Neoplasm Staging; Prognosis; Remission Induction; Survival Rate; Thioguanine; Treatment Outcome; Tretinoin; Young Adult

A risk-adapted strategy based on all-trans retinoic acid (ATRA) and anthracycline monochemotherapy (PETHEMA LPA99 trial) has demonstrated a high antileukemic efficacy in acute promyelocytic leukemia. We designed a new trial (LPA2005) with the objective of achieving stepwise improvements in outcome. Between July 2005 and April 2009, low- and intermediate-risk patients (leukocytes < 10 x 10(9)/L) received a reduced dose of mitoxantrone for the second consolidation course, whereas high- risk patients younger than 60 years of age received cytarabine combined with ATRA and idarubicin in the first and third consolidation courses. Of 372 patients attaining complete remission after ATRA plus idarubicin (92.5%), 368 proceeded to consolidation therapy. For low- and intermediate-risk patients, duration of neutropenia and thrombocytopenia and hospital stay were significantly reduced without sacrificing antileukemic efficacy, compared with the previous LPA99 trial. For high-risk patients, the 3-year relapse rate was significantly lower in the LPA2005 trial (11%) than in the LPA99 (26%; P = .03). Overall disease-free survival was also better in the LPA2005 trial (P = .04). In conclusion, the lower dose of mitoxantrone resulted in a significant reduction of toxicity and hospital stay while maintaining the antileukemic activity, and the combination of ATRA, idarubicin, and cytarabine for high-risk acute promyelocytic leukemia significantly reduced the relapse rate in this setting. Registered at http://www.clinicaltrials.gov as NCT00408278.

Topics: Adolescent; Adult; Aged; Anthracyclines; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Disease-Free Survival; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Risk Adjustment; Risk Factors; Survival Rate; Tretinoin

In this Phase 2 study, we evaluated the efficacy of combination of 5-azacitidine (AZA), valproic acid (VPA), and all-trans retinoic acid (ATRA) in patients with high-risk acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Treatment consisted of six cycles of AZA and VPA for 7 days, followed by ATRA for 21 days. Sixty-five patients were enrolled (median age, 72 years; 55 AML including 13 relapsed/refractory patients, 10 MDS; 30 unfavorable karyotypes). Best responses included 14 CR and 3 PR (26%), 75% of the responders and 36% of the non-responders achieving an erythroid response. Median overall survival (OS) was 12.4 months. Untreated patients had a longer OS than relapsed/refractory patients. In patients who fulfilled the 6 planned cycles, OS did not appear to depend on CR/PR achievement, suggesting that stable disease while on-treatment would be a surrogate for survival with this approach. During therapy, early platelet response and demethylation of the FZD9, ALOX12, HPN, and CALCA genes were associated with clinical response. Finally, there was no evidence for the restoration of an ATRA-induced differentiation during therapy. Epigenetic modulation deserves prospective comparisons to conventional care in patients with high-risk AML, at least in those presenting previously untreated disease and low blast count.

Topics: Aged; Aged, 80 and over; Anticonvulsants; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; DNA Methylation; DNA, Neoplasm; Epigenomics; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Polymerase Chain Reaction; Survival Rate; Treatment Outcome; Tretinoin; Valproic Acid

In a previous randomized trial, AML HD98B, we showed that administration of all-trans retinoic acid in addition to intensive chemotherapy improved the outcome of older patients with acute myeloid leukemia. The objectives of this study were to evaluate the prognostic impact of gene mutations and to identify predictive genetic factors for the all-trans retinoic acid treatment effect.. Data from mutation analyses of the NPM1, CEBPA, FLT3, and MLL genes were correlated with outcome in patients 61 years and older treated within the AML HD98B trial.. The frequencies of mutations were: NPM1, 23%; CEBPA, 8.5% (analysis restricted to patients with a normal karyotype); FLT3 internal tandem duplications (ITD), 17%; FLT3 tyrosine kinase domain mutations, 5%; and MLL partial tandem duplications, 4.5%. The genotype mutant NPM1 was positively and adverse cytogenetics as well as higher white blood cell count negatively correlated with achievement of complete remission. In Cox regression analysis, a significant interaction between the genotype mutant NPM1 without FLT3-ITD and treatment with all-trans retinoic acid was identified, in that the beneficial effect of all-trans retinoic acid on relapse-free and overall survival was restricted to this subgroup of patients. Other significant factors for survival were age, adverse cytogenetics, and logarithm of white cell count.. In elderly patients with acute myeloid leukemia, NPM1 mutations are associated with achievement of complete remission, and the genotype 'mutant NPM1 without FLT3-ITD' appears to be a predictive marker for response to all-trans retinoic acid given as an adjunct to intensive chemotherapy (ClinicalTrials.gov Identifier: NCT00151242).

Topics: Aged; Aged, 80 and over; Biomarkers; Disease-Free Survival; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Nucleophosmin; Pilot Projects; Survival Rate; Treatment Outcome; Tretinoin

Preclinical data suggest that all-trans retinoic acid (ATRA) synergizing with granulocyte colony stimulating factor (G-CSF), can improve the effectiveness of chemotherapy in acute myeloid leukemia (AML). Fludarabine 15 mg/m(2) is the minimum dose able to optimize intensification with fludarabine-arabinosylcytosine regimen. In this study 52 patients with relapsed/refractory AML obtained a complete remission (CR) rate of 69.2% after FLAIRG regimen (Fludarabine and arabinosylcytosine twice daily, idarubicin, G-CSF, ATRA). This schedule resulted effective and tolerable enabling 53% of the responding patients to receive transplant procedure. FLAIRG regimen could be proposed as a "bridge" to transplant treatment in this poor risk setting.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Disease-Free Survival; Female; Granulocyte Colony-Stimulating Factor; Humans; Idarubicin; Leukemia, Myeloid, Acute; Male; Middle Aged; Prospective Studies; Remission Induction; Salvage Therapy; Survival Rate; Tretinoin; Vidarabine

All-trans retinoic acid (ATRA) is mandatory in the treatment of acute promyelocytic leukaemia (APL). Experimental studies suggest that ATRA can induce differentiation and apoptosis in leukaemia cells also for other acute myelogenous leukaemia (AML) subtypes, but the clinical observations are conflicting.. Twenty-two AML patients with non-APL disease received oral ATRA alone (22.5 mg/m2 twice daily) for two days, the patients thereafter continued ATRA together with valproic acid and theophylline. We investigated the biological effects of the initial 2 days treatment with ATRA alone. Serum/plasma samples were collected before and after 2 days of ATRA, peripheral blood AML cells were collected from all 12 patients with circulating leukaemia cells (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22).. AML cells collected during therapy had altered flow cytometric forward and right angle light scatters but no morphological signs of differentiation. ATRA increased the percentage of circulating AML cells in G0/G1 phase for 9 out of 12 patients (p = 0.043). Circulating leukaemia cells derived during therapy had increased intracellular levels of P21 (mean increase in mean fluorescence intensity (MFI) being 18.2%, p = 0.017), and decreased levels of Gata-2 (mean decrease in MFI 19%, p = 0.026), NF-kappaB p65 (mean decrease in MFI 15.4%, p = 0.033) and Bcl-2 (mean decrease in MFI 7.2%, p = 0.005). In addition, increased systemic levels of the endothelial marker endocan (plasma) and the angioregulatory mediator angiopoietin-2 (serum) were observed.. In vivo ATRA treatment in AML affects leukaemic cell morphology, regulation of cell cycle progression and apoptosis, and possibly also microvascular endothelial cell functions.

Topics: Aged; Aged, 80 and over; Biology; Cell Proliferation; Cell Separation; Cell Shape; Cyclin-Dependent Kinase Inhibitor p21; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Overexpression of wild-type MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We evaluated whether MN1 plays a functional role in leukemogenesis. We demonstrate using retroviral gene transfer and bone marrow (BM) transplantation that MN1 overexpression rapidly induces lethal AML in mice. Insertional mutagenesis and chromosomal instability were ruled out as secondary aberrations. MN1 increased resistance to all-trans retinoic acid (ATRA)-induced cell-cycle arrest and differentiation by more than 3000-fold in vitro. The differentiation block could be released by fusion of a transcriptional activator (VP16) to MN1 without affecting the ability to immortalize BM cells, suggesting that MN1 blocks differentiation by transcriptional repression. We then evaluated whether MN1 expression levels in patients with AML (excluding M3-AML) correlated with resistance to ATRA treatment in elderly patients uniformly treated within treatment protocol AMLHD98-B. Strikingly, patients with low MN1 expression who received ATRA had a significantly prolonged event-free (P = .008) and overall (P = .04) survival compared with patients with either low MN1 expression and no ATRA, or high MN1 expression with or without ATRA. MN1 is a unique oncogene in hematopoiesis that both promotes proliferation/self-renewal and blocks differentiation, and may become useful as a predictive marker in AML treatment.

Topics: Aged; Animals; Antineoplastic Agents; Biomarkers, Tumor; Bone Marrow Cells; Cell Cycle; Cell Differentiation; Cell Transformation, Viral; Chromosomal Instability; Disease-Free Survival; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Hematopoiesis; Herpes Simplex Virus Protein Vmw65; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutagenesis, Insertional; Predictive Value of Tests; Recombinant Fusion Proteins; Repressor Proteins; Retroviridae; Risk Factors; Survival Rate; Trans-Activators; Transduction, Genetic; Tretinoin; Tumor Suppressor Proteins

The combination of a DNA hypomethylating agent with a histone deacetylase inhibitor has synergistic antileukemia activity and may restore sensitivity to all-trans retinoic acid (ATRA). We conducted a phase 1/2 study of the combination of 5-azacitidine (5-AZA), valproic acid (VPA), and ATRA in patients with acute myeloid leukemia or high-risk myelodysplastic syndrome. 5-AZA was administered subcutaneously at a fixed dose of 75 mg/m(2) daily for 7 days. VPA was dose-escalated and given orally daily for 7 days concomitantly with 5-AZA. ATRA was given at 45 mg/m(2) orally daily for 5 days, starting on day 3. A total of 53 patients were treated. Their median age was 69 years (range, 5-84 years). The maximum tolerated dose of VPA in this combination was 50 mg/kg daily for 7 days. Dose-limiting toxicity was reversible neurotoxicity. The overall response rate was 42%. In previously untreated older patients, the response rate was 52%. Median number of courses to response was 1 (range, 1-3 courses). Median remission duration was 26 weeks, and median survival has not been reached. A significant decrease in global DNA methylation and induction of histone acetylation were achieved. VPA blood levels were higher in responders (P < .005). In conclusion, the combination studied is safe and has significant clinical activity. This clinical trial was registered at www.clinicaltrials.gov as no. NCT00326170.

Topics: Acetylation; Adolescent; Adult; Aged; Aged, 80 and over; Azacitidine; Child; Child, Preschool; DNA Methylation; Dose-Response Relationship, Drug; Drug Therapy, Combination; Drug-Related Side Effects and Adverse Reactions; Gene Expression Regulation; Histones; Humans; Leukemia, Myeloid, Acute; Middle Aged; Myelodysplastic Syndromes; RNA, Messenger; Tretinoin; Valproic Acid

The optimum chemotherapy schedule for reinduction of patients with high-risk acute myeloid leukemia (relapsed, resistant/refractory, or adverse genetic disease) is uncertain. The MRC AML (Medical Research Council Acute Myeloid Leukemia) Working Group designed a trial comparing fludarabine and high-dose cytosine (FLA) with standard chemotherapy comprising cytosine arabinoside, daunorubicin, and etoposide (ADE). Patients were also randomly assigned to receive filgrastim (G-CSF) from day 0 until neutrophil count was greater than 0.5 x 10(9)/L (or for a maximum of 28 days) and all-trans retinoic acid (ATRA) for 90 days. Between 1998 and 2003, 405 patients were entered: 250 were randomly assigned between FLA and ADE; 356 to G-CSF versus no G-CSF; 362 to ATRA versus no ATRA. The complete remission rate was 61% with 4-year disease-free survival of 29%. There were no significant differences in the CR rate, deaths in CR, relapse rate, or DFS between ADE and FLA, although survival at 4 years was worse with FLA (16% versus 27%, P = .05). Neither the addition of ATRA nor G-CSF demonstrated any differences in the CR rate, relapse rate, DFS, or overall survival between the groups. In conclusion these findings indicate that FLA may be inferior to standard chemotherapy in high-risk AML and that the outcome is not improved with the addition of either G-CSF or ATRA.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Disease-Free Survival; Etoposide; Female; Filgrastim; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Recombinant Proteins; Remission Induction; Retrospective Studies; Risk Factors; Survival Rate; Treatment Outcome; Tretinoin

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induced in vitro differentiation of primary acute myeloid leukemia (AML) blasts, an effect enhanced by all-trans retinoic acid (ATRA). Clinical responses to VPA were recently observed in patients with myelodysplastic syndrome (MDS). Herein, the authors have described results of a clinical trial with VPA plus ATRA in 26 patients with poor-risk AML.. VPA (5-10 mg/kg starting dose) and ATRA (45 mg/m(2)) were administered orally. Low-dose AraC or hydroxyurea were permitted to control leukocytosis. Biologic activity of VPA was confirmed by serial analysis of HDAC2 protein levels in peripheral blood (PB) mononuclear cells.. Nineteen of 26 patients completed at least 4 weeks of VPA/ATRA treatment; 7 patients were withdrawn prematurely because of rapidly progressive disease (n = 3) or unacceptable neurologic and cardiovascular toxicity (n = 4). Additional cytoreductive treatment was required in 58% of patients enrolled. Median treatment duration was 3 months. No patient achieved complete remission, one with de novo AML had a minor response, and two patients with secondary AML arising from myeloproliferative disorder (MPD) achieved a partial remission and clearance of PB blasts, respectively. The latter responses were accompanied by profound granulocytosis and erythrocytosis in both patients, reminiscent of the response pattern known from ATRA treatment of acute promyelocytic leukemia. However, cytogenetic analysis of isolated CD34(+) cells and granulocytes did not reveal terminal differentiation of leukemic blasts.. Treatment with VPA/ATRA results in transient disease control in a subset of patients with AML that has evolved from a myeloproliferative disorder but not in patients with a primary or MDS-related AML.

Topics: Administration, Oral; Aged; Aged, 80 and over; Blotting, Western; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Therapy, Combination; Female; Flow Cytometry; Follow-Up Studies; Humans; In Situ Hybridization, Fluorescence; Leukemia, Myeloid, Acute; Male; Maximum Tolerated Dose; Middle Aged; Prospective Studies; Risk Assessment; Severity of Illness Index; Survival Analysis; Treatment Outcome; Tretinoin; Valproic Acid

Clinical outcome in acute myeloid leukemia (AML) is unsatisfactory. One strategy to augment cytotoxicity is TST. All-trans retinoic acid (ATRA) down-regulates bcl-2 expression and heightens AML sensitivity to cytosine arabinoside (ara-C)-induced apoptosis in vitro. We designed a trial of ATRA plus ara-C-based TST in an attempt to enhance drug-induced apoptosis and clinical outcome. Between January 1998 and February 2000, 63 patients received induction TST (oral ATRA days 1-6, ara-C and idarubicin days 2-4, VP-16 days 9-11) followed by consolidation TST (ATRA, ara-C and idarubicin followed by a second ara-C infusion days 11-13). Complete remission (CR) was 60%, with higher rates for patients of <60 years (79%), de novo AML (70%), and non-adverse cytogenetics (81%). Median disease-free survival (DFS) for CR patients was 11.2 months (32% at 3+ years). For patients <60 years with de novo AML and non-adverse cytogenetics who underwent two-cycle TST, DFS was 67% at 3+ years. However, patients of age equal to 60 years and those with poor-risk disease features still have poor CR and DFS, despite the addition of ATRA.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Drug Administration Schedule; Etoposide; Female; Humans; Idarubicin; Leukemia, Myeloid, Acute; Male; Middle Aged; Remission Induction; Risk Factors; Survival Analysis; Treatment Outcome; Tretinoin

All-trans retinoic acid (ATRA) is synergistic with chemotherapy in leukaemia cell lines. We treated 53 patients with newly diagnosed acute myelogenous leukaemia (AML) with high-dose cytarabine-based chemotherapy followed by ATRA. Peripheral blood and bone marrow samples were obtained to study the effect of in vitro exposure to ATRA and to measure apoptosis and bcl-2. The response rate was 72% for patients under age 60 years and 46% for patients aged 60 years or above. There was no difference in the percentage of responding patients, time to recurrence or overall survival for patients receiving chemotherapy with ATRA vs. historical controls receiving chemotherapy without ATRA. After in vitro exposure of day 3 bone marrow samples to ATRA, there was an increase in apoptotic cells in 25% of patient samples compared with samples not exposed to ATRA. Later date of peak apoptosis in peripheral blood and higher percentage of apoptotic cells in bone marrow on day 3 of treatment were associated with lack of clinical response to treatment. Increased bcl-2 in patient samples was associated with shorter time to recurrence and poor cytogenetic risk. The addition of ATRA to chemotherapy did not improve patient outcome. However, evidence of in vitro response to ATRA in 25% of patients suggests that retinoid pathways should be studied further in patients with AML.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cytarabine; Female; Genes, bcl-2; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Recurrence; Survival Analysis; Treatment Outcome; Tretinoin

Treatment results were evaluated in 167 children with acute myeloblastic leukemia (AML) treated on four protocols (ANLL 861, 8912, 9205, APL-ATRA) of the Children's Cancer Leukemia Study Group. In the ANLL 9205 protocol, anthracycline was used with a continuous infusion of cytosine arabinoside, followed by an intensive sequential post remission chemotherapy of short duration, 42/46 patients (91.3%) achieved complete remission, and 58.8% of these patients projected a 3-year disease free survival. These results were apparently superior to those obtained with the ANLL 861 & 8912 protocols, which used conventional doses of multi drugs followed by a moderate post remission chemotherapy of long duration. This favorable response with the ANLL 9205 protocol was attributed mainly to the high induction rate of patients with the M4 and M5 FAB subtypes, as compared to those in the previous two protocols (91.3% in ANLL 9205 vs 57.9% in ANLL 861 + 8912; p < 0.05). No significant difference in the patients outcome was found between the chemotherapy group and allogenic bone marrow transplantation group in the ANLL 9205 study. The patients with the M3 FAB subtype treated with the APL-ATRA protocol which consisted of an alternative use of all-trans retinoic acid and chemotherapy significantly prolonged event free survival as compared with the patients treated with ANLL 861/8912 protocols without all-trans retinoic acid.

Topics: Anthracyclines; Antineoplastic Combined Chemotherapy Protocols; Child; Cytarabine; Humans; Leukemia, Myeloid, Acute; Remission Induction; Tretinoin

We administered liposome-encapsulated all-trans retinoic acid (L-ATRA) to 48 patients with refractory hematologic malignancies using an every-other-day schedule for 28 days and doses of 15 to 175 mg/m2. In 19 patients, pharmacology studies were conducted after the first (day 1) and seventh (day 15) doses. In contrast to the decline in tretinoin concentration seen within 3 to 4 days of administration of daily oral ATRA, there were no differences between the area under the curve (AUC) of tretinoin concentration versus time on day 1 and day 15 (P = .98, Wilcoxon signed-rank test). Peak day 1 concentrations after 15 mg/m2 were higher than those reported after 45 mg/m2 oral ATRA. Six patients with relapsed acute promyelocytic leukemia (APL) were treated. Three, each in first relapse and at least year from the last exposure to oral ATRA, achieved a complete response (CR). Disease recurred in two (one at 3 months despite maintenance L-ATRA and similarity in tretinoin AUC on days 1 and 85, and the other at 5 months, 2 months after discontinuation of L-ATRA) and the third was transplanted 1 month into CR. The three nonresponders were in at least a second relapse and failed to respond to oral ATRA before or immediately after receiving L-ATRA. Severe toxicity developed in three of eight patients treated at 175 mg/m2 (joint pains in two, skin in one). The maximum tolerated dose (MTD) was determined to be 140 mg/m2, at which dose grade 2 toxicity (primarily headache and skin) occurred in eight of eight patients, but grade 3 to 4 toxicity in none. Compared with oral ATRA, L-ATRA apparently results in greater exposure to tretinoin and for a longer time.

Topics: Drug Carriers; Hematologic Diseases; Humans; Leukemia, Myeloid, Acute; Liposomes; Neoplasms; Recurrence; Tretinoin

Thirty-three patients with 'poor prognosis' acute myeloid leukemia, no longer suitable for aggressive chemotherapy, were treated with daily oral all-trans retinoic acid (45 mg/m2) daily and subcutaneous cytosine arabinoside (20 mg standard dose twice a day, day 1 to 10, every 4 weeks). Seventeen patients were males and 16 females, the median age was 67 (range 39-82 years). Eleven patients were at onset of disease, 15 were refractory to previous conventional therapies, three were in first relapse and three in second relapse and one patient had a secondary AML. Seventeen patients had a bone marrow blast infiltration < 50% and 16 > or = 50%. A total of 16 (48%) patients entered complete remission; the rate of complete remission increased to 88% in those patients (n = 17) with < 50% blast infiltration at the time of entering the study. Seventeen patients (52%) were resistant. The difference in response to therapy, according to bone marrow blast percentage (< or > or = 50%), was statistically significant (P < 0.001). Median duration of complete remission was 34.4 weeks (range 6.4-62.8). Mild to moderate hematologic toxicity was the most common side-effect. In conclusion all-trans retinoic acid and low-dose cytosine arabinoside appears to be an effective regimen for inducing complete remission in 'poor prognosis' acute myeloid leukemia and patients with < 50% bone marrow infiltration are likely to represent the ideal target to receive this combination therapy.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Cytarabine; Drug Administration Schedule; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Prognosis; Remission Induction; Tretinoin

63 evaluable patients with myelodysplastic syndromes (MDS) and 15 with acute myelogenous leukemia (AML) were randomized between low-dose ara-C (arm A) and low dose ara-C in combination with 13-cis-retinoic acid (13-CRA) and 1 alpha-hydroxy-vitamin D3 (1 alpha D3) (arm B). 69 patients were evaluable and 18 (26.1%) responded to therapy. The addition of 13-CRA and 1 alpha D3 had no positive influence on survival of the patients, remission rates or duration of remissions. 12/27 patients in arm A and 6/29 patients in arm B progressed from MDS to AML during the course of the study (p = 0.0527). Arm B gave significantly more side-effects than arm A (p = 0.005). Therapeutic effects of 13-CRA and 1 alpha D3 on MDS is not supported by this study. However, an inhibiting effect on AML development in some MDS subgroups cannot be excluded.

Topics: Aged; Aged, 80 and over; Cytarabine; Drug Therapy, Combination; Female; Humans; Hydroxycholecalciferols; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Tretinoin

Human myeloid leukemia cells have a growth advantage over normal cells because they do not differentiate into functional end-cells but remain in the proliferative pool. Several agents have been identified as inducers of differentiation of leukemia cells; among them are the retinoids, 1 alpha,25-dihydroxyvitamin D3 and human tumor necrosis factor alpha, which will be discussed in the following chapter.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Calcitriol; Cell Differentiation; Clinical Trials as Topic; Humans; Leukemia, Experimental; Leukemia, Myeloid, Acute; Mice; Middle Aged; Myelodysplastic Syndromes; Recombinant Proteins; Retinoids; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Retinoic Acid Receptor gamma; Tretinoin

Topics: Antigens, Neoplasm; Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; RNA-Binding Proteins; Tretinoin

Decitabine (DAC) is used as the first-line therapy in patients with higher-risk myelodysplastic syndromes (HR-MDS) and elderly acute myeloid leukaemia (AML) patients unsuitable for intensive chemotherapy. However, the clinical outcomes of patients treated with DAC as a monotherapy are far from satisfactory. Adding all-trans retinoic acid (ATRA) to DAC reportedly benefitted MDS and elderly AML patients. However, the underlying mechanisms remain unclear and need further explorations from laboratory experiments.. We used MDS and AML cell lines and primary cells to evaluate the combined effects of DAC and ATRA as well as the underlying mechanisms. We used the MOLM-13-luciferase murine xenograft model to verify the enhanced cytotoxic effect of the drug combination.. The combination treatment reduced the viability of MDS/AML cells in vitro, delayed leukaemia progress, and extended survival in murine xenograft models compared to non- and mono-drug treated models. DAC application as a single agent induced Nrf2 activation and downstream antioxidative response, and restrained reactive oxygen species (ROS) generation, thus leading to DAC resistance. The addition of ATRA blocked Nrf2 activation by activating the RARα-Nrf2 complex, leading to ROS accumulation and ROS-dependent cytotoxicity.. These results demonstrate that combining DAC and ATRA has potential for the clinical treatment of HR-MDS/AML and merits further exploration.

Topics: Aged; Animals; Antineoplastic Agents; Azacitidine; Decitabine; Humans; Leukemia, Myeloid, Acute; Mice; Myelodysplastic Syndromes; NF-E2-Related Factor 2; Reactive Oxygen Species; Tretinoin

Early death remains a major factor in survival in APL. We aimed to analyze the risk factors for differentiation syndrome and early death in acute promyelocytic leukemia (APL).. The clinical data of APL patients who were newly diagnosed at Mianyang Central Hospital from January 2013 to January 2022 were retrospectively analyzed.. Eighty-six newly diagnosed APL patients (37 males and 49 females) were included in this study. The median age was 46 (17-75) years. Sixty-one patients (70.9%) had low/intermediate-risk APL, and 25 patients (29.1%) had high-risk APL. The incidence of differentiation syndrome (DS) was 62.4%. The multivariate analysis showed that a peak white blood cell (WBC) count ≥16 × 10^9/L was an independent risk factor (OR = 11.000, 95% CI: 2.830-42.756, P = 0.001) for DS in all APL patients, while a WBC count ≥10 × 10^9/L on Day 5 was an independent risk factor for DS in low-intermediate risk APL patients (OR = 9.114, 95% CI: 2.384-34.849, P = 0.001). There were 31 patients (36.5%) with mild DS and 22 patients (25.9%) with severe DS. The multivariate analysis showed that WBC count ≥23 × 10^9/L at chemotherapy was an independent risk factor for severe DS (OR = 10.500, 95% CI: 2.344-47.034, P = 0.002). The rate of early death (ED) was 24.4% (21/86). The multivariate analysis showed that male gender (OR = 7.578,95% CI:1.136-50.551, P = 0.036), HGB < 65 g/L (OR = 16.271,95% CI:2.012-131.594, P = 0.009) and WBC count ≥7 × 10^9/L on Day 3(OR = 23.359,95% CI:1.825-298.959, P = 0.015) were independent risk factors for ED. The WBC count at diagnosis, WBC count on Day 3 and WBC count on Day 5 had moderate positive correlations with tumor necrosis factor-α (TNF-α) at diagnosis, and the correlation coefficients were 0.648 (P = 0.012), 0.615 (P = 0.033), and 0.609 (P = 0.035), respectively. The WBC count had no correlation with IL-6.. During induction treatment, cytotoxic chemotherapy may need to be initiated to reduce the risk of DS for APL patients with a low-intermediate risk WBC count ≥10 × 10^9/L on Day 5 or for all patients with a peak WBC count ≥16 × 10^9/L. Patients with WBC > 7 × 10^9/L on Day 3 have a higher risk of ED. Leukocyte proliferation is associated with TNF-α rather than IL-6, and TNF-α may be a potential biomarker for predicting ED.

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Interleukin-6; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Leukocyte Count; Leukocytes; Leukopenia; Male; Middle Aged; Retrospective Studies; Syndrome; Thrombocytopenia; Tretinoin; Tumor Necrosis Factor-alpha; Young Adult

All-trans retinoic acid (ATRA) is only effective in acute promyelocytic leukemia (APL), but not in other subtype of acute myeloid leukemia (AML). Salinomycin targets tumor cells rather than non-tumorigenic cells, and WNT/β-catenin pathway inhibition is one of the mechanisms of its anti-tumor activity. There is a crosstalk between RA and WNT/β-catenin pathway. Here, we investigate the effect of the combination of salinomycin and ATRA (S+RA) in non-APL AML cells.. Apoptosis was evaluated by cell viability and Annexin-V assay. Cell differentiation was analyzed by CD11c expression and morphology. To explore the underlying mechanisms, Western blot analysis and mitochondrial transmembrane potentials (ΔΨm) were used.. S+RA induced differentiation and apoptosis in AML cell lines and AML primary cells. S+RA inhibited the β-catenin signal pathway as determined by the decreased protein levels of β-catenin, the low-density lipoprotein receptor-related proteins 6 (LRP6), and its downstream proteins such as survivin, c-Myc, caspase-3/7, cdc25A and cyclinD1 and reduced phosphorylation level of GSK3β S9. S+RA also increased the protein levels of CCAAT/enhancer-binding proteins (C/EBPs) and PU.1 and collapsed Δψm. The above molecular and cellular changes induced by S+RA were inhibited by β-catenin specific activator and promoted by β-catenin specific inhibitor.. S+RA induced differentiation by β-catenin-inhibition-mediated up-regulation of C/EBPs and PU.1 and suppression of c-Myc. S+RA triggered apoptosis through β-catenin-inhibition-regulated ΔΨm collapse and caspase-3/7 activation. Taken together, our findings may provide novel therapeutic strategies for AML patients by targeting the WNT/β-catenin pathway.

Topics: Apoptosis; beta Catenin; Caspase 3; Cell Differentiation; Cell Line, Tumor; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Wnt Signaling Pathway

Acute myeloid leukemia (AML) with retinoic acid receptor γ (RARG) rearrangement has clinical, morphologic, and immunophenotypic features similar to classic acute promyelocytic leukemia. However, AML with RARG rearrangement is insensitive to alltrans retinoic acid (ATRA) and arsenic trioxide (ATO) and carries a poor prognosis. We initiated a global cooperative study to define the clinicopathological features, genomic and transcriptomic landscape, and outcomes of AML with RARG rearrangements collected from 29 study groups/institutions worldwide. Thirty-four patients with AML with RARG rearrangements were identified. Bleeding or ecchymosis was present in 18 (54.5%) patients. Morphology diagnosed as M3 and M3v accounted for 73.5% and 26.5% of the cases, respectively. Immunophenotyping showed the following characteristics: positive for CD33, CD13, and MPO but negative for CD38, CD11b, CD34, and HLA-DR. Cytogenetics showed normal karyotype in 38% and t(11;12) in 26% of patients. The partner genes of RARG were diverse and included CPSF6, NUP98, HNRNPc, HNRNPm, PML, and NPM1. WT1- and NRAS/KRAS-mutations were common comutations. None of the 34 patients responded to ATRA and/or ATO. Death within 45 days from diagnosis occurred in 10 patients (∼29%). At the last follow-up, 23 patients had died, and the estimated 2-year cumulative incidence of relapse, event-free survival, and overall survival were 68.7%, 26.7%, and 33.5%, respectively. Unsupervised hierarchical clustering using RNA sequencing data from 201 patients with AML showed that 81.8% of the RARG fusion samples clustered together, suggesting a new molecular subtype. RARG rearrangement is a novel entity of AML that confers a poor prognosis. This study is registered with the Chinese Clinical Trial Registry (ChiCTR2200055810).

Topics: Arsenic Trioxide; HLA-DR Antigens; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin

The "Differentiation therapy" has been emerging as a promising and more effective strategy against acute leukemia relapses.. In extension to the revolutionising therapeutic outcomes of All Trans Retinoic Acid (ATRA) to induce terminal differentiation of Acute Promyelocytic Leukemic (APL) blast cells, we decipher the potential effect of a natural compound "Esculetin" to serve as a differentiating agent in Acute Myeloid Leukemia (AML). Underlaying role of Wnt signaling pathways in esculetin mediated blast cell differentiation was also evaluated.. Human acute myeloid leukemic cells (Kasumi-1) with t(8;21/AML-ETO) translocation were used as a model system. Growth inhibitory and cytotoxic activity of esculetin were analysed using growth kinetics and MTT assay. Morphological alterations, cell scatter characteristics, NBT reduction assay and cell surface marker expression patterns were analysed to detect terminally differentiated phenotypes. We employed RT. We illustrate cytotoxic as well as blast cell differentiation potential of esculetin on Kasumi-1 cells. Morphological alterations akin to neutrophilic differentiation as well as the corresponding acquisition of myeloid lineage markers indicate terminal differentiation potential of esculetin in leukemic blast cells. Exposure to esculetin also resulted in downregulation of canonical Wnt axis while upto ~ 21 fold upregulation of non-canonical axis associated genes.. Our study highlights the importance of selective use of calcium pools as well as "axis shift" of the canonical to non-canonical Wnt signaling upon esculetin treatment which might abrogate the inherent proliferation to release maturation arrest and induce the differentiation in leukemic blast cells. The current findings provide further therapeutic interventions to consider esculetin as a potent differentiating agent to counteract AML relapses.

Topics: Antineoplastic Agents; Calcium; Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Tretinoin; Wnt Signaling Pathway

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML) which is characterized by specific clinical and biological features. Typical APL cases are caused by PML::RARA fusion gene and are exquisitely sensitive to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Rarely, APLs are caused by atypical fusions involving RARA or, in fewer cases still, fusions involving other members of the retinoic acid receptors (RARB or RARG). To date, seven partner genes of RARG have been reported in a total of 18 cases of variant APL. Patients with RARG fusions showed distinct clinical resistance to ATRA and had poor outcomes. Here, we report PRPF19 gene as a novel partner of RARG and identify a rare interposition-type gene fusion in a variant APL patient with a rapidly fatal clinical course. The incomplete ligand-binding domain of RARG in the fusion protein may account for the clinical ATRA resistance in this patient. These results broaden the spectrum of variant APL associated molecular aberrations. Accurately and timely identification of these rare gene fusions in variant APL is essential to guide therapeutic decisions.

Topics: Arsenic Trioxide; DNA Repair Enzymes; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Nuclear Proteins; Receptors, Retinoic Acid; RNA Splicing Factors; Tretinoin

Topics: Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Tretinoin

The promyelocytic leukemia-retinoic acid receptor-α (PML::RARA) fusion is the hallmark of acute promyelocytic leukemia (APL) and is observed in over 95% of APL cases. RARA and homologous receptors RARB and RARG are occasionally fused to other gene partners, which differentially affect sensitivity to targeted therapies. Most APLs without RARA fusions have rearrangements involving RARG or RARB, both of which frequently show resistance to all-trans-retinoic acid (ATRA) and/or multiagent chemotherapy for acute myeloid leukemia (AML). We present a 13-year-old male diagnosed with variant APL with a novel FNDC3B::RARB in-frame fusion that showed no response to ATRA but responded well to conventional AML therapy. While FNDC3B has been identified as a rare RARA translocation partner in ATRA-sensitive variant APL, it has never been reported as a fusion partner with RARB and it is only the second known fusion partner with RARB in variant APL. We also show that this novel fusion confers an RNA expression signature that is similar to APL, despite clinical resistance to ATRA monotherapy.

Topics: Adolescent; Fibronectins; Genomics; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Oncogene Proteins, Fusion; Retinoic Acid Receptor alpha; Translocation, Genetic; Tretinoin

Acute myeloid leukemia (AML) is afflicted by a high-mortality rate and few treatment options. The lack of specific surface antigens severely hampers the development of targeted therapeutics and cell therapy. Here, it is shown that exogenous all-trans retinoic acid (ATRA) mediates selective and transient CD38 upregulation on leukemia cells by up to 20-fold, which enables high-efficiency targeted nanochemotherapy of leukemia with daratumumab antibody-directed polymersomal vincristine sulfate (DPV). Strikingly, treatment of two CD38-low expressing AML orthotopic models with ATRA and DPV portfolio strategies effectively eliminates circulating leukemia cells and leukemia invasion into bone marrow and organs, leading to exceptional survival benefits with 20-40% of mice becoming leukemia-free. The combination of exogenous CD38 upregulation and antibody-directed nanotherapeutics provides a unique and powerful targeted therapy for leukemia.

Topics: ADP-ribosyl Cyclase 1; Animals; Antibodies; Antigens; Antineoplastic Agents; Humans; Leukemia, Myeloid, Acute; Mice; Tretinoin; Up-Regulation

In this study, we present a case of acute myeloid leukemia characterized by the t(11;12)(p15;q13) translocation, exhibiting clinical, immunophenotypical, and morphological features consistent with acute promyelocytic leukemia (APL). The RNA sequencing analysis of the patient's bone marrow samples revealed the presence of the NUP98-retinoic acid receptor gamma (RARG) (NUP98::RARG) gene resulting from the translocation. Furthermore, the presence of a mutation in the ARID1B gene in the patient under study indicates a potential association with resistance to all-trans retinoic acid (ATRA).

Topics: DNA-Binding Proteins; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mutation; Transcription Factors; Translocation, Genetic; Tretinoin

One of the defining features of acute myeloid leukemia (AML) is an arrest of myeloid differentiation whose molecular determinants are still poorly defined. Pharmacological removal of the differentiation block contributes to the cure of acute promyelocytic leukemia (APL) in the absence of cytotoxic chemotherapy, but this approach has not yet been translated to non-APL AMLs. Here, by investigating the function of hypoxia-inducible transcription factors HIF1α and HIF2α, we found that both genes exert oncogenic functions in AML and that HIF2α is a novel regulator of the AML differentiation block. Mechanistically, we found that HIF2α promotes the expression of transcriptional repressors that have been implicated in suppressing AML myeloid differentiation programs. Importantly, we positioned HIF2α under direct transcriptional control by the prodifferentiation agent all-trans retinoic acid (ATRA) and demonstrated that HIF2α blockade cooperates with ATRA to trigger AML cell differentiation. In conclusion, we propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and leukemia debulking.

Topics: Cell Differentiation; Gene Expression Regulation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Transcription Factors; Tretinoin

Elderly patients with AML ineligible for induction have a dismal prognosis; hence disease stabilization is a primary treatment goal. This case of a 75-year-old patient with secondary AML receiving the combination of decitabine and ATRA (within the DECIDER trial, NCT00867672) demonstrates an above-average survival. The therapy administered over 52 cycles led to complete molecular and hematological remission and resulted in 5.3 years overall survival. Clonal evolution of the leukemic clone could be demonstrated using DNA sequencing methods. According to the literature, this case constitutes the longest continued HMA exposure in an elderly AML patient ineligible for standard chemotherapy.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Decitabine; DNA Methylation; Humans; Leukemia, Myeloid, Acute; Prognosis; Treatment Outcome; Tretinoin

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

Topics: Antineoplastic Agents; Blast Crisis; CD11b Antigen; Cell Differentiation; Cell Line, Tumor; Chromosome Inversion; Chromosomes, Human, Pair 16; Core Binding Factor beta Subunit; Gene Fusion; Gene Rearrangement; Histone-Lysine N-Methyltransferase; Humans; Leukemia, Myeloid, Acute; Myeloid-Lymphoid Leukemia Protein; Myosin Heavy Chains; Tretinoin

Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia (AML) characterized by block of differentiation at the promyelocytic stage and the presence of PML-RARA fusion. In rare instances, RARA is fused with other partners in variant APL. More infrequently, non-RARA genes are rearranged in AML patients resembling APL. However, the underlying disease pathogenesis in these atypical cases is largely unknown. Here, we report the identification and characterization of a NUP98- JADE2 fusion in a pediatric AML patient showing APL-like morphology and immunophenotype. Mechanistically, we showed that NUP98-JADE2 could impair all-trans retinoic acid (ATRA)-mediated transcriptional control and myeloid differentiation. Intriguingly, NUP98-JADE2 was found to alter the subcellular distribution of wild-type JADE2, whose down-regulation similarly led to attenuated ATRA-induced responses and myeloid activation, suggesting that NUP98-JADE2 may mediate JADE2 inhibition. To our knowledge, this is the first report of a NUP98-non-RAR rearrangement identified in an AML patient mimicking APL. Our findings suggest JADE2 as a novel myeloid player involved in retinoic acid-induced differentiation. Despite lacking a rearranged RARA, our findings implicate that altered retinoic acid signaling by JADE2 disruption may underlie the APL-like features in our case, corroborating the importance of this signaling in APL pathogenesis.

Topics: Child; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Nuclear Pore Complex Proteins; Receptors, Retinoic Acid; Tretinoin

Topics: Antineoplastic Agents; Cell Differentiation; Enzyme Inhibitors; Humans; Leukemia, Myeloid, Acute; Pyrimidines; Strobilurins; Tretinoin

Acute myeloid leukaemia (AML) is a biologically heterogeneous disease with an overall poor prognosis; thus, novel therapeutic approaches are needed. Our previous studies showed that 4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a new derivative of all-trans retinoic acid (ATRA), could induce AML cell differentiation and cycle arrest. The current study aimed to determine the potential pharmacological mechanisms of ATPR therapies against AML. Our findings showed that E2A was overexpressed in AML specimens and cell lines, and mediate AML development by inactivating the P53 pathway. The findings indicated that E2A expression and activity decreased with ATPR treatment. Furthermore, we determined that E2A inhibition could enhance the effect of ATPR-induced AML cell differentiation and cycle arrest, whereas E2A overexpression could reverse this effect, suggesting that the E2A gene plays a crucial role in AML. We identified P53 and c-Myc were downstream pathways and targets for silencing E2A cells using RNA sequencing, which are involved in the progression of AML. Taken together, these results confirmed that ATPR inhibited the expression of E2A/c-Myc, which led to the activation of the P53 pathway, and induced cell differentiation and cycle arrest in AML.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Leukemia, Myeloid, Acute; Tretinoin

This study aimed to investigate the biological impacts and possible mechanisms of a novel lncRNA, LncSIK1, in AML progression and retinoic acid-regulated AML cell development.. The expression pattern of LncSIK1 was evaluated by qPCR and fluorescence in situ hybridization. CCK-8 assay, immunofluorescence, Wright-Giemsa staining, flow cytometry and Western blotting were performed to assess cell proliferation and differentiation. Bioluminescence imaging and H&E staining were used to detect AML progression in vivo. RNA or chromatin immunoprecipitation assays were conducted to measure the interaction of E2F1 and LncSIK1 or the LC3 and DRAM promoters. Autophagy was measured by transmission electron microscopy and Western blotting.. LncSIK1 was silenced in bone marrow mononuclear cells from AML patients compared with those from healthy donors. LncSIK1 strengthened the effect of retinoic acid in inducing cell differentiation and inhibiting cell proliferation in AML cells. Moreover, the silencing of LncSIK1 was critical to maintaining AML leukaemogenesis, as LncSIK1 enhancement retarded AML progression in vivo. Mechanistically, in NB4 cells, LncSIK1 recruited the E2F1 protein to the promoters of LC3 and DRAM and induced autophagy-dependent degradation of the oncoprotein PML-RARa. However, LncSIK1 blocked E2F1 expression and the E2F1-mediated transcription of LC3 and DRAM, thereby relieving aggressive autophagy in Molm13 cells.. Taken together, these data indicated that LncSIK1 was an important regulator of AML development through regulating the E2F1/autophagy signalling pathway.

Topics: Animals; Antineoplastic Agents; Autophagy; Cell Differentiation; Cell Proliferation; E2F1 Transcription Factor; Humans; Leukemia, Myeloid, Acute; Mice, Transgenic; RNA, Long Noncoding; Tretinoin

To investigate the effects of decitabine (DEC) combined with all-trans retinoic acid (ATRA) on the number of immune cells, efficacy and adverse reactions in the treatment of myeloid neoplasms patients.. Eighty-four patients with myeloid tumors, including AML, MDS-EB-1 or MDS-EB-2 treated by the regimen containing decitabine in our hospital from January 2009 to October 2019 were enrolled and retrospectively analyzed, among the patients, 21 patients treated with DEC alone, 24 patients treated with DEC combined with ATRA (DEC/ATRA) and 39 patients treated with DEC combined with G-CSF priming regimen (DEC/priming). The changes of peripheral blood immune cell levels before and after treatment of the patients between the three groups were compared, and the differences in clinical efficacy and adverse reactions of the patients between the three groups were also compared.. There was no statistical differences in the number of immune cells among the patients in the three groups before treatment (P>0.05). NK cell levels decreased significantly in the patients in DEC and DEC/ATRA group after treatment (P<0.05); After treatment, the levels of CD8+ and CD3+T cells in the patients treated by DEC /priming regimen significantly increased (P<0.05), while the levels of CD3-HLA-DR+ B cells significantly decreased (P<0.05). The overall response rate (ORR) of the patients in DEC/ATRA group (75%) and DEC/priming group (74.36%) was significantly higher than 42.86% in DEC monotherapy group, and the differences showed statistically significant (P<0.05), while the ORR between the patients in DEC/ATRA and DEC/priming group showed no statistic differences (P>0.05). There were no statistical differences in overall survival (OS) and incidence of bleeding between the patients in the three groups (P>0.05). The incidences of grade 3 to 4 bone marrow suppression and the infection rate of the patients in DEC monotherapy and DEC/ATRA group were significantly lower than that in DEC/priming regimen group after treatment (all P<0.05), however, there was no statistical difference between DEC monotherapy and the DEC/ATRA group.. The efficacy of DEC/ATRA on myeloid neoplasms is comparable to that of DEC/priming regimen, and the anti-myeloid tumor effect of DEC/ATRA regimen may be related to the regulation of NK cells and T cells.. 地西他滨联合全反式维甲酸治疗髓系肿瘤患者 免疫细胞水平变化的研究.. 探讨地西他滨（DEC）联合全反式维甲酸（ATRA）治疗髓系肿瘤患者过程中，免疫细胞数量变化及疗效与安全性.. 选取并回顾性分析2009年1月至2019年10月于常州市第一人民医院住院接受DEC治疗的髓系肿瘤患者84例，包括急性髓系白血病（AML）、骨髓增生异常综合征伴原始细胞增多（MDS-EB-1或MDS-EB-2），其中DEC单药组21例、DEC联合ATRA组24例及DEC联合预激方案组39例。比较3组患者治疗前后外周血免疫细胞水平的变化、临床疗效及不良反应差异.. 3组患者治疗前免疫细胞数量比较无统计学差异（P>0.05）。DEC单药组及DEC联合ATRA组患者治疗后NK细胞水平明显下降（P<0.05），DEC联合预激方案组患者治疗后CD8+及CD3+T细胞水平明显上升，而CD3-HLA-DR+的B细胞水平下降，且上述差异均有统计学意义（P<0.05）。DEC联合ATRA组及DEC联合预激组的总体有效率（ORR）分别为75%和74.36%，均显著高于DEC单药组的42.86%（P<0.05），而DEC联合ATRA组与DEC联合预激组的ORR无统计学差异（P>0.05）。3组患者OS无统计学差异（P>0.05）。3组患者治疗后出血发生率均无统计学差异（P>0.05）。DEC单药组及DEC联合ATRA组治疗后3-4级骨髓抑制发生率及感染率明显低于DEC联合预激组（P<0.05），而DEC单药组与DEC联合ATRA组治疗后3-4级骨髓抑制发生率及感染率无统计学差异.. DEC联合ATRA方案治疗髓系肿瘤的疗效与DEC联合预激方案相当，其抗髓系肿瘤的作用可能与其对NK细胞及T细胞的调控有关.

Topics: Antineoplastic Combined Chemotherapy Protocols; Decitabine; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Retrospective Studies; Treatment Outcome; Tretinoin

Aberrant activity of the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR [PAM]) pathway, as well as suppressed retinoic acid signalling, contribute to enhanced proliferation and the differentiation blockade of immature myeloid cells in acute myeloid leukaemia (AML). Inhibition of the PAM pathway was shown to affect especially mixed-lineage leukaemia-rearranged AML. Here, we sought to test a combined strategy using small molecule inhibitors against members of the PAM signalling pathway in conjunction with all-trans retinoic acid (ATRA) to target a larger group of different AML subtypes. We find that ATRA treatment in combination with inhibition of PI3K (ZSTK474), mTOR (WYE132) or PI3K/mTOR (BEZ235, dactolisib) drastically reduces protein levels of the proto-oncogene MYC. In combination with BEZ235, ATRA treatment led to almost complete eradication of cellular MYC, G1 arrest, loss of clonal capacity and terminal granulocytic differentiation. We demonstrate that PAM inhibitor/ATRA treatment targets MYC via independent mechanisms. While inhibition of the PAM pathway causes MYC phosphorylation at threonine 58 via glycogen synthase kinase 3 beta and subsequent degradation, ATRA reduces its expression. Here, we present an approach using a combination of known drugs to synergistically reduce aberrant MYC levels, thereby effectively blocking proliferation and enabling differentiation in various AML subtypes.

Topics: Cell Line, Tumor; Cell Proliferation; Humans; Leukemia, Myeloid, Acute; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Tretinoin

Acute promyelocytic leukaemia (APL) is a unique subtype of acute myeloid leukaemia (AML) characterized by haematopoietic failure caused by the accumulation of abnormal promyelocytic cells in bone marrow (BM). However, indispensable BM biopsy frequently afflicts patients in leukaemia surveillance, which increases the burden on patients and reduces compliance. This study aimed to explore whether the novel circulating long noncoding RNA LOC100506453 (lnc-LOC) could be a target in diagnosis, assess the treatment response and supervise the minimal residual disease (MRD) of APL, thereby blazing a trail in noninvasive lncRNA biomarkers of APL.. Our study comprised 100 patients (40 with APL and 60 with non-APL AML) and 60 healthy donors. BM and peripheral blood (PB) sample collection was accomplished from APL patients at diagnosis and postinduction. Quantitative real-time PCR (qRT-PCR) was conducted to evaluate lnc-LOC expression. A receiver operating characteristic (ROC) analysis was implemented to analyse the value of lnc-LOC in the diagnosis of APL and treatment monitoring. For statistical analysis, the Mann-Whitney U test, a t test, and Spearman's rank correlation test were utilized.. Our results showed that BM lnc-LOC expression was significantly different between APL and healthy donors and non-APL AML. lnc-LOC was drastically downregulated in APL patients' BM after undergoing induction therapy. Lnc-LOC was upregulated in APL cell lines and downregulated after all-trans retinoic acid (ATRA)-induced myeloid differentiation, preliminarily verifying that lnc-LOC has the potential to be considered a treatment monitoring biomarker. PB lnc-LOC was positively correlated with BM lnc-LOC in APL patients, non-APL AML patients and healthy donors and decreased sharply after complete remission (CR). However, upregulated lnc-LOC was manifested in relapsed-refractory patients. A positive correlation was revealed between PB lnc-LOC and PML-RARα transcript levels in BM samples. Furthermore, we observed a positive correlation between PB lnc-LOC and BM lnc-LOC expression in APL patients, suggesting that lnc-LOC can be utilized as a noninvasive biomarker for MRD surveillance.. Our study demonstrated that PB lnc-LOC might serve as a novel noninvasive biomarker in the treatment surveillance of APL, and it innovated the investigation and application of newly found lncRNAs in APL noninvasive biomarkers used in diagnosis and detection.

Topics: Biomarkers; Bone Marrow; Case-Control Studies; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Neoplasm, Residual; RNA, Long Noncoding; Tretinoin

Topics: Cell Differentiation; Humans; Interferon-Stimulated Gene Factor 3, gamma Subunit; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Phenotype; Tretinoin; U937 Cells

Midostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations.. Cell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells.. In this study, 0.1 - 0.25 μM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 - 0.5 μM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPβ, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation.. Midostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin.

Topics: Animals; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Staurosporine; Tretinoin

The suspicion of acute myeloid leukemia (AML) is a haematological emergency that requires a rapid diagnostic workup. Symptoms are usually caused by cytopenias of all blood cell lines. The differentiation of acute promyelocytic leukemia (APL) is important because of the early death rate caused by thrombembolic and bleeding events. Rapid immunophenotypic and genetic characterization is necessary for risk stratification and therapy selection. For this purpose, a center with appropriate expertise should be contacted. Therapy has become more complex due to numerous new approvals. For certain patients, the established intensive induction therapy with cytarabine and anthracycline is now combined with targeted agents, like the antibody conjugate Gemtuzumab-Ozogamicin or the FLT3 inhibitor Midostaurin. Patients with secondary AML benefit from the liposomal chemotherapy combination CPX-351. Therapy with the hypomethylating agent Azacitidine and the BCL2-inhibitor Venetoclax (Aza/Ven) represents the standard for patients who are not fit for intensive therapy. Here, it is important to consider interactions with CYP3A4-effective drugs.In most cases, APL is treated "chemotherapy-free" with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). In high-risk patients, the combination of chemotherapy and ATRA is still standard.Moreover, maintenance therapies were (re)established as an important therapeutic element of post-remission therapy. For example, Midostaurin is used in patients with FLT3 mutations, as is the multikinase inhibitor sorafenib after allogeneic stem cell transplantation. In addition, oral azacitidine is available for non-allogeneic transplant eligible patients in first complete remission. These new drugs have improved prognosis and resulted in a more individualized therapy mostly driven by genetic aberrations. This development will continue in the next years and will significantly improve treatment options, especially for older patients.. Für die Risikostratifizierung und Therapie der akuten myeloischen Leukämie (AML) ist eine Klassifikation nach zytogenetischen und molekulargenetischen Merkmalen erforderlich. Die Panel-Sequenzierung mittels Next-Generation-Sequencing ist inzwischen in der initialen Diagnostik der AML Routine. Durch die Bestimmung der sogenannten messbaren Resterkrankung steht ein Werkzeug zur Verfügung, das auch innerhalb der kompletten Remission noch Abstufungen hinsichtlich der Tiefe des Ansprechens zulässt.. Die Therapie der AML ist in den letzten Jahren durch zahlreiche Neuzulassungen deutlich komplexer geworden. Die etablierte intensive Induktionstherapie mit Cytarabin und Anthracyclin wird inzwischen für bestimmte Patient*innen um zielgerichtete Substanzen wie das Antikörperkonjugat Gemtuzumab-Ozogamicin (GO) oder den FLT3-Inhibitor Midostaurin ergänzt. Insbesondere Patient*innen mit einer sekundären AML profitieren von der liposomalen Chemotherapie-Fixkombination CPX-351. Die Therapie mit der hypomethylierenden Substanz Azacitidin und dem BCL2-Inhibitor Venetoclax (Aza/Ven) hat sich als Standard für Patient*innen etabliert, die nicht fit genug für eine intensive Induktionstherapie sind. Bei dieser Therapie ist die Beachtung von Interaktionen mit CYP3A4-wirksamen Medikamenten besonders wichtig. Ob ältere Patienten*innen eher von einer intensiven Chemotherapie oder Aza/Ven profitieren, ist derzeit unklar.. Auch in der Postremissionstherapie hat sich mit der (erneuten) Etablierung von Erhaltungstherapien viel verändert. So wird Midostaurin bei Patient*innen mit FLT3-Mutation eingesetzt oder der Multikinase-Inhibitor Sorafenib nach allogener Stammzell-Transplantation. Zudem steht für nicht allogen transplantierbare Patient*innen orales Azacitidin zur Verfügung. AKUTE PROMYELOZYTEN-LEUKäMIE:  Für die Therapie der APL erfolgt in den meisten Fällen eine „Chemotherapie-freie“ Behandlung mit All-trans-Retinsäure (ATRA) und Arsentrioxid (ATO). Bei hohem Risiko ist weiterhin die Kombination aus Chemotherapie und ATRA Standard.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Azacitidine; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin

The coincident downregulation of NR4A1 and NR4A3 has been implicated in myeloid leukemogenesis, but it remains unknown how these two genes function in myeloid cells and how their combined downregulation promotes myeloid leukemogenesis. Since NR4A1 abrogation is thought to confer a survival and proliferation advantage to myeloid cells, we hypothesized that downregulation of NR4A3 may have a complementary effect on myeloid cell differentiation. First, we tested the association between differentiation status of leukemic cells and NR4A3 expression using two large clinical datasets from patients with different acute myeloid leukemia (AML) subtypes. The analysis revealed a close association between differentiation status and different subtypes of AML Then, we probed the effects of differentiation-inducing treatments on NR4A3 expression and NR4A3 knockdown on cell differentiation using two myeloid leukemia cell lines. Differentiation-inducing treatments caused upregulation of NR4A3, while NR4A3 knockdown prevented differentiation in both cell lines. The cell culture findings were validated using samples from chronic myeloid leukemia (CML) patients at chronic, accelerated and blastic phases, and in acute promyelocytic leukemia (APL) patients before and after all trans-retinoic acid (ATRA)-based differentiation therapy. Progressive NR4A3 downregulation was coincident with impairments in differentiation in patients during progression to blastic phase of CML, and NR4A3 expression was increased in APL patients treated with ATRA-based differentiating therapy. Together, our findings demonstrate a tight association between impaired differentiation status and NR4A3 downregulation in myeloid leukemias, providing a plausible mechanistic explanation of how myeloid leukemogenesis might occur upon concurrent downregulation of NR4A1 and NR4A3.

Topics: Cell Differentiation; DNA-Binding Proteins; Down-Regulation; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Receptors, Steroid; Receptors, Thyroid Hormone; Tretinoin

Acute myeloid leukemia (AML) is prone to relapse. Targeted therapy with a specific inhibitor of the anti-apoptotic protein Bcl-2 ABT-199 is an effective method for relapsed and refractory patients, but drug resistance is likely, which is primarily related to high Mcl-1 and S100A8 expression. All-trans retinoic acid (ATRA) can inhibit Bcl-2 and Mcl-1 expression. The study purpose was to determine whether ATRA can enhance the antileukemia effect of ABT-199 on AML cells. Our data showed that ATRA combined with ABT-199 exerts a synergistic antileukemic effect by inducing apoptosis and cell cycle arrest in AML. In vivo, combination therapy prolonged the survival of AML xenograft mice. The possible mechanism involves promoting apoptosis through downregulation of S100A8 expression by inhibiting the PI3K/AKT signaling pathway. This study provides a potential treatment strategy and theoretical support for overcoming the clinical ABT-199 resistance problem in AML patients.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Humans; Leukemia, Myeloid, Acute; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tretinoin

Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify

Topics: Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Network Pharmacology; Oncogene Proteins, Fusion; Transcription Factors; Tretinoin

Internal tandem duplications of the Fms-like tyrosine kinase 3 gene (. This was a retrospective cohort study including 60 patients with APL treated with all-trans retinoic acid (ATRA) and chemotherapy. Five-year overall survival (OS) and progression-free survival (PFS) were analyzed in patient groups according to the presence of

Topics: Chromosome Aberrations; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mutation; Prognosis; Retrospective Studies; Tretinoin

In the present study, we determined the effects of the Src family kinase (SFK) inhibitor, Bosutinib, and the engineered loss of the Lyn SFK on all-trans retinoic acid-induced leukemic cell differentiation. Retinoic acid (RA) is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing differentiation of a non-APL AML cell model, the HL-60 human myeloblastic (FAB-M2) leukemia cell line, via activation of a novel signalsome containing an ensemble of signaling molecules that drive differentiation. Bosutinib is an inhibitor of SFKs used to treat myeloid leukemias where prominent high expression of SFKs, in particular Lyn, has been observed. Using either Bosutinib or loss of Lyn expression due to shRNA promoted RA-induced phenotypic differentiation, G0 arrest, and respiratory burst (functional differentiation) of HL-60 cells. Signaling events putatively seminal to RA-induced differentiation, the expression of Fgr, Cbl, Slp-76 and Vav, and the phosphorylation of c-Raf (pS259), Vav (p-tyr), and Slp76 (p-tyr) were not inhibited by Bosutinib or loss of Lyn. Nor was RA-induced upregulation of p-tyr phosphorylation of p47phox, a member of the NADPH complex that produces ROS, a putative phosphorylation dependent signaling regulator. Surprisingly, Bosutinib still works in the absence of Lyn to enhance RA-induced differentiation and neither compromised RA-induced expression, nor phosphorylation of signaling molecules that drive differentiation. These findings suggested there is a novel, off-target, Lyn-independent effect of Bosutinib that is of therapeutic significance to differentiation therapy.

Topics: Aniline Compounds; Cell Differentiation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Nitriles; Quinolines; Tretinoin

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Blood Coagulation Disorders; Chromosomes, Human, Pair 16; Chromosomes, Human, Pair 8; Cytarabine; Daunorubicin; Dexamethasone; Gemtuzumab; Humans; Leukemia, Myeloid, Acute; Male; Translocation, Genetic; Tretinoin

Topics: Cell Differentiation; Cell Line; Dexrazoxane; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin

Differentiation therapies with all-trans retinoic acid (ATRA) have been successful in treating acute promyelocytic leukemia, a rare subtype of acute myeloid leukemia (AML). However, their efficacy is limited in the case of other AML subtypes. Here, we show that the combination of ATRA with salt-inducible kinase (SIK) inhibition significantly enhances ATRA-mediated AML differentiation. SIK inhibition augmented the ability of ATRA to induce growth inhibition and G1 cell cycle arrest of AML cells. Moreover, combining ATRA and SIK inhibition synergistically activated the Akt signaling pathway but not the MAPK pathway. Pharmacological blockade of Akt activity suppressed the combination-induced differentiation, indicating an essential role for Akt in the action of the combination treatment. Taken together, our study reveals a novel role for SIK in the regulation of ATRA-mediated AML differentiation, implicating the combination of ATRA and SIK inhibition as a promising approach for future differentiation therapy.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Immunohistochemistry; Immunophenotyping; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Tretinoin

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow, comprising various subtypes. We have investigated seven different AML cell lines that showed different sensitivities toward the inducer of apoptosis ABT-737, with IC

Topics: Apoptosis; Biphenyl Compounds; Cell Differentiation; Fluorodeoxyglucose F18; Humans; Leukemia, Myeloid, Acute; Nitrophenols; Piperazines; Sulfonamides; Tretinoin

A little number of current autophagy inhibitors may have beneficial effects on the acute myeloid leukemia (AML) patients. However, there is a strong need to figure out which settings should be activated or inhibited in autophagy pathway to prevail drug resistance and also to improve current treatment options in leukemia. Therefore, this study aimed to compare the effects of well-known inhibitors of autophagy (as 3-MA, BafA1, and HCQ) in leukemia KG-1 and HL-60 cells exposed to arsenic trioxide (ATO) and/or all-trans retinoic acid (ATRA). Cell proliferation and cytotoxicity of cells were examined by MTT assay. Autophagy was studied by evaluating the development of acidic vesicular organelles, and the autophagosomes formation was investigated by acridine orange staining and transmission electron microscopy. Moreover, the gene and protein expressions levels of autophagy markers (ATGs, p62/SQSTM1, and LC-3B) were also performed by qPCR and western blotting, respectively. The rate of apoptosis and cell cycle were evaluated using flow cytometry. We compared the cytotoxic and apoptotic effects of ATO and/or ATRA in both cell lines and demonstrated that some autophagy markers upregulated in this context. Also, it was shown that autophagy blockers HCQ and/or BafA1 could potentiate the cytotoxic effects of ATO/ATRA, which were more pronounced in KG-1 cells compared to HL-60 cell line. This study showed the involvement of autophagy during the treatment of KG-1 and HL-60 cells by ATO/ATRA. This study proposed that therapy of ATO/ATRA in combination with HCQ can be considered as a more effective strategy for targeting leukemic KG-1 cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arsenic Trioxide; Autophagy; Cell Proliferation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured

Long non-coding RNA HOTAIR has been reported to play a key role in regulating various biological processes in various cancers. However, the roles and mechanisms of HOTAIR in acute myeloid leukaemia (AML) are still unclear and need to be investigated. In this study, we induced differentiation of four AML cell lines by all-trans retinoic acid (ATRA) and found HOTAIR was significantly upregulated in the process. Chromatin immunoprecipitation (ChIP) assays indicated that C/EBPβ upregulated HOTAIR during ATRA induced differentiation in HL-60 cells. By gain- and loss-of-function analysis, we then observed that HOTAIR expression was positively correlated with ATRA-induced differentiation and negatively regulated G1 phase arrest in HL-60 cells. In addition, we found that HOTAIR promoted ATRA-induced differentiation via the regulation of the cell cycle regulator p21 via miR-17-5p. Moreover, we detected the expression of HOTAIR in 84 de novo AML patients, HOTAIR was found significantly downregulated in the AML patients compared to the iron deficiency anaemia (IDA) control group, negatively correlated with the platelet level in M2 patients. In all, our data suggest that HOTAIR may be subtype-specific in AML-M2 patients, also HOTAIR regulates AML differentiation by C/EBPBβ/HOTAIR/miR-17-5p/p21 pathway. The findings of the present study provide a novel insight into the mechanism of lncRNA-mediated differentiation and indicate that HOTAIR may be a promising therapeutic target for leukaemia, especially for AML with M2 type.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Case-Control Studies; Cell Cycle; Cell Differentiation; Child; Child, Preschool; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Infant; Infant, Newborn; Leukemia, Myeloid, Acute; Male; MicroRNAs; Middle Aged; RNA, Long Noncoding; Tretinoin; Young Adult

Acute myeloid leukemia (AML) is the most universal type and fatal disease of hematological malignancy, with poor outcomes despite chemotherapy and bone marrow transplantations. Benefited from the narrow tissue specificity of folate receptor β (FRβ) aberrantly expressed on hematological linage cell lines, NPs modified with folate acid (FA) has been widely applied for crossing cell membrane barriers in FR-targeted therapies for AML. Thus, the biomimetic nanoparticles (NPs) mediated by FRβ were conducted by an albumin modifier as previously synthesized and cationic liposomes. However, how to further enhance the tumor-targeting and cellular uptake of NPs have been great challenges in cancer therapy. It was reported that FRβ could be selectively augmented by all-trans retinoic acid (ATRA). Herein, we demonstrated the enhanced active tumor-targeting of FA-modified siRNA-loaded biomimetic albumin NPs (Lip-S@FBH) could be achieved by upregulating FRβ expression via ATRA NPs. And the systematic administration of ATRA NPs significantly promoted endocytosis and thereby increased the intracellular concentration of Lip-S@FBH. This strategy combined the FRβ amplification effect with the effective delivery of siRNA, is mostly desirable for the AML-targeting therapy.

Topics: Albumins; Biomimetics; Drug Delivery Systems; Folate Receptor 2; Folic Acid; Humans; Leukemia, Myeloid, Acute; Nanoparticles; RNA, Small Interfering; Tretinoin

The ZBTB16-RARA fusion gene, resulting from the reciprocal translocation between ZBTB16 on chromosome 11 and RARA genes on chromosome 17 [t(11;17)(q23;q21)], is rarely observed in acute myeloid leukemia (AML), and accounts for about 1% of retinoic acid receptor-α (RARA) rearrangements. AML with this rare translocation shows unusual bone marrow (BM) morphology, with intermediate aspects between acute promyelocytic leukemia (APL) and AML with maturation. Patients may have a high incidence of disseminated intravascular coagulation at diagnosis, are poorly responsive to all-trans retinoic acid (ATRA) and arsenic tryoxyde, and are reported to have an overall poor prognosis.. The mutational profile of ZBTB16-RARA rearranged AML has not been described so far.. We performed targeted next-generation sequencing of 24 myeloid genes in BM diagnostic samples from seven ZBTB16-RARA+AML, 103 non-RARA rearranged AML, and 46 APL. The seven ZBTB16-RARA-positive patients were then screened for additional mutations using whole exome sequencing (n = 3) or an extended cancer panel including 409 genes (n = 4).. ZBTB16-RARA+AML showed an intermediate number of mutations per patient and involvement of different genes, as compared to APL and other AMLs. In particular, we found a high incidence of ARID1A mutations in ZBTB16-RARA+AML (five of seven cases, 71%). Mutations in ARID2 and SMARCA4, other tumor suppressor genes also belonging to SWI/SNF chromatin remodeling complexes, were also identified in one case (14%).. Our data suggest the association of mutations of the ARID1A gene and of the other members of the SWI/SNF chromatin remodeling complexes with ZBTB16-RARA+AMLs, where they may support the peculiar disease phenotype.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Arsenic Trioxide; Bone Marrow; Child; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Disseminated Intravascular Coagulation; DNA Helicases; DNA-Binding Proteins; Female; High-Throughput Nucleotide Sequencing; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Nuclear Proteins; Oncogene Proteins, Fusion; Prognosis; Promyelocytic Leukemia Zinc Finger Protein; Retinoic Acid Receptor alpha; Transcription Factors; Translocation, Genetic; Tretinoin

Retinoic acid (RA) was proposed to increase survival of chemotherapy- treated patients with nucleophosmin-1 (NPM-1c)-mutated acute myeloid leukemia. We reported that, ex vivo, RA triggers NPM-1c degradation, P53 activation and growth arrest. PML organizes domains that control senescence or proteolysis. Here, we demonstrate that PML is required to initiate RA-driven NPM-1c degradation, P53 activation and cell death. Mechanistically, RA enhances PML basal expression through inhibition of activated Pin1, prior to NPM-1c degradation. Such PML induction drives P53 activation, favoring blast response to chemotherapy or arsenic in vivo. This RA/PML/P53 cascade could mechanistically explain RA-facilitated chemotherapy response in patients with NPM-1c mutated acute myeloid leukemia.

Topics: Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; NIMA-Interacting Peptidylprolyl Isomerase; Nuclear Proteins; Oncogene Proteins, Fusion; Tretinoin; Tumor Suppressor Protein p53

All-trans retinoic acid (ATRA), a derivate of vitamin A, has been successfully used as a therapy to induce differentiation in M3 acute promyelocytic leukemia (APML), and has led to marked improvement in outcomes. Previously, attempts to use ATRA in non-APML in the clinic, however, have been underwhelming, likely due to persistent signaling through other oncogenic drivers. Dysregulated JAK/STAT signaling is known to drive several hematologic malignancies, and targeting JAK1 and JAK2 with the JAK1/JAK2 inhibitor ruxolitinib has led to improvement in survival in primary myelofibrosis and alleviation of vasomotor symptoms and splenomegaly in polycythemia vera and myelofibrosis.. While dose-dependent anemia and thrombocytopenia limit the use of JAK2 inhibition, selectively targeting JAK1 has been explored as a means to suppress inflammation and STAT-associated pathologies related to neoplastogenesis. The objective of this study is to employ JAK1 inhibition (JAK1i) in the presence of ATRA as a potential therapy in non-M3 acute myeloid leukemia (AML).. Efficacy of JAK1i using INCB52793 was assessed by changes in cell cycle and apoptosis in treated AML cell lines. Transcriptomic and proteomic analysis evaluated effects of JAK1i. Synergy between JAK1i+ ATRA was assessed in cell lines in vitro while efficacy in vivo was assessed by tumor reduction in MV-4-11 cell line-derived xenografts.. Here we describe novel synergistic activity between JAK1i inhibition and ATRA in non-M3 leukemia. Transcriptomic and proteomic analysis confirmed structural and functional changes related to maturation while in vivo combinatory studies revealed significant decreases in leukemic expansion.. JAK1i+ ATRA lead to decreases in cell cycle followed by myeloid differentiation and cell death in human leukemias. These findings highlight potential uses of ATRA-based differentiation therapy of non-M3 human leukemia.

Topics: Cell Differentiation; Humans; Janus Kinase 1; Leukemia; Leukemia, Myeloid, Acute; Proteomics; STAT5 Transcription Factor; Tretinoin

All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib-ATRA (tra-ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra-ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.

Topics: Cell Differentiation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Tretinoin

All-trans retinoic acid (ATRA) serves as the backbone of the management of patients with acute promyelocytic leukemia (APL), with guidelines recommending the initiation of ATRA as soon as APL is suspected. As a regional referral center for patients with acute leukemia, those who are suspected of having APL are often transferred to our facility. However, many referring centers are unable to initiate treatment using ATRA. We conducted an exploratory analysis of the clinical availability of ATRA and the factors limiting access to this critical drug.. The United States was divided into 6 geographic regions: Northwest, Southwest, Central, Southeast, Northeast, and the Great Lakes. Twenty hospitals were randomly selected from states within each of these regions and were surveyed as to whether they typically treated patients with acute leukemia, the availability of ATRA at their institution, and reported reasons for not stocking ATRA (if not available).. Less than one-third of hospitals queried (31%) had ATRA in stock. Neither the size of the hospital nor the hospital's status as academic versus nonacademic (53% vs 31%; P=.08) influenced ATRA availability. Of the hospitals that referred patients with APL, only 14% (7/49) had ATRA readily available. Hospitals that treated patients with APL were more likely to have ATRA available than referring centers (58% vs 14%; P=.000002).. Nearly two-thirds of the hospitals surveyed that cared for patients with acute leukemia do not have ATRA immediately available. Moreover, the vast majority of hospitals that refer patients to other centers do not have ATRA. These findings should spur investigation into the impact of immediate ATRA availability on the morbidity and mortality of patients with APL. A call by hematologists nationwide to their formulary committees is warranted to ensure that this lifesaving medication is available to patients suspected of having APL.

Topics: Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin

A characteristic feature of leukemia cells is a blockade of differentiation in cellular maturation. All-trans-retinoic acid (ATRA) has been successfully applied for the treatment of M3-type AML (APL, 10 %), but it fails to demonstrate a significant efficacy on the remaining patients with non-APL AML (90 %). Therefore, the research for strategies to extend the efficacy of ATRA-based therapy to non-APL AML is a key avenue of investigation. Here, we evaluate the synergetic effect of CDK2 inhibition and ATRA in AML both in vitro and in vivo. We have determined that both the CDK2 depletion and pharmacological inhibitor of CDK2 significantly sensitize three subtypes of AML cells (including two non-APL cells) to ATRA-induced cell differentiation. RNA-sequence results indicate that transcription activation of differentiation and maturation pathways plays an important role in this synergetic effect. Furthermore, the down-regulation of CDK2 sensitized AML cells to ATRA-induced engraftment prevention of leukemia cells in NOD-SCID mice and promotes the primary AML blasts differentiation when combined with ATRA. Thus, our work not only provides relevant experimental evidence for further validating CDK2 as a target for differentiation therapy, but also uncovers the future clinical application of CDK2 inhibitors in ATRA-based differentiation therapeutics for AML.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Female; Humans; Leukemia, Myeloid, Acute; Mice, Inbred NOD; Mice, SCID; Myeloid Cells; RNAi Therapeutics; Tretinoin

All-trans retinoic acid (ATRA) is known to be a potent inhibitor of FLT3-ITD acute myeloid leukemia (AML) cells, although the exact mechanism remains unclear. In this work, we report that ATRA causes fatal mitotic catastrophe in FLT3-ITD AML cells by degrading Chk1 kinase, and therefore preventing DNA damage repair. In order to explore a further enhancement in the inhibitory effect of ATRA on FLT3-ITD AML cells, we investigated the suitability of a combination of ATRA and DNA damage drug SN38. In vitro experiments showed that this combinatorial approach effectively inhibited the proliferation of FLT3-ITD cells and induced cell apoptosis in AML. In vivo experiments confirmed that the combination could substantially improve the anti-tumor effect of SN38. Taken together, our results indicate that ATRA down-regulates Chk1 in FLT3-ITD AML cells, and the combination of ATRA and SN38 significantly improves the anti-tumor effect of either ATRA or SN38 when used alone.

Topics: Adult; Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Checkpoint Kinase 1; Child, Preschool; DNA Damage; DNA Repair; Down-Regulation; Drug Synergism; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Humans; Irinotecan; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Mitosis; Primary Cell Culture; Protein Kinase Inhibitors; Tandem Repeat Sequences; Topoisomerase I Inhibitors; Tretinoin; Xenograft Model Antitumor Assays

All-trans retinoic acid (ATRA)-based differentiation therapy has been unsuccessful in treating t(15;17) negative acute myeloid leukemia (AML) patients, motivating interest in combination therapies using ATRA plus other agents. Using the t (15, 17) negative HL-60 human myeloblastic leukemia model, we find that the cyclin-dependent kinase (CDK) inhibitor, roscovitine, augments signaling by an ATRA-induced macromolecular signalsome that propels differentiation and enhances ATRA-induced differentiation. Roscovitine co-treatment enhanced ATRA-induced expression of pS259- pS289/296/301- pS621-c-Raf, pS217/221-Mek, Src Family Kinases (SFKs) Lyn and Fgr and SFK Y416 phosphorylation, adaptor proteins c-Cbl and SLP-76, Vav, and acetylated 14-3-3 in the signalsome. Roscovitine enhanced ATRA-induced c-Raf interaction with Lyn, Vav, and c-Cbl. Consistent with signalsome hyper-activation, roscovitine co-treatment enhanced ATRA-induced G1/0 arrest and expression of differentiation markers, CD11b, ROS and p47 Phox. Because roscovitine regulated Lyn expression, activation and partnering, a stably transfected Lyn knockdown was generated from wt-parental cells to investigate its function in ATRA-induced differentiation. Lyn-knockdown enhanced ATRA-induced up-regulation of key signalsome molecules, c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, Vav1, SLP-76, and Fgr, but with essentially total loss of pY416-SFK. Compared to ATRA-treated wt-parental cells, differentiation markers p47 phox, CD11b, G1/G0 arrest and ROS production were enhanced in ATRA-treated Lyn-knockdown stable transfectants, and addition of roscovitine further enhanced these ATRA-inducible markers. The Lyn-knockdown cells expressed slightly higher c-Raf, pS259-c-Raf, pS289/296/301-c-Raf, and SLP-76 than wt-parental cells, and this was associated with enhanced ATRA-induced upregulation of Fgr and cell differentiation, consistent with heightened signaling, suggesting that enhanced Fgr may have compensated for loss of Lyn to enhance differentiation in the Lyn-knockdown cells.

Topics: Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Down-Regulation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Myeloid Cells; NADPH Oxidases; Phosphorylation; Principal Component Analysis; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins c-vav; Respiratory Burst; Roscovitine; Signal Transduction; src-Family Kinases; Tretinoin; Up-Regulation

A concomitant change of nucleus shape and chromosome conformation often happens in all-trans retinoic acid (ATRA)-induced differentiation of acute myeloid leukemia cells. However, the relation between the 3D chromosome architecture and the genome-wide epigenetic pattern for transcriptional regulation is poorly understood. In this study, high-throughput chromosome conformation capture (Hi-C) and chromosome immunoprecipitation (ChIP-seq) were employed to investigate the landscape of chromosome distal interaction and H3K4/27me3 in HL-60 cells treated with ATRA. We observed a general loss of topological associated domains (TADs) at PTPN11 during the differentiation of HL-60 cells. Furthermore, the significantly reduced enrichment of CCCTC binding factor (CTCF) near the boundary where PTPN11 located, as well as the decreased H3K4me3 and increased H3K27me3 enrichment at PTPN11 upon ATRA treatment was observed. Taken together, our study indicated a regulatory mechanism behind the silenced PTPN11 in HL-60 cells differentiation.

Topics: Cell Differentiation; Chromatin Immunoprecipitation; Chromosomes, Human; Histones; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Tretinoin

The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors. In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells, triggering degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knockout, but targeting LSD1 sensitizes them to physiological doses of RA without altering of PML-RAR levels, and extends survival of leukemic mice upon RA treatment. The combination of RA with LSD1 inhibition (or knockout) is also effective in other non-APL, acute myeloid leukemia (AML) cells. Nonenzymatic activities of LSD1 are essential to block differentiation, while RA with targeting of LSD1 releases a differentiation gene expression program, not strictly dependent on changes in histone H3K4 methylation. Integration of proteomic/epigenomic/mutational studies showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin, inhibiting the interaction between LSD1 and the transcription factor GFI1.

Topics: Antineoplastic Agents; Catalysis; Cell Differentiation; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Histone Demethylases; Histones; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Tretinoin; Tumor Cells, Cultured

Topics: Cell Differentiation; Co-Repressor Proteins; Gene Expression; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Myeloid Cells; Receptors, Retinoic Acid; Repressor Proteins; Retinoic Acid Receptor alpha; Tretinoin; Tumor Suppressor Proteins

Ferroptosis, a newly discovered form of non-apoptotic cell death, is induced by an excessive degree of iron-dependent lipid peroxide. ATPR, a novel all-trans retinoic acid (ATRA) derivative, has been extensively developed to show superior anticancer effect than ATRA in acute myeloid leukemia (AML). However, whether ferroptosis exists during ATPR treatment of AML remains unclear. Herein, we found that ferroptosis occurred in an AML xenograft mouse model of ATPR treatment. In vitro, ATPR was verified to induce ferroptosis in a dose-dependent manner by proferroptotic protein marker, lipid peroxidation, and lipid ROS, which could be significantly reversed by ferrostatin-1. Using lysosomal inhibitor chloroquine and iron chelator desferrioxamine, we further revealed that ATPR-induced ferroptosis was regulated by autophagy via iron homeostasis, especially Nrf2. Furthermore, targeting ferroptosis contributes to ATPR-induced AML differentiation. In conclusion, these results indicated that ferroptosis play an important role in ATPR-induced differentiation, and suggested that ATPR would provide a potential therapeutic value for AML treatment.

Topics: Animals; Antineoplastic Agents; Autophagy; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Female; Ferroptosis; Homeostasis; Humans; Iron; Leukemia, Myeloid, Acute; Mice; Mice, Nude; Reactive Oxygen Species; Retinoids; Signal Transduction; Tretinoin; Xenograft Model Antitumor Assays

The prognosis of patients with acute myeloid leukemia (AML) caused by the FLT3-ITD mutation is poor. Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) can down-regulate FLT3-ITD level and selectively kill leukemia cells carrying the FLT3-ITD mutation. However, the mechanisms of action of these two compounds are unknown. Here, we found that ATO could bind FLT3-ITD at Lys91 and Asp225, whereas ATRA could bind FLT3-ITD at Lys5 and Gln6. Both compounds could not bind wild-type FLT3. Further studies revealed that ATO/ATRA may suppress the Expression of FLT3-ITD by promoting the UBE2L6-mediated ubiquitination pathway and decreasing the expression of C-MYC. However, further studies are needed to define the mechanisms of these compounds on AML. Our research provides an experimental basis for the use of ATO/ATRA in FLT3-ITD AML in clinical practice.

Topics: Arsenic Trioxide; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mutation; Prognosis; Tretinoin

MicroRNA (miRNA) expression has been implicated in leukaemia. In recent years, miRNAs have been under investigation for their potential as non-invasive biomarkers in acute promyelocytic leukaemia (APL). We investigated whether miR-638 in circulating leukaemia cells is a non-invasive biomarker in diagnosis, assessment of the treatment response and minimal residual disease (MRD) surveillance of APL.. Sixty cases of acute myeloid leukaemia (AML), including 30 cases of APL and 30 cases of non-APL AML, were selected. Thirty healthy controls were also selected. Bone marrow (BM) and peripheral blood (PB) samples were collected from APL patients at diagnosis and post-induction. Microarray analysis and quantitative real-time PCR (qRT-PCR) were performed for miRNA profiling and miR-638 expression analysis, respectively. For statistical analysis, Mann-Whitney U test, Wilcoxon Signed Rank test, receiver operating characteristic (ROC) curve analysis and Spearman's rho correlation test were used.. Both microarray and qRT-PCR data showed that miR-638 was significantly upregulated in BM after APL patients received induction therapy. Moreover, miR-638, which is specifically downregulated in APL cell lines, was upregulated after all-trans retinoic acid (ATRA)-induced myeloid differentiation. Receiver operating characteristic (ROC) curve analyses revealed that miR-638 could serve as a valuable biomarker for differentiating APL from controls or non-APL AML. Furthermore, miR-638 expression was sharply increased after induction therapy and complete remission (CR). An inverse correlation was observed between miR-638 and PML-RARα transcripts levels in BM samples, while a positive correlation was revealed between PB miR-638 and BM miR-638 levels in APL patients after induction therapy.. Our study suggested that miR-638 may serve as a potential APL biomarker for diagnosis and assessment of the response to targeted therapy, and PB miR-638 could be used for non-invasive MRD surveillance in APL.

Topics: Biomarkers, Tumor; Bone Marrow Cells; Female; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; MicroRNAs; Middle Aged; Neoplasm, Residual; Neoplastic Cells, Circulating; Remission Induction; Tretinoin

Pharmacological inhibition of mutant isocitrate dehydrogenase (IDH) reduces R-2-hydroxyglutarate (2-HG) levels and restores cellular differentiation in vivo and in vitro. The IDH2 inhibitor enasidenib (AG-221) has been approved by the FDA as a first-in-class inhibitor for the treatment of relapsed or refractory (R/R) IDH2-mutant acute myeloid leukemia (AML). In this study, the effects of a combination of all-trans retinoic acid (ATRA) and AG-221 on AML cell differentiation was explored, along with the mechanisms employed by IDH2-mutant cells in AML.. We treated the human AML cell line, IDH2-mutant-TF-1, and primary human AML cells carrying IDH2 mutation with 30 μM AG-221 and 100 nM ATRA, alone or in combination.. Combined treatment with AG-221 and ATRA inhibited 2-HG production and resulted in synergistic effects on differentiation among IDH2-mutant AML cells and primary AML cells expressing IDH2 mutation. Combined treatment with AG-221 and ATRA altered autophagic activity. AG-221 and ATRA treatment-induced differentiation of IDH2-mutant AML cells was associated with autophagy induction, without suppressing autophagy flux at maturation and degradation stages. A RAF-1/MEK/ERK pathway was founded to be associated with AG-221 and ATRA-induced differentiation in IDH2-mutant AML cells. IDH-associated changes in histone methylation markers decreased after AG-221 and ATRA combination treatment.. Our preliminary evidence indicates that the addition of ATRA to treatments with IDH2 inhibitor may lead to further improvements or increases in response rates in IDH2-mutant AML patients who do not appear to benefit from treatments with IDH2 inhibitor alone.

Topics: Aminopyridines; Antineoplastic Agents; Cell Differentiation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Mutation; Tretinoin; Triazines

Despite the availability of various treatment protocols, response to therapy in patients with Acute Myeloid Leukemia (AML) remains largely unpredictable. Transcriptomic profiling studies have thus far revealed the presence of molecular subtypes of AML that are not accounted for by standard clinical parameters or by routinely used biomarkers. Such molecular subtypes of AML are predicted to vary in response to chemotherapy or targeted therapy. The Renin-Angiotensin System (RAS) is an important group of proteins that play a critical role in regulating blood pressure, vascular resistance and fluid/electrolyte balance. RAS pathway genes are also known to be present locally in tissues such as the bone marrow, where they play an important role in leukemic hematopoiesis. In this study, we asked if the RAS genes could be utilized to predict drug responses in patients with AML. We show that the combined in silico analysis of up to five RAS genes can reliably predict sensitivity to Doxorubicin as well as Etoposide in AML. The same genes could also predict sensitivity to Doxorubicin when tested in vitro. Additionally, gene set enrichment analysis revealed enrichment of TNF-alpha and type-I IFN response genes among sensitive, and TGF-beta and fibronectin related genes in resistant cancer cells. However, this does not seem to reflect an epithelial to mesenchymal transition per se. We also identified that RAS genes can stratify patients with AML into subtypes with distinct prognosis. Together, our results demonstrate that genes present in RAS are biomarkers for drug sensitivity and the prognostication of AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Cell Line, Tumor; Computer Simulation; Cytarabine; Datasets as Topic; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Gene Ontology; Humans; Inhibitory Concentration 50; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Nonlinear Dynamics; Precision Medicine; Prognosis; Real-Time Polymerase Chain Reaction; Renin-Angiotensin System; Tretinoin

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) in combination with low doses of arsenic trioxide or chemotherapy leads to exceptionally high cure rates (>90%). ATRA forces APL cells into differentiation and cell death. Unfortunately, ATRA-based therapy has not been effective among any other acute myeloid leukemia (AML) subtype, and long-term survival rates remain unacceptably low; only 30% of AML patients survive 5 years after diagnosis. Here, we identified insulin-like growth factor binding protein 7 (IGFBP7) as part of ATRA-induced responses in APL cells. Most importantly, we observed that addition of recombinant human IGFBP7 (rhIGFBP7) increased ATRA-driven responses in a subset of non-APL AML samples: those with high RARA expression. In nonpromyelocytic AML, rhIGFBP7 treatment induced a transcriptional program that sensitized AML cells for ATRA-induced differentiation, cell death, and inhibition of leukemic stem/progenitor cell survival. Furthermore, the engraftment of primary AML in mice was significantly reduced following treatment with the combination of rhIGFBP7 and ATRA. Mechanistically, we showed that the synergism of ATRA and rhIGFBP7 is due, at least in part, to reduction of the transcription factor GFI1. Together, these results suggest a potential clinical utility of IGFBP7 and ATRA combination treatment to eliminate primary AML (leukemic stem/progenitor) cells and reduce relapse in AML patients.

Topics: Animals; Cell Differentiation; Humans; Insulin-Like Growth Factor Binding Proteins; Leukemia, Myeloid, Acute; Mice; Retinoids; Tretinoin

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Discovery; Drug Evaluation, Preclinical; Drug Screening Assays, Antitumor; Glycogen Synthase Kinase 3; Humans; Leukemia, Myeloid, Acute; Molecular Docking Simulation; Molecular Structure; Protein Kinase Inhibitors; Structure-Activity Relationship

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring.

Topics: Alkynes; Antineoplastic Agents; Apoptosis; CCAAT-Enhancer-Binding Protein-alpha; Cell Differentiation; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Molecular Structure; Quinazolinones; Structure-Activity Relationship

Interferon (IFN)-γ is the major mediator of anti-tumor immune responses; nevertheless, cancer cells use intrigue strategies to alter IFN-γ signaling and avoid elimination. Understanding the immune regulatory mechanisms employed by acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells upon exposure to IFN-γ is critical for development of immunotherapy and checkpoint blockade therapy approaches. This study aims to explore the influence of myeloid maturation on IFN-γ-induced PD-L1 and PD-L2 expression and on pro-leukemogenic transcription factor STAT3 signaling in AML and MDS. Stimulation of myeloid blasts' maturation by all-trans retinoic acid (ATRA) or 1α,25-dihydroxyvitamin D3 (vitamin D) increased the CD11b

Topics: Aged; B7-H1 Antigen; Calcitriol; CD11b Antigen; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Programmed Cell Death 1 Ligand 2 Protein; Rho Guanine Nucleotide Exchange Factors; Signal Transduction; THP-1 Cells; Tretinoin

Topics: Antineoplastic Agents; Biomarkers, Tumor; CCAAT-Enhancer-Binding Proteins; Chromosome Inversion; Decision Trees; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mutation; Precision Medicine; Predictive Value of Tests; Prognosis; Translocation, Genetic; Treatment Outcome; Tretinoin

To date, only one subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) can be effectively treated by differentiation therapy utilizing all-trans retinoic acid (ATRA). Non-APL AMLs are resistant to ATRA. Here we demonstrate that the acetyltransferase GCN5 contributes to ATRA resistance in non-APL AML via aberrant acetylation of histone 3 lysine 9 (H3K9ac) residues maintaining the expression of stemness and leukemia associated genes. We show that inhibition of GCN5 unlocks an ATRA-driven therapeutic response. This response is potentiated by coinhibition of the lysine demethylase LSD1, leading to differentiation in most non-APL AML. Induction of differentiation was not correlated to a specific AML subtype, cytogenetic, or mutational status. Our study shows a previously uncharacterized role of GCN5 in maintaining the immature state of leukemic blasts and identifies GCN5 as a therapeutic target in AML. The high efficacy of the combined epigenetic treatment with GCN5 and LSD1 inhibitors may enable the use of ATRA for differentiation therapy of non-APL AML. Furthermore, it supports a strategy of combined targeting of epigenetic factors to improve treatment, a concept potentially applicable for a broad range of malignancies.

Topics: Apoptosis; Bone Marrow; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Drug Resistance, Neoplasm; Epigenesis, Genetic; Genotype; HEK293 Cells; Histone Demethylases; Histones; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; p300-CBP Transcription Factors; Tretinoin

Acute myeloid leukemia (AML) is an aggressive malignancy, and many elderly/unfit patients cannot receive intensive and potentially curative therapy. These patients receive low-toxicity disease-stabilizing treatment. The combination of all-trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid can stabilize the disease for a subset of such patients. We performed untargeted serum metabolomic profiling for 44 AML patients receiving treatment based on ATRA and valproic acid combined with low-dose cytotoxic drugs (cytarabine, hydroxyurea, 6-mercaptopurin) which identified 886 metabolites. When comparing pretreatment samples from responders and non-responders, metabolites mainly belonging to amino acid and lipid (i.e., fatty acid) pathways were altered. Furthermore, patients with rapidly progressive disease showed an extensively altered lipid metabolism. Both ATRA and valproic acid monotherapy also altered the amino acid and lipid metabolite profiles; however, these changes were only highly significant for valproic acid treatment. Twenty-three metabolites were significantly altered by seven-day valproic acid treatment (

Topics: Aged; Aged, 80 and over; Amino Acids; Cytarabine; Drug Therapy; Female; Humans; Hydroxyurea; Leukemia, Myeloid, Acute; Lipid Metabolism; Male; Mercaptopurine; Metabolomics; Middle Aged; Treatment Outcome; Tretinoin; Valproic Acid

Ecotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1

Topics: Animals; Apoptosis; Carcinogenesis; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; MDS1 and EVI1 Complex Locus Protein; Mice; Myeloid Cells; Neoplastic Stem Cells; Receptor, Notch4; Tretinoin

Acute myeloid leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) stress and oxidative stress that could otherwise be overcome. Here, we show that the triple combination of the differentiating agent retinoic acid (RA), the ER stress-inducing drug tunicamycin (Tm), and arsenic trioxide (ATO), able to generate oxidative stress, leads to the death of AML cell lines expressing fusion proteins involving the gene MLL and the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, the combination of RA, Tm, and ATO decreased the colony-forming capacity of primary leukemic blasts bearing the FLT-ITD mutation without affecting healthy hematopoietic progenitor cells. We demonstrate in cell lines that combination of these drugs generates ER and oxidative stresses and impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stresses combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation.

Topics: Cell Death; Cell Line, Tumor; Endoplasmic Reticulum Stress; fms-Like Tyrosine Kinase 3; Gene Duplication; Humans; Leukemia, Myeloid, Acute; Oxidative Stress; Tandem Repeat Sequences; Tretinoin; Unfolded Protein Response

Lysine specific demethylase 1 (LSD1) is a histone modifying enzyme that suppresses gene expression through demethylation of lysine 4 on histone H3. The anti-tumor activity of GSK2879552 and GSK-LSD1, potent, selective irreversible inactivators of LSD1, has previously been described. Inhibition of LSD1 results in a cytostatic growth inhibitory effect in a range of acute myeloid leukemia cell lines. To enhance the therapeutic potential of LSD1 inhibition in this disease setting, a combination of LSD1 inhibition and all-

Topics: Antineoplastic Agents; Apoptosis; Benzoates; Caspases; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclopropanes; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Demethylases; Humans; Leukemia, Myeloid, Acute; Treatment Outcome; Tretinoin

In non-acute promyelotic leukemia (APL)- non myelocytic leukemia (AML), identification of a signaling signature would predict potentially actionable targets to enhance differentiation effects of all-trans-retinoic acid (RA) and make combination differentiation therapy realizable. Components of such a signaling machine/signalsome found to drive RA-induced differentiation discerned in a FAB M2 cell line/model (HL-60) were further characterized and then compared against AML patient expression profiles. FICZ, known to enhance RA-induced differentiation, was used to experimentally augment signaling for analysis. FRET revealed novel signalsome protein associations: CD38 with pS376SLP76 and caveolin-1 with CD38 and AhR. The signaling molecules driving differentiation in HL-60 cluster in non-APL AML

Topics: Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Carbazoles; Cell Differentiation; Drug Resistance, Neoplasm; Drug Synergism; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Signal Transduction; Tretinoin

Primary myelofibrosis (PMF) is a type of cloned myeloproliferative neoplasm stemming from haematopoietic stem cells, and tends to transform to acute myeloid leukaemia (AML) in approximately 10-20% of cases over a 10-year period. The transformation into AML has a poor prognosis, with a median overall survival of only 2.6 months in patients receiving supportive treatment. To date, treatment of AML transformation remains poor. The case of a 58-year-old female patient with AML transformed from PMF, who was treated with decitabine combined with all-trans retinoic acid, is reported. The patient had complete remission and a 17-month overall survival from initial diagnosis of transformed AML, with tolerated haematologic toxicity during the treatment period.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Decitabine; Female; Humans; Leukemia, Myeloid, Acute; Middle Aged; Primary Myelofibrosis; Prognosis; Tretinoin

Topics: Animals; Arsenic; fms-Like Tyrosine Kinase 3; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mice; Tretinoin

RAS-responsive element-binding protein 1 (RREB1) is a transcription factor that is implicated in RAS signaling and multiple tumors. However, the role of RREB1 in acute myeloid leukemia has not been studied. We found that RREB1 is overexpressed in AML patients and myeloid leukemia cell lines (NB4 and HL-60), and RREB1 expression was significantly decreased during granulocytic differentiation of myeloid leukemia cells induced by all-

Topics: Cell Differentiation; DNA-Binding Proteins; Gene Knockdown Techniques; Granulocytes; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MicroRNAs; ras Proteins; Signal Transduction; Transcription Factors; Tretinoin

The RARG gene is a member of the nuclear hormone receptor superfamily and shares high homology with RARA and RARB. RARA is involved in translocation with PML in acute promyelocytic leukaemia (APL). Little is known about RARB or RARG rearrangement. RARG fusions were reported in only five APL patients and the partner genes were NUP98, PML and CPSF6. Here, we report NPM1 as a new partner gene of RARG and identify a unique NPM1-RARG-NPM1 chimeric fusion for the first time in an old male with morphological and immunophenotypical features of hypergranular APL but lacking response to all-trans retinoic acid (ATRA) and arsenic trioxide (As

Topics: Aged; Arsenic Trioxide; Drug Resistance, Neoplasm; Gene Fusion; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Nuclear Proteins; Nucleophosmin; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Tretinoin

Although targeted therapies have proven effective and even curative in human leukaemia, resistance often ensues. IDH enzymes are mutated in ~20% of human AML, with targeted therapies under clinical evaluation. We here characterize leukaemia evolution from mutant IDH2 (mIDH2)-dependence to independence identifying key targetable vulnerabilities of mIDH2 leukaemia that are retained during evolution and progression from early to late stages. Mechanistically, we find that mIDH2 leukaemia are metastable and vulnerable at two distinct levels. On the one hand, they are characterized by oxidative and genotoxic stress, in spite of increased 1-carbon metabolism and glutathione levels. On the other hand, mIDH2 leukaemia display inhibition of LSD1 and a resulting transcriptional signature of all-trans retinoic acid (ATRA) sensitization, in spite of a state of suppressed ATRA signalling due to increased levels of PIN1. We further identify GSH/ROS and PIN1/LSD1 as critical nodes for leukaemia maintenance and the combination of ATRA and arsenic trioxide (ATO) as a key therapeutic modality to target these vulnerabilities. Strikingly, we demonstrate that the combination of ATRA and ATO proves to be a powerfully synergistic and effective therapy in a number of mouse and human mIDH1/2 leukemic models. Thus, our findings pave the way towards the treatment of a sizable fraction of human AMLs through targeted APL-like combinatorial therapies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Disease Models, Animal; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neoplasms, Experimental; Tretinoin; Tumor Cells, Cultured; U937 Cells

Acute myeloid leukemia (AML) is characterized by the accumulation of immature white blood cell precursors in the bone marrow and peripheral circulation. In essence, leukemic cells fail to differentiate and are stalled at a particular step of hematopoietic maturation and are unable to complete their development into functional blood cells with a finite life cycle. They are thus said to possess a "differentiation block." Pharmacological override of this block is one attractive avenue of therapy, termed "differentiation therapy." The most successful example of this therapeutic strategy is the use of the physiologic retinoid all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). In this chapter, we will outline the methods used to characterize the mechanisms mobilized by retinoid signaling and will use the activation of a key regulator of autophagy, ATG7, as an example of the functional characterization of a retinoid regulated gene during differentiation. We will discuss how lentiviral delivery of shRNA constructs into cultured APL cells, such as NB4, can be used to functionally deplete key proteins. We will also describe how the effect of protein knockdown on ATRA-induced differentiation and autophagy can be assessed using quantitative PCR, Western blotting, and flow cytometry.

Topics: Autophagy; Autophagy-Related Protein 7; Cell Differentiation; Flow Cytometry; Gene Expression Profiling; Gene Knockdown Techniques; Humans; Leukemia, Myeloid, Acute; Proteomics; RNA, Small Interfering; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.

Topics: Animals; Carcinogenesis; Cell Differentiation; Cell Plasticity; Cell Transdifferentiation; Cells, Cultured; Humans; Leukemia, Myeloid, Acute; Mice; Neoplastic Stem Cells; Proto-Oncogene Proteins; Trans-Activators; Tretinoin

Histone lysine specific demethylase 1 (LSD1) is overexpressed in diverse hematologic disorders and recognized as a promising target for blood medicines. In this study, molecular docking-based virtual screening united with bioevaluation was utilized to identify novel skeleton of 5-arylidene barbiturate as small-molecule inhibitors of LSD1. Among the synthesized derivatives, 12a exhibited reversible and potent inhibition (IC

Topics: Antineoplastic Agents; Barbiturates; Cell Line, Tumor; DNA Methylation; Enzyme Inhibitors; Histone Demethylases; Humans; Inhibitory Concentration 50; Leukemia, Myeloid, Acute; Molecular Docking Simulation; Spectrum Analysis

KDM3B reportedly shows both tumor-suppressive and tumor-promoting activities in leukemia. The function of KDM3B is likely cell-type dependent and its seeming functional discordance may reflect its phenotypic dependence on downstream targets. Here, we first showed the underexpression of KDM3B in acute myeloid leukemia (AML) patients and AML cell lines with MLL-AF6/9 or PML-RARA translocations. Overexpression of KDM3B repressed colony formation of AML cell line with 5q deletion. We then performed global microarray profiling to identify potential downstream targets of KDM3B, notably HOXA1, which was verified by real time PCR and Western blotting. We further showed KDM3B binding at retinoic acid response elements (RARE) but not at the promoter region of HOXA1 gene. KDM3B knockdown resulted in increased mono-methylation but decreased di-methylation of H3K9 at RARE while eschewing the promoter region of HOXA1. Collectively, we found that KDM3B exhibits potential tumor-suppressive activity and transcriptionally modulates HOXA1 expression via RARE in AML.

Topics: Cell Line, Tumor; Gene Expression Regulation, Leukemic; Histones; Homeodomain Proteins; Humans; Jumonji Domain-Containing Histone Demethylases; Leukemia, Myeloid, Acute; Methylation; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Response Elements; Transcription Factors; Translocation, Genetic; Tretinoin

Homeobox (HOX) genes are frequently dysregulated in leukemia. Previous studies have shown that aberrant HOX gene expression accompanies leukemogenesis and affects disease progression and leukemia patient survival. Patients with acute myeloid leukemia (AML) bearing PML-RARα fusion gene have distinct HOX gene signature in comparison to other subtypes of AML patients, although the mechanism of transcription regulation is not completely understood. We previously found an association between the mRNA levels of HOX genes and those of the histone demethylases JMJD3 and UTX in PML-RARα- positive leukemia patients. Here, we demonstrate that the release of the PML-RARα-mediated block in PML-RARα-positive myeloid leukemia cells increased both JMJD3 and HOX gene expression, while inhibition of JMJD3 using the specific inhibitor GSK-J4 reversed the effect. This effect was driven specifically through PML-RARα fusion protein since expression changes did not occur in cells with mutated RARα and was independent of differentiation. We confirmed that gene expression levels were inversely correlated with alterations in H3K27me3 histone marks localized at HOX gene promoters. Furthermore, data from chromatin immunoprecipitation followed by sequencing broaden a list of clustered HOX genes regulated by JMJD3 in PML-RARα-positive leukemic cells. Interestingly, the combination of GSK-J4 and all-trans retinoic acid (ATRA) significantly increased PML-RARα-positive cell apoptosis compared with ATRA treatment alone. This effect was also observed in ATRA-resistant NB4 clones, which may provide a new therapeutic opportunity for patients with acute promyelocytic leukemia (APL) resistant to current treatment. The results of our study reveal the mechanism of HOX gene expression regulation and contribute to our understanding of APL pathogenesis.

Topics: Benzazepines; Cell Differentiation; Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Leukemic; Genes, Homeobox; Histone Demethylases; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; Leukemia, Myeloid, Acute; Methylation; Nuclear Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Pyrimidines; Tretinoin

The success of all-trans retinoic acid (ATRA) in differentiation therapy for patients with acute promyelocytic leukemia (APL) highly encourages researches to apply a new combination therapy based on ATRA. Therefore, research strategies to further sensitize cells to retinoids are urgently needed. In this study, we showed that Dihydromyricetin (DMY), a 2,3-dihydroflavonol compound, exhibited a strong synergy with ATRA to promote APL NB4 cell differentiation. We observed that DMY sensitized the NB4 cells to ATRA-induced cell growth inhibition, CD11b expression, NBT reduction and myeloid regulator expression. PML-RARα might not be essential for DMY-enhanced differentiation when combined with ATRA, while the enhanced differentiation was dependent on the activation of p38-STAT1 signaling pathway. Taken together, our study is the first to evaluate the synergy of DMY and ATRA in NB4 cell differentiation and to assess new opportunities for the combination of DMY and ATRA as a promising approach for future differentiation therapy.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Drug Synergism; Flavonols; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; MAP Kinase Signaling System; Oncogene Proteins, Fusion; Proteolysis; Signal Transduction; STAT1 Transcription Factor; Tretinoin

High-mobility group AT-hook 2 (HMGA2) may serve as an architectural transcription factor, and it can regulate a range of normal biological processes including proliferation and differentiation. Upregulation of HMGA2 expression is correlated to the undifferentiated phenotype of immature leukaemic cells. However, the underlying mechanism of HMGA2-dependent myeloid differentiation blockage in leukaemia is unknown.. To reveal the role and mechanism of HMGA2 in differentiation arrest of myeloid leukaemia cells, the quantitative expression of HMGA2 and homeobox A9 (HOXA9) was analysed by real-time PCR (qRT-PCR). The regulatory function of HMGA2 in blockage of differentiation in human myeloid leukaemia was investigated through in vitro assays (XTT assay, May-Grünwald-Giemsa, flow cytometry analysis and western blot).. We found that the expression of HMGA2 and HOXA9 was reduced during the process of granulo-monocytic maturation of acute myeloid leukaemia (AML) cells, knockdown of HMGA2 promotes terminal (granulocytic and monocytic) differentiation of myeloid leukaemia primary blasts and cell lines, and HOXA9 was significantly downregulated in leukaemic cells with knockdown of HMGA2. Downregulation of HOXA9 in myeloid leukaemia cells led to increased differentiation capacity in vitro.. Our data suggest that increased expression of HMGA2 represents a possible new mechanism of myeloid differentiation blockage of leukaemia. Aberrant expression of HMGA2 may enhance HOXA9-dependent leukaemogenesis and myeloid leukaemia phenotype. Disturbance of the HMGA2-HOXA9 pathway is probably a therapeutic strategy in myeloid leukaemia.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Proliferation; Cell Survival; Dimethyl Sulfoxide; Free Radical Scavengers; Gene Expression; Gene Knockdown Techniques; Granulocytes; HL-60 Cells; HMGA2 Protein; Homeodomain Proteins; Humans; K562 Cells; Leukemia, Myeloid, Acute; Monocytes; Primary Cell Culture; RNA, Messenger; Tretinoin; Up-Regulation

Differentiation therapies using all-

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Male; Mice; Neoplasm Transplantation; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transplantation, Heterologous; Tretinoin; U937 Cells

The increasing genomic complexity of acute myeloid leukemia (AML), the most common form of acute leukemia, poses a major challenge to its therapy. To identify potent therapeutic targets with the ability to block multiple cancer-driving pathways is thus imperative. The unique peptidyl-prolyl cis-trans isomerase Pin1 has been reported to promote tumorigenesis through upregulation of numerous cancer-driving pathways. Although Pin1 is a key drug target for treating acute promyelocytic leukemia (APL) caused by a fusion oncogene, much less is known about the role of Pin1 in other heterogeneous leukemia.. The mRNA and protein levels of Pin1 were detected in samples from de novo leukemia patients and healthy controls using real-time quantitative RT-PCR (qRT-PCR) and western blot. The establishment of the lentiviral stable-expressed short hairpin RNA (shRNA) system and the tetracycline-inducible shRNA system for targeting Pin1 were used to analyze the biological function of Pin1 in AML cells. The expression of cancer-related Pin1 downstream oncoproteins in shPin1 (Pin1 knockdown) and Pin1 inhibitor all-trans retinoic acid (ATRA) treated leukemia cells were examined by western blot, followed by evaluating the effects of genetic and chemical inhibition of Pin1 in leukemia cells on transformed phenotype, including cell proliferation and colony formation ability, using trypan blue, cell counting assay, and colony formation assay in vitro, as well as the tumorigenesis ability using in vivo xenograft mouse models.. First, we found that the expression of Pin1 mRNA and protein was significantly increased in both de novo leukemia clinical samples and multiple leukemia cell lines, compared with healthy controls. Furthermore, genetic or chemical inhibition of Pin1 in human multiple leukemia cell lines potently inhibited multiple Pin1 substrate oncoproteins and effectively suppressed leukemia cell proliferation and colony formation ability in cell culture models in vitro. Moreover, tetracycline-inducible Pin1 knockdown and slow-releasing ATRA potently inhibited tumorigenicity of U937 and HL-60 leukemia cells in xenograft mouse models.. We demonstrate that Pin1 is highly overexpressed in human AML and is a promising therapeutic target to block multiple cancer-driving pathways in AML.

Topics: Animals; Antineoplastic Agents; Carcinogenesis; Case-Control Studies; Cell Proliferation; Heterografts; Humans; Leukemia, Myeloid, Acute; Mice; NIMA-Interacting Peptidylprolyl Isomerase; RNA, Messenger; RNA, Small Interfering; Tretinoin

Acute myeloid leukemia (AML) remains a significant health problem, with poor outcomes despite chemotherapy and bone marrow transplants. Although one form of AML, acute promyelocytic leukemia (APL), is successfully treated with all-trans retinoic acid (ATRA), this drug is seemingly ineffective against all other forms of AML. Here, we show that ATRA up-regulates CD38 expression on AML blasts to sufficient levels that promote antibody-mediated fratricide following the addition of anti-CD38 daratumumab (DARA). The combination of ATRA plus DARA induced Fc-dependent conjugate formation and cytotoxicity among AML blasts in vitro. Combination treatment also led to reduction in tumor volume and resulted in increased overall survival in murine engraftment models of AML. These results suggest that, although ATRA does not induce differentiation of non-APL, it may be effective as a therapy in conjunction with DARA.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Cell Proliferation; Drug Screening Assays, Antitumor; Drug Therapy, Combination; Humans; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured

To investigate the effect of all transretinoicacid(ATRA) combined with decitabine (5-Aza-2'-deoxycytidine;DAC) on DNA methylation and gene expression of p16INK4a (p16) and retinoic acid receptor β (RARβ), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients.. The expression levels of p16 and RARβ were determined by qRT-PCR and Western blot. Methylation-specific PCR was used to analyze their methylation status. WST-1 and flow cytometry were performed to detect growth inhibition, differentiation, apoptosis and cell cycle of U937 cells respectively.. The expression p16 and RARβ was down-regulated by promoter hypermethylation in newly diagnose delder AML patients and U937 cells. Combination treatment of ATRA and DAC induced DNA hypomethylation as well as gene expression of p16 and RARβ, which contributed to the growth inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells. In addition for elder AML patients intolerable to standard chemotherapy, the combination regimen of ATRA and DAC showed antineoplastic activity accompamied by up-regulation of p16 and RARβ expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen.. The regimen of ATRA combined with DAC as the combination therapeutic strategy for inducing differentiation and demethylation possesses the anti-AML potency, and contributes to optimizing the therapeutic strategy for elder AML patients and promoting the clinical prognosis.

Topics: Azacitidine; Decitabine; Humans; Leukemia, Myeloid, Acute; Tretinoin; U937 Cells

Acute promyelocytic leukemia, a form of acute myeloid leukemia, is characterized by cell differentiation arrest at the promyelocyte stage. Current therapeutic options include administration of all trans-retinoic acid (ATRA), but this treatment produces many side effects. ATRA is known to induce differentiation of leukemic cells into granulocytes, but the mechanism of this process is poorly studied. We performed comparative proteomic profiling of HL-60 promyelocytic cells at different stages of ATRA-induced differentiation to identify differentially expressed proteins by high-resolution mass spectrometry and relative quantitative analysis without isotope labels. A total of 1162 proteins identified by at least two unique peptides were analyzed, among them 46 and 172 differentially expressed proteins were identified in the nuclear and cytosol fractions, respectively. These differentially expressed proteins can represent candidate targets for combination therapy of acute promyelocytic leukemia.

Topics: Cell Differentiation; Cell Proliferation; Flow Cytometry; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mass Spectrometry; Tretinoin

Purpose Most anemic patients with non-deleted 5q lower-risk myelodysplastic syndromes (MDS) are treated with erythropoiesis-stimulating agents (ESAs), with a response rate of approximately 50%. Second-line treatments, including hypomethylating agents (HMAs), lenalidomide (LEN), and investigational drugs, may be used after ESA failure in some countries, but their effect on disease progression and overall survival (OS) is unknown. Here, we analyzed outcome after ESA failure and the effect of second-line treatments. Patients and Methods We examined an international retrospective cohort of 1,698 patients with non-del(5q) lower-risk MDS treated with ESAs. Results Erythroid response to ESAs was 61.5%, and median response duration was 17 months. Of 1,147 patients experiencing ESA failure, 653 experienced primary failure and 494 experienced relapse after a response. Primary failure of ESAs was associated with a higher risk of acute myeloid leukemia (AML) progression, which did not translate into an OS difference. Of 450 patients (39%) who received second-line treatment, 194 received HMAs, 148 received LEN, and 108 received other treatments (MISC), whereas 697 received RBC transfusions only. Five-year AML cumulative incidence was 20.3%, 20.3%, and 11.3% for those receiving HMAs, LEN, and MISC, respectively ( P = .05). Five-year OS for patients receiving HMA, LEN, and MISC was 36.5%, 41.7%, and 51%, respectively ( P = .21). In a multivariable analysis adjusted for age, sex, revised International Prognostic Scoring System score, and progression at ESA failure, there was no significant OS difference among the three groups. Conclusion In this large, multicenter, retrospective cohort of patients with non-del(5q) lower-risk MDS treated with ESAs, none of the most commonly used second-line treatments (HMA and LEN) significantly improved OS. Early failure of ESAs was associated with a higher risk of AML progression.

Topics: Aged; Aged, 80 and over; Anemia; Antilymphocyte Serum; Antineoplastic Agents; Arsenic; Azacitidine; Chromosome Deletion; Chromosomes, Human, Pair 5; Cyclosporine; Cytarabine; Decitabine; Disease Progression; Enzyme Inhibitors; Erythrocyte Transfusion; Female; Hematinics; Humans; Hydroxyurea; Immunologic Factors; Lenalidomide; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Recurrence; Retreatment; Retrospective Studies; Risk Factors; Survival Rate; Thalidomide; Treatment Failure; Tretinoin; Valproic Acid

Topics: Active Transport, Cell Nucleus; Animals; Calcium; Calcium Signaling; Cell Differentiation; Cell Nucleus; Female; Flavanones; Glucosides; Humans; Inositol 1,4,5-Trisphosphate Receptors; Leukemia, Myeloid, Acute; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Phospholipid Transfer Proteins; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of

Topics: Animals; Blood Cells; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Gene Expression Regulation; Hematopoiesis; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Receptors, Calcitriol; Retinoic Acid 4-Hydroxylase; Tretinoin; Vitamin D

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-

Topics: Cells, Cultured; Female; Humans; Karyotyping; Leukemia, Myeloid, Acute; Membrane Proteins; ras-GRF1; Retinoids; Transcription Factors; Translocation, Genetic; Tretinoin

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that

Topics: Actins; Animals; Cell Differentiation; Cell Line, Tumor; Cell Self Renewal; Cytoskeletal Proteins; Dendritic Cells; Humans; Immunity, Innate; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Neoplastic Stem Cells; Oculocerebrorenal Syndrome; Phagocytosis; Phagosomes; Phosphoproteins; Phosphoric Monoester Hydrolases; Polymerization; Receptor Aggregation; Tretinoin

Acute myeloid leukemia (AML) is an aggressive malignancy only cured by intensive therapy. However, many elderly and unfit patients cannot receive such treatment due to an unacceptable risk of treatment-related morbidity and mortality. Disease-stabilizing therapy is then the only possible strategy, one alternative being treatment based on all-trans retinoic acid (ATRA) combined with the histone deacetylase inhibitor valproic acid and possibly low-toxicity conventional chemotherapy.. Primary AML cells were derived from 43 patients included in two clinical studies of treatment based on ATRA, valproic acid and theophyllamine; low toxicity chemotherapy (low-dose cytarabine, hydroxyurea, 6-mercaptopurin) was also allowed. Pretreatment leukemic cells were analyzed by mutation profiling of 54 genes frequently mutated in myeloid malignancies and by global gene expression profiling before and during in vivo treatment.. Patients were classified as responders and non-responders to the treatment, however response to treatment showed no significant associations with karyotype or mutational profiles. Significance analysis of microarray (SAM) showed that responders and non-responders significantly differed with regard to the expression of 179 different genes. The differentially expressed genes encoding proteins with a known function were further classified based on the PANTHER (protein annotation through evolutionary relationship) classification system. The identified genes encoded proteins that are involved in several important biological functions, but a main subset of the genes were important for transcriptional regulation. These pretherapy differences in gene expression were largely maintained during treatment. Our analyses of primary AML cells during in vivo treatment suggest that ATRA modulates HOX activity (i.e. decreased expression of HOXA3, HOXA4 and HOXA5 and their regulator PBX3), but altered function of DNA methyl transferase 3A (DNMT3A) and G-protein coupled receptor signaling may also contribute to the effect of the overall treatment.. Responders and non-responders to AML stabilizing treatment based on ATRA and valproic acid differ in the pretreatment transcriptional regulation of their leukemic cells, and these differences may be important for the clinical effect of this treatment.. ClinicalTrials.gov no. NCT00175812 ; EudraCT no. 2004-001663-22, registered September 9, 2005 and ClinicalTrials.gov no. NCT00995332 ; EudraCT no. 2007-2007-001995-36, registered October 14, 2009.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Computational Biology; Female; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Karyotype; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Pharmacogenomic Testing; Transcriptome; Treatment Outcome; Tretinoin; Valproic Acid

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cell Transformation, Neoplastic; Down-Regulation; Genes, Homeobox; Humans; Leukemia, Myeloid, Acute; Octamer Transcription Factor-3; Tretinoin

Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that are blocked in differentiation. Acute promyelocytic leukemia (APL) is a rare form of AML, which generally presents with a t(15;17) translocation causing expression of the fusion protein PML-RARA. Pharmacological doses of all-trans retinoic acid (ATRA) induce granulocytic differentiation of APL cells leading to cure rates of >80% if combined with conventional chemotherapy. Autophagy is a lysosomal degradation pathway for the removal of cytoplasmic content and recycling of macromolecules. ATRA induces autophagy in ATRA-sensitive AML and APL cells and autophagy inhibition attenuates ATRA-triggered differentiation. In this study, we aimed at identifying if the autophagy-linked FYVE-domain containing protein (ALFY/WDFY3) is involved in autophagic degradation of protein aggregates contributes to ATRA therapy-induced autophagy. We found that ALFY mRNA levels increase significantly during the course of ATRA-induced differentiation of APL and AML cell lines. Importantly ALFY depletion impairs ATRA-triggered granulocytic differentiation of these cells. In agreement with its function in aggrephagy, knockdown of ALFY results in reduced ATRA-induced proteolysis. Our data further suggest that PML-RARα is an autophagy substrate degraded with the help of ALFY. In summary, we present a crucial role for ALFY in retinoid triggered maturation of AML cells.

Topics: Adaptor Proteins, Signal Transducing; Autophagy; Autophagy-Related Proteins; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Leukemic; HEK293 Cells; Humans; Leukemia, Myeloid, Acute; Lysosomes; Membrane Proteins; MicroRNAs; Neutrophils; Proteolysis; RNA, Messenger; Transcription Factors; Tretinoin; Up-Regulation

Altered transforming growth factor-beta (TGF-β) signaling has been implicated in the pathogenesis of leukemia. Although TGF-β type II receptor (TβRII) isoforms have been isolated from human leukemia cells, their expression patterns and functions of these variants are unclear. In this study, we determined that two TβRII isoforms (TβRII and TβRII-B) are abnormally expressed in leukemic cells, as compared to normal hematopoietic cells. TβRII-B, but not TβRII, was found to promote cell cycle arrest, apoptosis, and differentiation of leukemic cells. TβRII-B also enhanced TGF-β1 binding and downstream signaling and reduced tumorigenicity in vivo. By contrast, TβRII blocked all-trans retinoic acid-induced differentiation through inhibition of TβRII-B. Overall survival was significantly lower in acute myeloid leukemia (AML) patients with high compared to low TβRII expression. Thus, whereas TβRII-B is a potent inducer of cell cycle arrest, apoptosis, and differentiation, higher TβRII expression correlates with poor clinical prognosis in AML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Female; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; K562 Cells; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Male; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prognosis; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad Proteins; Time Factors; Transfection; Transforming Growth Factor beta1; Tretinoin; U937 Cells

FLT3-ITD mutations occur in approximately 30% of acute myeloid leukemia (AML) and are associated with a poor outcome. Currently available FLT3 inhibitors have in vitro but limited clinical activity in FLT3-ITD AML. Reports have shown that an arsenic trioxide (ATO)/all-trans-retinoic acid (ATRA) combination improves prognosis in acute promyelocytic leukemia, especially with FLT3-ITD, and ATO or ATRA alone enhances apoptosis in FLT3-ITD AML cells treated with FLT3 inhibitors, providing a rationale to investigate the role of ATO/ATRA in FLT3-ITD AML. Here, we demonstrate that an ATO/ATRA combination selectively exerts synergistic cytotoxicity against FLT3-ITD AML cell lines (MV4;11/MOLM-13). The signaling pathways affected by ATO/ATRA include FLT3/STAT5/MYC, FLT3/STAT5/E2F1, FLT3/ERK/ATF5 and FLT3/AKT/ATF5.ATF5 may function as an oncogene in FLT3-ITD AML. Our findings provide experimental evidence that supports further exploration of ATO/ATRA in FLT3-ITD AML in vivo and warrants a clinical evaluation of regimens comprising an ATO/ATRA combination.

Topics: Apoptosis; Arsenic Trioxide; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Signal Transduction; Tretinoin

The ETS transcription factor ERG has been implicated as a major regulator of both normal and aberrant hematopoiesis. In acute myeloid leukemias harboring t(16;21), ERG function is deregulated due to a fusion with FUS/TLS resulting in the expression of a FUS-ERG oncofusion protein. How this oncofusion protein deregulates the normal ERG transcription program is unclear. Here, we show that FUS-ERG acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR:RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-trans retinoic acid treatment of t(16;21) cells as well as FUS-ERG knockdown alleviate the myeloid-differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway.

Topics: Amino Acid Motifs; Cell Line, Tumor; Chromosomes, Human, Pair 16; Chromosomes, Human, Pair 21; Dimerization; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Multiprotein Complexes; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Protein Binding; Protein Interaction Mapping; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; RNA Interference; RNA-Binding Protein FUS; RNA, Small Interfering; Signal Transduction; Trans-Activators; Transcription Factors; Translocation, Genetic; Tretinoin; U937 Cells

Large, comprehensive population-based studies in acute myeloid leukemia (AML) are scarce. We conducted a nationwide population-based study on treatment, trial participation and survival among all adult patients diagnosed with AML (n=12,032) and acute promyelocytic leukemia (APL; n=585) in the Netherlands between 1989-2012. Patients were categorized into four periods and four age groups (18-40, 41-60, 61-70 and >70 years). The application of allogeneic stem cell transplantation increased over time among AML patients up to age 70 years. For APL patients, the use of chemotherapy increased across all age groups. When a clinical trial was open for accrual in the Netherlands, the inclusion rates were 68%, 57%, 30% and 12% for AML patients in the four age groups, respectively (data for APL unavailable). Relative survival improved over time among AML (up to age 70 years) and APL patients. In the period 2007-2012, 5-year relative survival rates were 54%, 38%, 14% and 2% for AML patients and 84%, 75%, 54% and 37% for APL patients in the four age groups, respectively. As survival remained poor for older AML patients over the last two decades, clinical trials and active participation in those trials, are warranted that explore innovative treatment strategies for this elderly population.

Topics: Adolescent; Adult; Aged; Clinical Trials as Topic; Female; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Netherlands; Patient Participation; Time Factors; Tretinoin; Young Adult

All trans-retinoic acid (ATRA) is successful in treating acute promyelocytic leukemia (APL) by inducing terminal differentiation-mediated cell death, but it has limited activity in non-APL acute myeloid leukemia (AML). We aim to improve ATRA therapy of AML by enhancing apoptosis through repression of the antiapoptotic proteins Bcl-2 and Mcl-1.. APL and AML cell lines, as well as primary AML samples, were used to explore the mechanisms regulating differentiation and apoptosis during ATRA treatment. Stable transfection and gene silencing with siRNA were used to identify the key factors that inhibit apoptosis during induction of differentiation and drugs that accelerate apoptosis.. In differentiation-responsive AML cells, ATRA treatment induces long-lasting repression of Bcl-2 while first upmodulating and then reducing the Mcl-1 level. The Mcl-1 level appears to serve as a gatekeeper between differentiation and apoptosis. During differentiation induction, activation of MEK/ERK and PI3K/Akt pathways by ATRA leads to activation of p90RSK and inactivation of glycogen synthase kinase 3β (GSK3β), which increase Mcl-1 levels by increasing its translation and stability. Sorafenib blocks ATRA-induced Mcl-1 increase by reversing p90RSK activation and GSK3β inactivation, maintains the repressed Bcl-2 level, and enhances ATRA induced apoptosis in non-APL AML cell lines and in primary AML cells.. Inhibition of Mcl-1 is required for apoptosis induction in ATRA differentiation-responsive AML cells. ATRA and sorafenib can be developed as a novel drug combination therapy for AML patients because this drug combination augments apoptosis by inhibiting Bcl-2 and Mcl-1.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Differentiation; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sorafenib; Tretinoin; Xenograft Model Antitumor Assays

Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; DNA-Binding Proteins; Female; Humans; Leukemia, Myeloid, Acute; Male; MDS1 and EVI1 Complex Locus Protein; Mice, SCID; Myeloid Cells; Proto-Oncogenes; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Chemotherapy is the only therapy option for the majority of AML patients, however, there are several limitations for this treatment. Our aim was to find a new chemotherapy strategy that is more effective and less toxic.. MTT assays and a xenograft mouse model were employed to evaluate the synergistic activity of all-trans retinoic acid (ATRA) combined with topotecan (TPT). Drug-induced DNA damage and apoptosis were determined by flow cytometry analysis with PI and DAPI staining, the comet assay and Western blots. Short hairpin RNA (shRNA) and a RARα plasmid were used to determine whether RARα expression influenced DNA damage and apoptosis.. We found that ATRA exhibited synergistic activity in combination with Topotecan in AML cells, and the enhanced apoptosis induced by Topotecan plus ATRA resulted from caspase pathway activation. Mechanistically, ATRA dramatically down regulated RARα protein levels and led to more DNA damage and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of Topotecan combined with ATRA was further validated in the HL60 xenograft mouse model.. Our data demonstrated, for the first time, that the combination of TPT and ATRA showed potential benefits in AML, providing a novel insight into clinical treatment strategies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Differentiation; Cell Line, Tumor; DNA Damage; Drug Synergism; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mice; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Small Interfering; Topotecan; Tretinoin; Xenograft Model Antitumor Assays

Diagnosis of acute promyelocytic leukemia (APL) has been accelerated by multiparameter flow cytometry (MFC). However, diagnostic interpretation of MFC readouts for APL depends on individual experience and knowledge, which inevitably increases the risk of arbitrariness. We appraised the feasibility of using stepwise discriminant function analysis (SDFA) based on MFC to optimize the minimal variables needed to distinguish APL from other acute myeloid leukemia (AML) without complicated data interpretation. Samples from 327 patients with APL (n = 51) and non-APL AML (n = 276) were randomly allocated into training (243 AML) and test sets (84 AML) for SDFA. The discriminant functions from SDFA were examined by correct classification, and the final variables were validated by differential expression. Finally, additional 20 samples from patients with atypical APL and AML confusable with APL were also identified by SDFA method and morphological analysis. The weighed discriminant function reveals seven differentially expressed variables (CD2/CD9/CD11b/CD13/CD34/HLA-DR/CD117), which predict a molecular result for APL characterization with an accuracy that approaches 99% (99.6 and 98.8% for AML samples in training and test sets, respectively). Furthermore, the SDFA outperformed either single variable analysis or the more limited 3-component analysis (CD34/CD117/HLA-DR) via separate SDFA, and was also superior to morphological analysis in terms of diagnostic efficacy. The established SDFA based on MFC with seven variables can precisely and rapidly differentiate APL and non-APL AML, which may contribute to the urgent initiation of all-trans-retinoic acid-based APL therapy.

Topics: Antineoplastic Agents; Diagnosis, Differential; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Pilot Projects; Tretinoin

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPβ, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPβ-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.

Topics: 3' Untranslated Regions; Antagomirs; Antineoplastic Agents; Base Sequence; Bcl-2-Like Protein 11; CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; Cell Line, Tumor; E2F Transcription Factors; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Mutagenesis; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Transcriptional Activation; Tretinoin; Up-Regulation

Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D.

Topics: Calcitriol; Enzyme Induction; Gene Expression; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Vitamin D3 24-Hydroxylase

Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Down-Regulation; Gene Silencing; Hematopoietic Stem Cells; Leukemia, Myeloid, Acute; Mice, Inbred C57BL; Mutant Proteins; Mutation; Polycomb Repressive Complex 1; Polycomb-Group Proteins; Protein Binding; Tretinoin

Leukemia is the most common malignant disease in children with high incidence and mortality rates, and a poor treatment effect. The aim of the present study was to examine the changes in the expression of homeobox (Hox) A5 gene and its relationship with cell cycle and apoptosis through the intervention of human K562 myeloid leukemia cell line by all-trans retinoic acid (ATRA), to analyze the role of HOXA5 in the pathogenesis and development process of myeloid leukemia. The optimal concentration of ATRA to be used with K562 cells was determined using a cell counting kit‑8 (CCK‑8). After 24, 72 and 48 h following treatment of K562 cells with 10 µmol/l ATRA, cell cycle events and apoptosis were measured using flow cytometry. HOXA5 mRNA and protein expression in K562 cells was assessed by RT‑PCR and western blot analysis, and the relationship between HOXA5 expression and cell cycle and apoptosis was analyzed. The HOXA5 mRNA and protein expression levels were increased following treatment with ATRA in K562 cells. Apoptosis was increased significantly. The cell cycle was inhibited in G0/G1 phase. Cell proliferation was also inhibited. HOXA5 mRNA and protein expression rates positively correlated with cell apoptosis and the increased percentage and cell cycle of the G0/G1 phase. However, HOXA5 negatively correlated with the reduced percentage of S stage. In conclusion, the expression of HOXA5 in cells was increased following treatment with ATRA in K562 cells, in a time-dependent manner. Additionally, ATRA may inhibit the proliferation of K562 cells and promote apoptosis by upregulating the HOXA5 mRNA and protein expression.

Topics: Apoptosis; G1 Phase; Gene Expression Regulation, Leukemic; Homeodomain Proteins; Humans; K562 Cells; Leukemia, Myeloid, Acute; Neoplasm Proteins; Resting Phase, Cell Cycle; Tretinoin

The success of all-trans retinoic acid (ATRA) in differentiation therapy for patients with acute promyelocytic leukemia (APL) highly encourages researches to apply this therapy to other types of acute myeloid leukemia (AML). However, AML, with the exception of APL, fails to respond to differentiation therapy. Therefore, research strategies to further sensitize cells to retinoids and to extend the range of AMLs that respond to retinoids beyond APLs are urgently needed. In this study, we showed that TAK165, a HER2 inhibitor, exhibited a strong synergy with ATRA to promote AML cell differentiation. We observed that TAK165 sensitized the AML cells to ATRA-induced cell growth inhibition, G0/G1 phase arrest, CD11b expression, mature morphologic changes, NBT reduction and myeloid regulator expression. Unexpectedly, HER2 pathway might not be essential for TAK165-enhanced differentiation when combined with ATRA, while the enhanced differentiation was dependent on the activation of the RARα/STAT1 axis. Furthermore, the MEK/ERK cascade regulated the activation of STAT1. Taken together, our study is the first to evaluate the synergy of TAK165 and ATRA in AML cell differentiation and to assess new opportunities for the combination of TAK165 and ATRA as a promising approach for future differentiation therapy.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Humans; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Protein Interaction Maps; Receptor, ErbB-2; Retinoic Acid Receptor alpha; STAT1 Transcription Factor; Tretinoin

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)(+) LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD(+) cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD(+) cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD(+) LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD(+) patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drug's apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD(+) LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.

Topics: Animals; Cell Death; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; fms-Like Tyrosine Kinase 3; Gene Duplication; Humans; Leukemia, Myeloid, Acute; Mice; Mutant Proteins; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Sorafenib; Tandem Repeat Sequences; Tretinoin; Xenograft Model Antitumor Assays

In our previous in vitro trials, decitabine and all-trans retinoic acid (ATRA) demonstrated synergistic effects on growth inhibition, differentiation, and apoptosis in SHI-1 cells; in K562 cells, ATRA enhanced the effect of decitabine on p16 demethylation, and the combination of the two drugs was found to activate RAR-β expression (p16 and RAR-β are two tumor suppressor genes). On the rationale of our in vitro trials, we used low-dose decitabine and ATRA to treat 31 myeloid neoplasms deemed ineligible for intensive chemotherapy. The regimen consisted of decitabine at the dose of 15 mg/m(2) intravenously over 1 h daily for consecutive 5 days and ATRA at the dose of 20 mg/m(2) orally from day 1 to 28 except day 4 to 28 in the first cycle, and the regimen was repeated every 28 days. After 6 cycles, decitabine treatment was stopped, and ATRA treatment was continued for maintenance treatment. Treated with a median of 2 cycles (range 1-6), 7 patients (22.6 %) achieved complete remission (CR), 7 (22.6 %) marrow CR (mCR), and 4 (12.9 %) partial remission (PR). The overall remission (CR, mCR, and PR) rate was 58.1 %, and the best response (CR and mCR) rate was 45.2 %. The median overall survival (OS) was 11.0 months, the 1-year OS rate was 41.9 %, and the 2-year OS rate was 26.6 %. In univariate analyses, age, performance status, comorbidities, white blood cell counts and platelets at diagnosis, percentage of bone marrow blasts, karyotype, and treatment efficacy demonstrated no impacts on OS (P > 0.05, each). Main side effects were tolerable hematologic toxicities. In conclusion, low-dose decitabine plus ATRA is a promising treatment for patients with myeloid neoplasms judged ineligible for intensive chemotherapy.

Topics: Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Decitabine; Dose-Response Relationship, Drug; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Tretinoin

All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest of HL-60 cells. Responding to an RA-induced cytosolic signaling machine, c-Raf translocates to the nucleus, providing propulsion for RA-induced differentiation. This novel mechanism is not understood, but presumably reflects c-Raf binding with nuclear gene regulatory proteins. RRD-251 is a small molecule that prevents the interaction of c-Raf and RB, the retinoblastoma tumor suppressor protein. The involvement of c-Raf and RB in RA-induced differentiation motivates interest in the effects of combined RA and RRD-251 treatment on leukemic cell differentiation. We demonstrate that RRD-251 enhances RA-induced differentiation. Mechanistically, we find that nuclear translocated c-Raf associates with pS608 RB. RA causes loss of pS608 RB, where cells with hypophosphorylated S608 RB are G0/G1 restricted. Corroborating the pS608 RB hypophosphorylation, RB sequestration of E2F increased with concomitant loss of cdc6 expression, which is known to be driven by E2F. Hypophosphorylation of S608 RB releases c-Raf from RB sequestration to bind other nuclear targets. Release of c-Raf from RB sequestration results in enhanced association with GSK-3 which is phosphorylated at its S21/9 inhibitory sites. c-Raf binding to GSK-3 is associated with dissociation of GSK-3 and RARα, thereby relieving RARα of GSK-3 inhibition. RRD-251 amplifies each of these RA-induced events. Consistent with the posited enhancement of RARα transcriptional activity by RRD-251, RRD-251 increases the RARE-driven CD38 expression per cell. The RA/c-Raf/GSK-3/RARα axis emerges as a novel differentiation regulatory mechanism susceptible to RRD-251, suggesting enhancing RA-effects with RRD-251 in therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Thiourea; Tretinoin

The E3 ubiquitin ligase and tumor suppressor APC/CCdh1 is crucial for cell cycle progression, development and differentiation in many cell types. However, little is known about the role of Cdh1 in hematopoiesis. Here we analyzed Cdh1 expression and function in malignant hematopoiesis. We found a significant decrease of Cdh1 in primary acute myeloid leukemia (AML) blasts compared to normal CD34+ cells. Thus, according to its important role in connecting cell cycle exit and differentiation, decreased expression of Cdh1 may be a mechanism contributing to the differentiation block in leukemogenesis. Indeed, knockdown (kd) of Cdh1 in HL-60 cell line (AML with maturation, FAB M2) led to less differentiated cells and a delay in PMA-induced differentiation. Acute promyelocytic leukemia (APL, FAB M3) is an AML subtype which is highly vulnerable to differentiation therapy with all-trans retinoic acid (ATRA). Accordingly, we found that APL is resistant to a Cdh1-kd mediated differentiation block. However, further depletion of Cdh1 in APL significantly reduced viability of leukemia cells upon ATRA-induced differentiation. Thus, low Cdh1 expression may be important in AML biology by contributing to the differentiation block and response to therapy depending on differences in the microenvironment and the additional genetic background.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Cadherins; Cell Cycle Checkpoints; Cell Differentiation; Female; Gene Knockdown Techniques; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Tetradecanoylphorbol Acetate; Tretinoin; Young Adult

Abnormal cell differentiation, in particular suppression of terminal cell differentiation, exists in all tumors. Therapeutic interventions to restore terminal differentiation ("differentiation therapy") are a very attractive way to treat cancer, especially leukemia. A variety of chemicals stimulates differentiation of leukemic cells, such as dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA). Tumor suppressor genes have a vital role in the gateway to terminal cell differentiation. In this study, we inhibited PTEN tumor suppressor gene expression by siRNA to investigate the effect of potentiating cell survival and inhibiting apoptosis on HL-60 cell differentiation by DMSO and ATRA. Our results show that PTEN siRNA increases HL-60 cell differentiation in the presence of DMSO and ATRA. At the same time, the presence of siRNA hampers accumulation of apoptotic cells during incubation. Our study suggests that manipulation of PTEN could hold promise for enhancing efficacy of differentiation therapy of acute myelogenous leukemia.

Topics: Apoptosis; Cell Differentiation; Cell Proliferation; Cell Survival; Dimethyl Sulfoxide; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Neutrophils; PTEN Phosphohydrolase; RNA, Small Interfering; Tretinoin

Although all-trans retinoic acid (ATRA) is regarded as a prominent example of differentiation therapy, it is not effective for the treatment of other subtypes of acute myeloid leukemia (AML) beyond acute promyelocytic leukemia (APL). Therefore, new strategies need to be explored to extend the efficacy of ATRA-based therapy to non-APL AML patients. In the present study, staurosporine, a protein kinase C (PKC) pan-inhibitor, exhibited synergism with ATRA to promote granulocytic differentiation in poorly ATRA-sensitive U937 cells but not in ATRA unresponsive K562 and Kasumi cells. Staurosporine or the combined treatment did not affect PKC activity in U937 cells. Moreover, other selective PKC inhibitors, UCN-01, Go6976 or rottlerin failed to enhance ATRA‑induced granulocytic differentiation in U937 cells. Therefore, staurosporine-enhanced ATRA-induced granulocytic differentiation in U937 cells may be independent of PKC. Staurosporine activated mitogen‑activated protein kinase kinase (MEK) and extracellular signal‑regulated kinase (ERK). Meanwhile, staurosporine also enhanced ATRA-promoted upregulation of the protein level of CCAAT/enhancer‑binding protein β (C/EBPβ) and C/EBPε in U937 cells. Furthermore, blockade of MEK activation suppressed staurosporine‑enhanced differentiation as well as the elevated protein level of C/EBPs. Taken together, we concluded that staurosporine enhanced ATRA‑induced granulocytic differentiation in U937 cells via MEK/ERK-mediated modulation of the protein level of C/EBPs.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; Staurosporine; Tretinoin; U937 Cells

Individuals who are exposed to cytotoxic agents are at risk of developing therapyrelated myeloid neoplasms (t-MN). Cytogenetic findings of a neoplasm play an important role in stratifying patients into different risk groups and thus predict the response to treatment and overall survival.. A 59-year-old man was diagnosed with acute promyelocytic leukaemia. Following this, he underwent all-trans retinoic acid (ATRA) based chemotherapy and achieved remission. Four years later, the disease relapsed and he was given idarubicin, mitoxantrone and ATRA followed by maintenance chemotherapy (ATRA, mercaptopurine and methotrexate). He achieved a second remission for the next 11 years. During a follow-up later, his full blood picture showed leucocytosis, anaemia and leucoerythroblastic picture. Bone marrow examination showed hypercellular marrow with trilineage dysplasia, 3% blasts but no abnormal promyelocyte. Fluorescence in-situ hybridisation (FISH) study of the PML/RARA gene was negative. Karyotyping result revealed complex abnormalities and monosomal karyotype (MK). A diagnosis of therapy-related myelodysplastic syndrome/myeloproliferative neoplasm with unfavourable karyotypes and MK was made. The disease progressed rapidly and transformed into therapy-related acute myeloid leukaemia in less than four months, complicated with severe pneumonia. Despite aggressive treatment with antibiotics and chemotherapy, the patient succumbed to the illness two weeks after the diagnosis.. Diagnosis of t-MN should be suspected in patients with a history of receiving cytotoxic agents. Karyotyping analysis is crucial for risk stratification as MK in addition to complex aberrant karyotypes predicts unfavourable outcome. Further studies are required to address the optimal management for patients with t-MN.

Topics: Abnormal Karyotype; Antineoplastic Combined Chemotherapy Protocols; Humans; Idarubicin; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Mercaptopurine; Methotrexate; Middle Aged; Mitoxantrone; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Tretinoin

MicroRNAs have been shown to play an important role in normal hematopoisis and leukemogenesis. Here, we report function and mechanisms of miR-181 family in myeloid differentiation and acute myeloid leukemia (AML). The aberrant overexpression of all the miR-181 family members (miR-181a/b/c/d) was detected in French-American-British M1, M2 and M3 subtypes of adult AML patients. By conducting gain- and loss-of-function experiments, we demonstrated that miR-181a inhibits granulocytic and macrophage-like differentiation of HL-60 cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) by directly targeting and downregulating the expression of PRKCD (which then affected the PRKCD-P38-C/EBPα pathway), CTDSPL (which then affected the phosphorylation of retinoblastoma protein) and CAMKK1. The three genes were also demonstrated to be the targets of miR-181b, miR-181c and miR-181d, respectively. Significantly decreases in the expression levels of the target proteins were detected in AML patients. Inhibition of the expression of miR-181 family members owing to Lenti-miRZip-181a infection in bone marrow blasts of AML patients increased target protein expression levels and partially reversed myeloid differentiation blockage. In the mice implanted with AML CD34+ HSPCs, expression inhibition of the miR-181 family by Lenti-miRZip-181a injection improved myeloid differentiation, inhibited engraftment and infiltration of the leukemic CD34+ cells into the bone marrow and spleen, and released leukemic symptoms. In conclusion, our findings revealed new mechanism of miR-181 family in normal hematopoiesis and AML development, and suggested that expression inhibition of the miR-181 family could provide a new strategy for AML therapy.

Topics: Animals; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cattle; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Gene Expression Regulation, Neoplastic; Granulocytes; Hematopoietic Stem Cells; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Macrophages; Mice; MicroRNAs; Molecular Targeted Therapy; Myeloid Cells; Protein Kinase C-delta; Rats; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transduction, Genetic; Tretinoin; Tumor Suppressor Proteins

Topics: Aged; Antimetabolites, Antineoplastic; Antineoplastic Agents; Azacitidine; Drug Resistance, Neoplasm; Drug Therapy, Combination; Female; Follow-Up Studies; Humans; Hypoglycemic Agents; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Pioglitazone; Prognosis; Remission Induction; Salvage Therapy; Survival Rate; Thiazolidinediones; Tretinoin

Acute myeloid leukemia (AML) is a heterogeneous disease. Numerous molecular abnormalities have been identified in AML and, amongst these, FMS‑like tyrosine kinase 3 (FLT3) mutations are one of the most common somatic alterations detected. In the present study, an in vitro investigation was performed to evaluate the effects of all‑trans retinoic acid (ATRA) and PKC412, alone and in combination, in FLT3‑mutated AML cell lines. Trypan blue exclusion test, as well as morphological, western blot and isobologram analyses were conducted. The results indicated that the combined ATRA and PKC412 treatment exhibited additive or synergistic effects in FLT3‑mutated AML cell lines. These results provided in vitro evidence for the future clinical trials evaluating the effects of a combination treatment using PKC412 and ATRA on AML patients with FLT3‑mutations.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Mutation; Protein Kinase Inhibitors; Staurosporine; Tretinoin

The success of all-trans retinoic acid (ATRA) therapy in acute promeylocytic leukemia (APL) has spawned numerous attempts to translate the paradigm of differentiation therapy to non-APL acute myelocytic leukemia (AML). However, the results of clinical trials have been overall disappointing. In this review we discuss the mechanism of retinoic acid signaling and the results of major clinical trials that have attempted to incorporate ATRA into AML regimens. We discuss recent evidence that indicate that the retinoic acid signaling pathway may be dysfunctional in AML. Preliminary studies suggest that targeting the pathways that modify retinoic acid receptor activity may reactivate the dormant retinoic acid-signaling pathway. Such strategies may revive the ability of ATRA to induce myeloid differentiation and apoptosis in non-APL AML.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Signal Transduction; Tretinoin

All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Female; Gene Knockdown Techniques; Hematopoietic Stem Cells; Histone Demethylases; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Small Interfering; Transcription Factors; Tretinoin; Xenograft Model Antitumor Assays

Nucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (∼30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)-retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1 mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics.

Topics: Animals; Apoptosis; Arsenic Trioxide; Arsenicals; Humans; Leukemia, Myeloid, Acute; Mice; Mice, SCID; Mutant Proteins; Mutation; Nuclear Proteins; Nucleophosmin; Oncogene Proteins; Oxides; Tretinoin; Tumor Cells, Cultured; U937 Cells; Xenograft Model Antitumor Assays

Nucleophosmin-1 (NPM1) is the most frequently mutated gene in acute myeloid leukemia (AML). Addition of retinoic acid (RA) to chemotherapy was proposed to improve survival of some of these patients. Here, we found that RA or arsenic trioxide synergistically induce proteasomal degradation of mutant NPM1 in AML cell lines or primary samples, leading to differentiation and apoptosis. NPM1 mutation not only delocalizes NPM1 from the nucleolus, but it also disorganizes promyelocytic leukemia (PML) nuclear bodies. Combined RA/arsenic treatment significantly reduced bone marrow blasts in 3 patients and restored the subnuclear localization of both NPM1 and PML. These findings could explain the proposed benefit of adding RA to chemotherapy in NPM1 mutant AMLs, and warrant a broader clinical evaluation of regimen comprising a RA/arsenic combination.

Topics: Aged; Aged, 80 and over; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Mutant Proteins; Mutation; Nuclear Proteins; Nucleophosmin; Oxides; Proteolysis; Tretinoin; Tumor Cells, Cultured

MDI 301 is a novel 9-cis retinoic acid derivative in which the terminal carboxylic acid group has been replaced by a picolinate ester. MDI 301, a retinoic acid receptor-α - agonist, suppressed the growth of several human myeloid leukemia cell lines (HL60, NB4, OCI-M2, and K562) in vitro and induced cell-substrate adhesion in conjunction with upregulation of CD11b. Tumor growth in HL60-injected athymic nude mice was reduced. In vitro, MDI 301 was comparable to all-trans retinoic acid (ATRA) whereas in vivo, MDI 301 was slightly more efficacious than ATRA. Most importantly, unlike what was found with ATRA treatment, MDI 301 did not induce a cytokine response in the treated animals and the severe inflammatory changes and systemic toxicity seen with ATRA did not occur. A retinoid with these characteristics might be valuable in the treatment of promyelocytic leukemia, or, perhaps, other forms of myeloid leukemia.

Topics: Animals; Antineoplastic Agents; CD11b Antigen; CD18 Antigens; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Humans; Leukemia, Myeloid, Acute; Mice, Nude; Retinoids; Tretinoin

In this issue of Blood, Martelli et al and El Hajj et al independently report that nucleophosmin-1 (NPM1)-mutant leukemia is particularly vulnerable to a novel strategy combining all-trans retinoic acid (ATRA) with arsenic trioxide (ATO). The era of targeted therapy has seen some of its greatest successes in the hematologic arena (eg, breakpoint cluster region [BCR]/Abelson [ABL] kinase inhibitors in chronic myeloblastic leukemia and ATRA in acute promyelocytic leukemia [APL]). Moreover, addition of ATO, an agent that induces oxidative stress and interferes with protein translation, to ATRA sharply increases APL cell killing to the extent that cures in this disease are no longer unrealistic. A theoretical (and practical) basis for translating ATRA/ATO-based strategies to non-APL acute myelocytic leukemia (AML) is currently lacking.

Topics: Animals; Apoptosis; Arsenicals; Humans; Leukemia, Myeloid, Acute; Nuclear Proteins; Nucleophosmin; Oxides; Proteolysis; Tretinoin

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved outcome in acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of resistance have been previously reported, yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, single agent atRA induces remission without cure. The microenvironment expression of cytochrome P450 (CYP)26, a retinoid-metabolizing enzyme was shown to determine normal hematopoietic stem cell fate. Accordingly, we hypothesized that the bone marrow (BM) microenvironment is responsible for difference between in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. We observed that the pro-differentiation effects of atRA on APL and non-APL AML cells as well as on leukemia stem cells from clinical specimens were blocked by BM stroma. In addition, BM stroma produced a precipitous drop in atRA levels. Inhibition of CYP26 rescued atRA levels and AML cell sensitivity in the presence of stroma. Our data suggest that stromal CYP26 activity creates retinoid low sanctuaries in the BM that protect AML cells from systemic atRA therapy. Inhibition of CYP26 provides new opportunities to expand the clinical activity of atRA in both APL and non-APL AML.

Topics: ADP-ribosyl Cyclase 1; Antigens, CD34; Antineoplastic Agents; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Coculture Techniques; Cytochrome P-450 Enzyme System; Humans; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Retinoic Acid 4-Hydroxylase; Tretinoin

Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI), is approved for the second-line treatment of chronic myeloid leukemia (CML) in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML) are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA). We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b+ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b+ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨm) in CD11b+ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Benzamides; CD11b Antigen; Cell Line, Tumor; Female; Humans; Leukemia, Myeloid, Acute; Leukocytes; Male; Middle Aged; Protein Kinase Inhibitors; Pyrazines; src-Family Kinases; Tretinoin

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.

Topics: Amantadine; Antigens, CD; Antineoplastic Agents; CCAAT-Enhancer-Binding Proteins; Cell Cycle; Cell Differentiation; Cell Proliferation; Gene Expression; HL-60 Cells; Humans; Hypoxanthine Phosphoribosyltransferase; K562 Cells; Leukemia, Myeloid, Acute; Macrophages; Piperidines; Protein Interaction Maps; Receptors, Calcitriol; Tamoxifen; Tretinoin

Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future.

Topics: Antineoplastic Agents; Apoptosis; Base Sequence; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Drug Screening Assays, Antitumor; Gene Expression Regulation, Leukemic; Genes, Reporter; High-Throughput Screening Assays; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Transcriptional Activation; Tretinoin

Topics: Aged, 80 and over; Antineoplastic Agents; Bone Marrow; Granulocyte Precursor Cells; Humans; Karyotype; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Tretinoin

6-Formylindolo(3,2-b)carbazole (FICZ) is a photoproduct of tryptophan and an endogenous high affinity ligand for aryl hydrocarbon receptor (AhR). It was previously reported that, in patient-derived HL-60 myeloblastic leukemia cells, retinoic acid (RA)-induced differentiation is driven by a signalsome containing c-Cbl and AhR. FICZ enhances RA-induced differentiation, assessed by expression of the membrane differentiation markers CD38 and CD11b, cell cycle arrest and the functional differentiation marker, inducible oxidative metabolism. Moreover, FICZ augments the expression of a number of the members of the RA-induced signalsome, such as c-Cbl, Vav1, Slp76, PI3K, and the Src family kinases Fgr and Lyn. Pursuing the molecular signaling responsible for RA-induced differentiation, we characterized, using FRET and clustering analysis, associations of key molecules thought to drive differentiation. Here we report that, assayed by FRET, AhR interacts with c-Cbl upon FICZ plus RA-induced differentiation, whereas AhR constitutively interacts with Cbl-b. Moreover, correlation analysis based on the flow cytometric assessment of differentiation markers and western blot detection of signaling factors reveal that Cbl-b, p-p38α and pT390-GSK3β, are not correlated with other known RA-induced signaling components or with a phenotypic outcome. We note that FICZ plus RA elicited signaling responses that were not typical of RA alone, but may represent alternative differentiation-driving pathways. In clusters of signaling molecules seminal to cell differentiation, FICZ co-administered with RA augments type and intensity of the dynamic changes induced by RA. Our data suggest relevance for FICZ in differentiation-induction therapy. The mechanism of action includes modulation of a SFK and MAPK centered signalsome and c-Cbl-AhR association.

Topics: ADP-ribosyl Cyclase 1; Antineoplastic Agents; Biomarkers; Carbazoles; CD11b Antigen; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-cbl; Receptors, Aryl Hydrocarbon; Signal Transduction; src-Family Kinases; Tretinoin

Use of all-trans retinoic acid (ATRA) as a differentiation agent has been limited to acute promyelocytic leukemia (APL) as non-APL leukemias are insensitive to ATRA. We recently demonstrated that the rexinoid, bexarotene, induces differentiation and therapeutic responses in patients with refractory AML. Rexinoids bind and activate retinoid X receptors (RXRs); however, rexinoids alone are incapable of activating retinoic acid receptor (RAR)/RXR complexes, suggesting that myeloid differentiation can occur independent of RAR. In this study, we demonstrate that rexinoid differentiation of AML cells is RAR independent and requires the expression of PU.1. Because of the promiscuousness of RXR with other nuclear receptors, myeloid differentiation by bexarotene with other nuclear receptor ligands was explored. Bexarotene cooperated with ATRA to enhance differentiation in some AML cell lines; however, the combination of bexarotene with the PPARγ agonist rosiglitazone did not. In contrast, bexarotene combined with liver X receptor (LXR) agonists, T0901317 or GW3965, induced potent differentiation and cytotoxicity in AML cell lines and primary human AML cells, but not in normal progenitor cells. These results suggest that RXR/LXR-regulated gene expression in normal cells is deregulated in AML cells and identifies a potential role for these agonists in differentiation therapy of non-APLs.

Topics: Bexarotene; CCAAT-Enhancer-Binding Protein-alpha; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Line, Tumor; Humans; Hydrocarbons, Fluorinated; Leukemia, Myeloid, Acute; Liver X Receptors; Nicotinic Acids; Orphan Nuclear Receptors; Proto-Oncogene Proteins; Retinoid X Receptors; Sulfonamides; Tetrahydronaphthalenes; Trans-Activators; Tretinoin

Acute myeloid leukemia (AML) is frequently linked to epigenetic abnormalities and deregulation of gene transcription, which lead to aberrant cell proliferation and accumulation of undifferentiated precursors. ZRF1, a recently characterized epigenetic factor involved in transcriptional regulation, is highly overexpressed in human AML, but it is not known whether it plays a role in leukemia progression. Here, we demonstrate that ZRF1 depletion decreases cell proliferation, induces apoptosis and enhances cell differentiation in human AML cells. Treatment with retinoic acid (RA), a differentiating agent currently used to treat certain AMLs, leads to a functional switch of ZRF1 from a negative regulator to an activator of differentiation. At the molecular level, ZRF1 controls the RA-regulated gene network through its interaction with the RA receptor α (RARα) and its binding to RA target genes. Our genome-wide expression study reveals that ZRF1 regulates the transcription of nearly half of RA target genes. Consistent with our in vitro observations that ZRF1 regulates proliferation, apoptosis, and differentiation, ZRF1 depletion strongly inhibits leukemia progression in a xenograft mouse model. Finally, ZRF1 knockdown cooperates with RA treatment in leukemia suppression in vivo. Taken together, our data reveal that ZRF1 is a key transcriptional regulator in leukemia progression and suggest that ZRF1 inhibition could be a novel strategy to be explored for AML treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Disease Progression; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Leukemia, Myeloid, Acute; Mice; Mice, SCID; Molecular Chaperones; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; RNA-Binding Proteins; Signal Transduction; Transcription, Genetic; Transfection; Tretinoin; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents; Bone Marrow Cells; Humans; Karyotyping; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oncogene Proteins, Fusion; Remission Induction; Tretinoin

Somatic mutations in isocitrate dehydrogenase 1 (IDH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique.. The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML.. Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDH1gene, codon 132 mutations screening.. The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p=0.03). Patients with IDH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p=0.08).. IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDH1 mutations in AML.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; DNA Mutational Analysis; Doxorubicin; Female; Genetic Association Studies; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Prognosis; Treatment Outcome; Tretinoin; Young Adult

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for treatment with other anticancer compounds. The major drawback of HDACi is the broad inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia (APL), repression by the PML-RARα oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic doses of all trans retinoic acid (ATRA) inducing differentiation and cell death at the expense of side effects and recurrence. We hypothesized that the context-specific close physical proximity of a retinoid and HDACi-binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275, yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of PML-RARα-binding sites. RNA-seq reveals that MC2392 alters expression of a number of stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive, caspase-8-dependent cell death accompanied by RIP1 induction and ROS production. Solid and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARα moiety and selectively inhibits the HDACs resident in the repressive complex responsible for the transcriptional impairment in APLs. Our findings provide proof-of-principle of the concept of a context-dependent targeted therapy.

Topics: Acetylation; Apoptosis; Caspase 8; Cell Death; Cell Differentiation; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Leukemia, Myeloid, Acute; Oncogene Proteins, Fusion; Reactive Oxygen Species; Retinoids; Signal Transduction; Transcription, Genetic; Transfection; Tretinoin; U937 Cells

Knowledge of the molecular basis of acute myeloid leukaemia has increased considerably in the past few years, and therapies targeting specific molecular defects of this disease are intensively investigated. Patients with both NPM1 and FLT3-ITD mutations encompass 20% of cytogenetically normal AML. The multikinase and FLT3 inhibitor, sorafenib, has shown some efficacy in patients with relapsed FLT3-ITD(+) AML. In addition, it is suggested that all-trans retinoic acid (ATRA) used in combination with chemotherapy has shown to improve outcome of patients harbouring NPM1 mutations. We report here the clinical course of three patients with refractory or relapsed FLT3-ITD(+) /NPM1(+) AML who achieved significant response upon sorafenib and ATRA combination.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Fatal Outcome; Female; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Niacinamide; Nuclear Proteins; Nucleophosmin; Phenylurea Compounds; Sorafenib; Treatment Outcome; Tretinoin; Young Adult

Folate and retinoic acid grafted/dextran (FA-RA/DEX) copolymers with different molecular weight of DEX were synthesized using carbonyldiimidazole and dimethylaminopyridine for targeted delivery of doxorubicin (DOX) in acute myelogenous leukemia (AML). The copolymers structure was confirmed by (1)H NMR and FTIR. Critical micelle concentration (CMC) of each copolymer was determined using pyrene as a fluorescent probe. DOX was loaded in micelles by the direct dissolution method. Physical properties of micelles, including particle size, zeta potential, drug loading efficiency, and drug release profiles, were examined. The orientation of the folate ligand on the surface of the micelles was studied by X-ray photoelectron spectroscopy (XPS) technique. The cytotoxicity of micelles loaded with DOX at different concentrations was studied in KG1 cells using MTT assay and their cellular uptake by flow cytometry technique. FTIR and (1)H NMR spectra confirmed successful production of the targeted micelles and XPS spectra showed the surface orientation of folate. R15D10F7 copolymer produced micelles with particle size of 82.86 nm, polydispersity index of 0.3, zeta potential of -4.68 mV, drug loading efficiency of 96%, and release efficiency of 63%. DOX loaded in folate-targeted micelles of RA/DEX was more toxic than that in nontargeted micelles and free drug and seems promising in reducing drug resistance in AML.

Topics: Antibiotics, Antineoplastic; Cell Line, Tumor; Dextrans; Doxorubicin; Drug Carriers; Folic Acid; Humans; Leukemia, Myeloid, Acute; Micelles; Tretinoin; Vitamin B Complex

Topics: Antineoplastic Agents; Drug Resistance, Neoplasm; Fatal Outcome; Humans; Leukemia, Myeloid, Acute; Male; Nuclear Pore Complex Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Tretinoin

All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML-RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPɛ and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Checkpoints; Cell Differentiation; Cytarabine; DNA-Binding Proteins; Drug Synergism; Histone-Lysine N-Methyltransferase; Histones; Humans; Leukemia, Myeloid, Acute; Myeloid-Lymphoid Leukemia Protein; Neoplasm Proteins; Nuclear Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Trans-Activators; Transcriptional Activation; Transcriptional Elongation Factors; Tretinoin

Acute myeloid leukemia is classified based upon recurrent cytogenetic abnormalities. The t(15;17)(q24.1;q21.1) abnormality is found in 5% to 8% of de novo acute myeloid leukemia and is diagnostic of acute promyelocytic leukemia (APL). The translocation results in fusion of the retinoic acid receptor-α (RARA) gene at 17q21.1 and the promyelocytic leukemia (PML) gene at 15q24.1. Standard APL therapy is a combination of all-trans retinoic acid and anthracycline-based chemotherapy. Anthracycline treatment is associated with secondary clonal chromosomal aberrations that can lead to therapy-related secondary myeloid neoplasms. We present a pediatric case of relapsed APL coexistent with treatment-associated secondary myeloid neoplasm with t(11;19)(q23;p13.1).

Topics: Anthracyclines; Antineoplastic Agents; Child; Chromosome Aberrations; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 19; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Neoplasms, Second Primary; Nuclear Proteins; Promyelocytic Leukemia Protein; Receptors, Retinoic Acid; Recurrence; Retinoic Acid Receptor alpha; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

Myeloid cell differentiation is the process by which stem cells develop into mature monocytes or granulocytes. This process is achieved by the sequential activation of variety of genes. Disruption of this process can result in immunodeficiency, bone marrow failure syndrome, or leukemia. Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation and can be treated by a combination of all-trans retinoic acid (ATRA) and anthracycline. This treatment can induce leukemic cell differentiation, leading to extremely high remission rates. XAB2, a molecule involved in nucleotide excision repair (NER), is downregulated during granulocyte differentiation and shows reduced expression in NB4 APL-derived cells in vitro. Differentiation of APL by ATRA treatment reduced XAB2 expression levels in vivo. These observations suggest that cellular differentiation is associated with reduced NER activity and provides new insights into combined differentiation induction. NB4 cells were more susceptible than the immature myeloid leukemic cell lines, Kasumi-3 and Kasumi-1, to the DNA interstrand crosslinking agent cisplatin.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cisplatin; DNA Repair; Gene Expression Regulation, Leukemic; Genes, Reporter; Granulocytes; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Luciferases; Monocytes; RNA Splicing Factors; Signal Transduction; Transcription Factors; Transcription, Genetic; Tretinoin

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34(+) progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Gene Expression; Gene Expression Regulation, Leukemic; Granulocytes; Humans; Leukemia, Myeloid, Acute; Sequestosome-1 Protein; Tretinoin; Ubiquitination; Up-Regulation

Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBPβ contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBPβ could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBPβ. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPβ in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.

Topics: Antineoplastic Agents; CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression; Humans; K562 Cells; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-myc; Thiazoles; Tretinoin

Emodin, an extracted natural compound from the root and rhizome of Rheum palmatum L, has been shown to have multiple biological activities including anticancer functions in previous studies. In this study, we investigated the anti-leukemic activity of emodin alone or emodin in the presence all-trans retinoic acid (ATRA) in acute myeloid leukemia (AML) cells and the potential signaling pathway involved. We demonstrated that emodin could significantly enhance the sensitivity to ATRA and present additive differentiation-inducing effects in AML cell line NB4 cells and, especially, in NB4-derived ATRA-resistant MR2 cells. Further study showed that increasing dose of emodin could effectively induce growth inhibition and apoptotic effects in both cell lines as well as in primary leukemic cells from AML patients. Moreover, the apoptotic induction in AML cells was associated with the activation of caspase cascades involving caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, leukemic cell response to emodin stimuli in vitro was observed through the decreased expression levels of Bcl-2 and retinoic acid receptor α (RARα). Importantly, emodin was demonstrated as a new inhibitor of PI3K/Akt in AML cells, even in primary AML cells. It inhibited Akt phosphoration (p-Akt) at Ser473 as efficiently as mTOR at Ser2448. Consistently, it exerted suppression effects on the phosphoration of mTOR downstream targets, 4E-BP1 and p70S6K. Taken together, these findings indicate that emodin might be developed as a promising anti-leukemic agent to improve the patient outcome in AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase 3; Cell Differentiation; Cell Proliferation; Emodin; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Tretinoin

We previously demonstrated that 6-benzylthioinosine (6-BT) could induce the differentiation of a subset of acute myeloid leukemia (AML) cell lines and primary AML cells regardless of their cytogenetics. In this study we investigated whether Wnt signaling pathways played roles in 6-BT-induced differentiation of AML cells.. We induced differentiation of HL-60 leukemic cells and primary AML cells in vitro using 6-BT. Real-time PCR (qPCR), western blot, and luciferase assays were used to examine the molecules' expression and biological activity in canonical and noncanonical Wnt signaling pathways. AML cell differentiation was measured by the Nitroblue tetrozolium (NBT) reduction assay.. 6-BT regulated the expression of both canonical and non-canonical Wnt signaling molecules in HL-60 cells. Both 6-BT and all-trans-retinoic-acid (ATRA) reduced canonical Wnt signaling and activated noncanonical Wnt/Ca2+ signaling in HL-60 cells. Pre-treatment of HL-60 cells with an inhibitor of glycogen synthase kinase-3β (GSK-3β), which activated canonical Wnt signaling, partly abolished the differentiation of HL-60 cells induced by 6-BT. Pre-treatment of HL-60 cells with an inhibitor of protein kinase C (PKC), resulting in inactivation of non-canonical Wnt/Ca2+ signaling, abolished 6-BT-induced differentiation of HL-60 cells. Several molecules in the non-canonical Wnt/Ca2+ pathway were detected in bone marrow samples from AML patients, and the expression of FZD4, FZD5, Wnt5a and RHOU were significantly reduced in newly diagnosed AML samples compared with normal controls.. Both canonical and non-canonical Wnt signaling were involved in 6-BT-induced differentiation of HL-60 cells, and played opposite roles in this process. Wnt signaling could be involved in the pathogenesis of AML not only by regulating self-renewal of hematopoietic stem cells, but also by playing a role in the differentiation of AML cells.

Topics: Calcium Signaling; Cell Differentiation; Cells, Cultured; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Thioinosine; Tretinoin; Wnt Signaling Pathway

In acute myeloid leukemia (AML) without retinoic acid receptor (RAR) rearrangement, the effect of all-trans-retinoic acid (ATRA) is still poorly understood despite an association of NPM1 mutation and ATRA response. Recently, preferentially expressed antigen in melanoma (PRAME) has been shown to be a dominant repressor of RAR signaling.. Thus, we further investigated ATRA response mechanisms, especially the impact of PRAME expression on ATRA responsiveness. We profiled gene expression in diagnostic samples derived from our AML HD98B trial, in which ATRA was administered in addition to intensive chemotherapy.. Our data revealed a PRAME expression-associated gene pattern to be significantly enriched for genes involved in the retinoic acid metabolic process. In leukemia cell line models, we could show that retinoic acid-regulated cell proliferation and differentiation are impacted by PRAME expression. In patients with primary AML, repressor activity of high-PRAME levels might be overcome by the addition of ATRA as indicated by better outcome in 2 independent studies (P = 0.029).. PRAME seems to impair differentiation and to increase proliferation likely via blocking RAR signaling, which might be reversed by ATRA. PRAME therefore represents a promising target for both ATRA treatment and possibly future immunotherapeutic approaches in AML.

Topics: Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cell Proliferation; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Clinical Trials, Phase III as Topic; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Humans; K562 Cells; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Nucleophosmin; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; RNA, Small Interfering; Signal Transduction; Transcriptome; Translocation, Genetic; Treatment Outcome; Tretinoin

To verify the differential expression of non-metastasis cell 3 (NME3) protein in HL-60 cells when they were induced to differentiate into monocyte and granulocyte like cells, and study its value in diagnosis of acute myeloid leukemia, all-trans retinoic acid (ATRA) and a new steroidal drug NSC67657 were employed to induce acute myeloid leukemia HL-60 cells into monocyte and granulocyte like cells. Then the cell differentiating direction was observed by chemical staining, the degree of differentiation was determined by surface antigen CD11b/CD14 detection, and the apoptosis was excluded by phosphatidylserine valgus analysis, by which cellular differentiating model was constructed. Furthermore, RT-PCR and Western blot were employed to verify the differentially expression of NME3 before and after differentiation of HL-60 cells. At last, samples from bone marrow nucleated cells of 26 patients with myeloid leukemia, which were diagnosed definitely by clinical doctors, and 5 normal people were chosen. Then the expressing trend of NME3 protein in these testing groups was analyzed by means of comparison. The results showed that ATRA (2 µmol/L for 5 d) and NSC67657 (10 µmol/L for 5 d) could induce HL-60 cells to differentiate into monocyte and granulocyte like cells above 90% without cell apoptosis. The expression of NME3 gene and protein were down-regulated by the inducers, which was accorded with the screening results that was got using proteomics technology in the former research. The expression of NME3 protein in bone marrow from acute myeloid leukemia patients was elevated significantly as compared to normal persons. It is concluded that the expression level of NME3 protein is down-regulated after cellular differentiation, according with the changing trend in leukemia patients, which imply that NME3 protein may be a potential biomarker for diagnosis of acute myeloid leukemia.

Topics: Adolescent; Adult; Aged; Bone Marrow; Case-Control Studies; Child; Child, Preschool; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mesylates; Middle Aged; NM23 Nucleoside Diphosphate Kinases; Steroids; Tretinoin; Young Adult

Alterations in cell cycle pathways and retinoic acid signaling are implicated in leukemogenesis. However, little is known about the roles of cyclin-dependent kinases (CDKs) in treatment response of leukemia. In this study, we observed that CDK1 expression was significantly higher in bone marrow from 42 patients with acute myeloid leukemia (AML) at recurrence than that at first diagnosis (p = 0.04). AML patients had higher level of nuclear CDK1 in their leukemic blasts tended to have poorer clinical outcome compared with those with lower levels. We showed that CDK1 function is required for all-trans retinoic acid (ATRA) to achieve the optimal effect in U-937 human leukemic cells. CDK1 modulates the levels of P27(kip) and AKT phosphorylation in response to ATRA treatment. Further, we show, for the first time, that RARγ in concert with ATRA regulates protein levels of CDK1 and its subcellular localization. The regulation of the subcellular content of CDK1 and RARγ by ATRA is an important process for achieving an effective response in treatment of leukemia. RARγ and CDK1 form a reciprocal regulatory circuit in the nucleus and influence the function and protein stability of each other and the level of P27(kip) protein. In addition, expression of wee1 kinase and Cdc25A/C phosphatases also coincide with CDK1 expression and its subcellular localization in response to ATRA treatment. Our study reveals a novel mechanism by which CDK1 and RARγ coordinate with ATRA to influence cell cycle progression and cellular differentiation.

Topics: Adult; Bone Marrow Cells; CDC2 Protein Kinase; Cell Cycle; Cell Differentiation; Cell Fractionation; Cyclin-Dependent Kinase Inhibitor p27; DNA Primers; Flow Cytometry; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Neoplasm Recurrence, Local; Oncogene Protein v-akt; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Addition of retinoic acid to chemotherapy improves survival of patients with acute myeloid leukemia. This effect is more pronounced in leukemias that express high levels of PRAME. PRAME is an inhibitor of retinoic acid signaling, which may prove to be an important marker for retinoic acid response.

Topics: Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Humans; Leukemia, Myeloid, Acute; Receptors, Retinoic Acid; Tretinoin

The American Society of Hematology Annual Meeting includes physicians, scientists, administrators, medical students, graduate students, allied health professionals and exhibitors. This year's meeting was held in Atlanta, GA, USA. Presentations included a wide variety of topics in benign and malignant hematology.

Topics: Anthracyclines; Antineoplastic Agents; fms-Like Tyrosine Kinase 3; Humans; Idarubicin; Leukemia, Myeloid, Acute; Societies, Scientific; Tretinoin; United States

Topics: Female; Humans; Leukemia, Myeloid, Acute; Middle Aged; Sweet Syndrome; Tretinoin

Potential prognostic biomarkers in acute myeloid leukemia (AML) can be identified by understanding the cellular pathway and molecular changes underlying leukemogenesis. Deregulation of apoptosis is one of the important features of AML and to understand the molecular mechanism underlying apoptosis and its contribution to tumor progression, this study aimed to evaluate anti-apoptotic Bcl2 protein expression in AML and correlate with FLT3 parameters for their role in prognosis of disease.Bcl2 and FLT3 protein expression was quantified by flow cytometry on leukemic blasts in total 174 de novo AML, myelodysplastic syndrome (MDS) and aplastic anemia patients. FLT3 internal tandem duplication (ITD), Tyrosine kinase domain (TKD) point mutations and quantification of mRNA level was carried out using PCR and RT-PCR methods. The incidence of Bcl2 positivity was 71% in AML patients. Bcl2 positivity was significantly associated with CD34+ and CD117+ AML. Bcl2 positivity tended to be associated with reduced DFS while Bcl2 positivity with FLT3 protein positivity was significantly associated with reduced DFS. In multivariate analysis, Bcl2+ and combined Bcl2+/FLT3 protein+ along with high WBC count emerged as poor prognostic factors for reduced DFS and high blast count for predicting reduced OS. In MDS patients, the incidence of Bcl2 expression was high while in aplastic anemia patients, incidence of Bcl2 expression was low.Patients with Bcl2 and FLT3 protein positivity showed significantly reduced DFS suggesting parallel role of these proteins in imparting chemoresistance to the leukemic cells.

Topics: Adolescent; Adult; Anemia, Aplastic; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Drug Resistance, Neoplasm; Female; Flow Cytometry; fms-Like Tyrosine Kinase 3; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Myelodysplastic Syndromes; Oxides; Prognosis; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Tandem Repeat Sequences; Tretinoin; Young Adult

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G0/G1 phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38(MAPK)) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38(MAPK) or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.

Topics: Antigens, CD34; CD11b Antigen; Cell Cycle Checkpoints; Cell Differentiation; Cholecalciferol; ErbB Receptors; Erlotinib Hydrochloride; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 14; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; src-Family Kinases; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.

Topics: Adult; Aged; Antineoplastic Agents; Benzamides; Cell Differentiation; Cell Line; DNA Methylation; Drug Interactions; Epigenesis, Genetic; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Promoter Regions, Genetic; Pyridines; Tretinoin

All-trans-retinoic acid (ATRA) has held great promise for differentiation-based therapy but reportedly downregulates retinoic acid receptor-α (RARα) in a proteasome-dependent manner, which leads to decreased acute myeloid leukemia (AML) cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond acute promyelocytic leukemia (APL) are key investigative avenues. Here, we show that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes, indicative of myeloid differentiation without cell death. In addition, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (P < 0.05). These enhanced differentiation effects were accompanied by RARα stabilization and STAT1 activation. Taken together, our study was the first to evaluate bortezomib and ATRA synergy in AML cell differentiation and to assess new opportunities for bortezomib and ATRA combination as a promising approach for future differentiation therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cell Differentiation; Cell Proliferation; Drug Synergism; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Nude; Pyrazines; Random Allocation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; STAT1 Transcription Factor; Tretinoin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Antibodies play a fundamental role in diagnostic immunophenotyping of leukemias and in cell-targeting therapy. However, this versatility is not reflected in imaging diagnostics. In the present study, we labeled anti–human mAbs monochromatically against selected human myeloid markers expressed on acute myeloid leukemia (AML) cells, all with the same near-infrared fluorochrome. In a novel “multiplexing” strategy, we then combined these mAbs to overcome the limiting target-to-background ratio to image multiple xenografts of AML. Time-domain imaging was used to discriminate autofluorescence from the distinct fluorophore-conjugated antibodies. Imaging with multiplexed mAbs demonstrated superior imaging of AML to green fluorescent protein or bioluminescence and permitted evaluation of therapeutic efficacy with the standard combination of anthracycline and cytarabine in primary patient xenografts. Multiplexing mAbs against CD11b and CD11c provided surrogate imaging biomarkers of differentiation therapy in an acute promyelocytic leukemia model treated with all-trans retinoic acid combined with the histone-deacetylase inhibitor valproic acid. We present herein an optimizedapplication of multiplexed immunolabeling in vivo for optical imaging of AML cellxenografts that provides reproducible, highly accurate disease staging and monitoring of therapeutic effects.

Topics: Animals; Anthracyclines; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; CD11b Antigen; CD11c Antigen; Cell Line, Tumor; Cytarabine; Fluorescent Dyes; Green Fluorescent Proteins; HL-60 Cells; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Luminescent Measurements; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Optical Imaging; Tretinoin; Valproic Acid; Xenograft Model Antitumor Assays

Acute myeloid leukemia (AML) is a rare complication observed after liver transplantation and only a handful of cases have been reported until now. We report a case of acute promyelocytic leukemia (APL) after liver transplantation in a 50-year-old man. The case presentation was postodontectomy bleeding with an associative abnormal coagulation test 85 months after liver transplantation. A routine blood test, bone marrow test, chromosome analysis and examination of PML/RARα chimeric gene confirmed the diagnosis of APL and disseminated intravascular coagulation (DIC). Induction chemotherapy with all-trans retinoic acid, arsenic trioxide and daunorubicin was given to this patient and complete remission was achieved. The patient was subjected to DA (daunorubicin combined with cytarabine) and MA (mitoxantrone combined with cytarabine) regimens after remission induction to consolidate the chemotherapy for two courses of treatment, and subsequently subjected to arsenous acid chemotherapy on a periodic basis. Twenty-two months into the follow-up, sustained bone marrow remission was observed with the adapted treatment regimen.

Topics: Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Bone Marrow; Cytarabine; Daunorubicin; Hepatitis B; Humans; Leukemia, Myeloid, Acute; Liver Cirrhosis; Liver Transplantation; Male; Middle Aged; Mitoxantrone; Oxides; Tretinoin

Topics: Animals; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Humans; Leukemia; Leukemia, Myeloid, Acute; Mice; Neoplasm Proteins; Trans-Activators; Tretinoin; Tumor Suppressor Proteins

MicroRNAs can influence hematopoietic cell lineage commitment and aberrant expression of hematopoietic miRNAs contributes to AML pathology. We found that miR-143 and miR-145 expression is significantly repressed in primary AML patient samples as compared to neutrophils of healthy donors. Further analysis revealed impaired neutrophil differentiation of APL cells upon inhibition of miR-145 expression. Lastly, we identified p73 as transcriptional regulator of miR-143/145 during neutrophil differentiation of APL cells. Our data suggest that low miR-145 levels in APL, possibly due to aberrant expression of p73 transcription factors, contribute to the differentiation block seen in this disease.

Topics: Antineoplastic Agents; Cell Differentiation; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Neutrophils; Nuclear Proteins; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured; Tumor Protein p73; Tumor Suppressor Proteins

Acute myeloid leukemia (AML) is the most common form of leukemia in adults. Unfortunately, the standard therapeutic agents used for this disease have high toxicities and poor efficacy. The one exception to these poor outcomes is the use of the retinoid, all-trans retinoic acid (ATRA), for a rare subtype of AML (APL). The use of the differentiation agent, ATRA, in combination with low-dose chemotherapy leads to the long-term survival and presumed cure of 75-85% of patients. Unfortunately ATRA has not been clinically useful for other subtypes of AML. Though many non-APL leukemic cells respond to ATRA, they require significantly higher concentrations of ATRA for effective differentiation. Here we show that the combination of ATRA with glycogen synthase kinase 3 (GSK3) inhibition significantly enhances ATRA-mediated AML differentiation and growth inhibition. These studies have revealed that ATRA's receptor, the retinoic acid receptor (RAR), is a novel target of GSK3 phosphorylation and that GSK3 can impact the expression and transcriptional activity of the RAR. Overall, our studies suggest the clinical potential of ATRA and GSK3 inhibition for AML and provide a mechanistic framework to explain the promising activity of this combination regimen.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Differentiation; Cells, Cultured; Drug Synergism; Enzyme Inhibitors; Female; Glycogen Synthase Kinase 3; Humans; Immunoprecipitation; Leukemia, Myeloid, Acute; Luciferases; Mice; Mice, Nude; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptors, Retinoic Acid; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcriptional Activation; Tretinoin

Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4(me2)) across the genome, but it did increase H3K4(me2) and expression of myeloid-differentiation-associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.

Topics: Animals; Antigens, CD34; Apoptosis; CD11b Antigen; Cell Differentiation; Cells, Cultured; Cytokines; Disease Models, Animal; Drug Interactions; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation; Histone Demethylases; Humans; Leukemia, Myeloid, Acute; Lysine; Mice; Mice, Inbred NOD; Mice, SCID; RNA, Small Interfering; Stem Cell Factor; Time Factors; Transplants; Tranylcypromine; Tretinoin

Although microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34(+) hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate-induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-β-activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.

Topics: Antineoplastic Agents; Carcinogens; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; HEK293 Cells; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Myeloid Cells; Tetradecanoylphorbol Acetate; Transfection; Tretinoin

The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is a member of a conserved superfamily of nuclear proteins whose functions are largely unknown. In our previous work, ANP32B was identified as a novel direct substrate for caspase-3 and acted as a negative regulator for leukemic cell apoptosis. In this work, we provided the first demonstration that ANP32B expression was down-regulated during differentiation induction of leukemic cells by all-trans retinoic acid (ATRA). Knockdown of ANP32B expression by specific shRNA enhanced ATRA-induced leukemic cell differentiation, while ectopic expression of ANP32B attenuated it, indicating an inhibitory role of ANP32B against leukemic cell differentiation. Furthermore, luciferase reporter assay revealed that ANP32B might exert this role through inhibiting the ATRA dependent transcriptional activity of retinoic acid receptor (RARα). These data will shed new insights into understanding the biological functions of ANP32B protein.

Topics: Apoptosis; Cell Line, Tumor; Down-Regulation; Gene Knockdown Techniques; Humans; Leukemia, Myeloid, Acute; Nuclear Proteins; Receptors, Retinoic Acid; RNA, Small Interfering; Signal Transduction; Tretinoin

All-trans-retinoic acid reverses malignant cell growth and induces cell differentiation and apoptosis. Poor aqueous solubility and uncertain bioavailability are the limiting factors for using all-trans-retinoic acid for tumor therapy. The objective of present study was to encapsulate the hydrophobic drug all-trans-retinoic acid in the polymer poly (lactide-coglycolide). The encapsulation was expected to improve the bioavailability and solubility of the drug. Oil in water single emulsion solvent evaporation technique used for the preparation efficiently encapsulated about 60% of the drug. The drug release profile showed a biphasic pattern with 70% of the drug being released in first 48 hrs and the residual drug showing a slow controlled release reaching up to 8 days. The particle size of 150-200 nm as determined with TEM was ideal for tumor targeting. All-trans-retinoic acid loaded nanoparticles were efficient to induce differentiation and blocked the proliferation of HL-60 cells invitro. These studies also revealed that the dosage of drug required for the therapeutic effects have been reduced efficiently. Our studies thereby demonstrate that Poly (lactide-co-glycolide) based nanoparticles may be efficient for parenteral administration of the drug.

Topics: Antineoplastic Agents; Cell Differentiation; Drug Carriers; Drug Compounding; Emulsions; HL-60 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Leukemia, Myeloid, Acute; Microscopy, Electron, Transmission; Nanoparticles; Particle Size; Polyglactin 910; Solubility; Tretinoin

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D(3) (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.

Topics: Cell Differentiation; Cytosol; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Receptors, Calcitriol; Steroid Hydroxylases; Tretinoin; Tumor Cells, Cultured; Vitamin D; Vitamin D3 24-Hydroxylase

The present study tested whether all-trans retinoic acid (ATRA) and 5-Aza-2'-deoxycitidine (5-Aza) affect AML cell differentiation and growth in vitro by acting on the CCAAT/enhancer binding protein α (C/EBPα) and c-Myc axis. After exposure to a combination of these agents, cell differentiation and growth arrest were significantly higher in human and murine MLL-AF9-expressing cells than in MLL-AF4/AF5q31-expressing cells, which were partly associated with increased expression of C/EBPα, C/EBPε, and PU.1, and decreased expression of c-Myc. These findings indicate that MLL-AF9-expressing cells are more sensitive to ATRA and 5-Aza, indicating that different MLL fusion proteins possess different epigenetic properties associated with retinoic acid pathway inactivation.

Topics: Animals; Azacitidine; CCAAT-Enhancer-Binding Protein-alpha; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Drug Resistance, Neoplasm; Hematopoietic Stem Cells; Histone-Lysine N-Methyltransferase; Humans; Leukemia, Myeloid, Acute; Mice; Myeloid-Lymphoid Leukemia Protein; Nuclear Proteins; Oncogene Proteins, Fusion; Tretinoin

(E)-4-[3'-(1-Adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces the cell cycle arrest and apoptosis of cancer cells. Because its pharmacologic properties-solubility, bioavailability, and toxicity-required improvement for translation, structural modifications were made by introducing nitrogen atoms into the cinnamyl ring and replacing its E-double bond with XCH(2) (X = O, N, and S) with the objective of enhancing these properties without impacting apoptosis-inducing activity. Analogues having nitrogen atoms in heterocyclic rings corresponding to the cinnamyl phenyl ring displayed equal or higher biological activities. The pyrimidine and pyridine analogues were more soluble in both phosphate-buffered saline and water. While the 2,5-disubstituted pyridine analogue was the most potent inducer of KG-1 acute myeloid leukemia cell apoptosis, on the basis of apoptotic activity in KG-1 cells and solubility, the 2,5-disubstituted pyrimidine proved to be the more promising candidate for treatment of acute myeloid leukemia.

Topics: Acrylates; Adamantane; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cinnamates; Drug Design; Humans; Leukemia, Myeloid, Acute; Ligands; Mice; Orphan Nuclear Receptors

All-trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation-based therapy in leukemia; however, the mechanisms of ATRA's biological effects remain largely unknown. Here we first reported that ATRA-induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin-protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA-induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA-elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRA's effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin-proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA-dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA-elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy.

Topics: Antineoplastic Agents; Blotting, Western; Cell Cycle; Cell Differentiation; Cell Proliferation; Humans; Immunoprecipitation; Leukemia, Myeloid, Acute; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Ubiquitination

We report the case of a 6-year-old boy diagnosed with acute promyelocytic leukemia (AML-M3V) when he presented with pallor, abdominal pain, anorexia, and fatigue. Induction chemotherapy was started according to the AML-BFM 98 protocol along with Vesanoid (ATRA, All-trans retinoic acid). On the sixth day of induction, he developed splenic and gallbladder infarcts. Splenectomy and cholecystectomy were performed while chemotherapy induction continued as scheduled. Four days later, he developed ischemic areas in the kidneys and ischemic colitis in the sigmoid colon. Hypercoagulation studies showed severe deficiency of protein C. Tests showed protein C 16% (reference range 70-140%), protein S 87% (reference range 70-140%), antithrombin III 122% (reference range 80-120%), prothrombin time 13.6 s (reference = 11.3), INR (international normalized ratio) 1.21, partial thromboplastin time 33 s (reference = 33), fibrinogen 214 mg/dl, D-dimer 970 μg/ml, factor II 98%, and that antinuclear antibody, antiphospholipid antibodies, mutation for factor II gene (G20210A), and mutation for Arg506 Gln of factor V were all negative (factor V Leiden). There was no evidence of clinical disseminated intravascular coagulation (DIC). He was treated with low molecular weight heparin and did well. He continues to be in complete remission 7 years later with normal protein C levels. Acquired protein C deficiency can occur in a variety of settings and has been reported in acute myelocytic leukemia. However, clinically significant thrombosis in the absence of clinical DIC, such as our case, remains extremely rare.

Topics: Antineoplastic Combined Chemotherapy Protocols; Child; Cholecystectomy; Cytarabine; Etoposide; Fibrin Fibrinogen Degradation Products; Fibrinogen; Gallbladder; Heparin, Low-Molecular-Weight; Humans; Idarubicin; Infarction; Kidney; Lebanon; Leukemia, Myeloid, Acute; Male; Protein C; Protein C Deficiency; Remission Induction; Splenectomy; Splenic Infarction; Thrombosis; Tretinoin

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- and phorbol myristate acetate (PMA)-stimulated cells more than PI3K inhibitor alone. These results demonstrate that, although the combination of PI3K inhibitor and rapamycin is more effective in inhibiting proliferation of AML, the concomitant inhibition of PI3K and mTOR by LY 294002 and rapamycin has more inhibitory effects on ATRA-mediated differentiation than the presence of PI3K-inhibitor alone, and diminishes positive effects of rapamycin on leukemia cell differentiation.

Topics: Blotting, Western; Cell Cycle; Cell Differentiation; Cell Proliferation; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tretinoin; Tumor Cells, Cultured

The known participation of Kruppel-like transcription factors (KLF) in cellular differentiation prompted us to investigate their expression in acute myeloid leukemia (AML) blast cells that are typically blocked in their differentiation. We determined the expression patterns of KLFs with a putative role in myeloid differentiation in a large cohort of primary AML patient samples, CD34+ progenitor cells and granulocytes from healthy donors. We found that KLF2, KLF3, KLF5 and KLF6 are significantly lower expressed in AML blast and CD34+ progenitor cells as compared to normal granulocytes. Moreover, we found markedly increased KLF levels in acute promyelocytic leukemia patients who received oral ATRA. Accordingly, we observed a strong induction of KLF5/6 upon ATRA-treatment in NB4 and HT93 APL but not in ATRA-resistant NB4-R cells. Lastly, knocking down KLF5 or KLF6 in NB4 cells significantly attenuated neutrophil differentiation. In conclusion, we found a significant repression of KLF transcription factors in primary AML samples as compared to mature neutrophils and further show that KLF5 and KLF6 are functionally involved in neutrophil differentiation of APL cells.

Topics: Antineoplastic Agents; Blotting, Western; Case-Control Studies; Cell Differentiation; Cells, Cultured; Granulocytes; Humans; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Neutrophils; Prognosis; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

Topics: Antineoplastic Agents; Cell Differentiation; Humans; Kruppel-Like Transcription Factors; Leukemia, Myeloid, Acute; Prognosis; Tretinoin

The expressions of β1,3-N-acetylglucosamonyltransferase-2 and -8 (β3GnT-2, β3GnT-8),-the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and β3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. β3GnT-2 and β3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while β3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of β3GnT-8 was more than β3GnT-2, while in NB4 cells treated with DMSO, the increase of β3GnT-2 was more than β3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of β3GnT-2 and β3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of β3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of β3GnT-2 and -8. The expression of β3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of β3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.

Topics: Cell Differentiation; Cell Line, Tumor; Dimethyl Sulfoxide; Flow Cytometry; Fluorescence; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; N-Acetylglucosaminyltransferases; Polysaccharides; Tetradecanoylphorbol Acetate; Tretinoin; Up-Regulation

Hematopoiesis is coordinated by a complex regulatory network of transcription factors that involves proliferation, differentiation and maturation of a very small population of pluripotent hematopoietic stem cells with self-renewing and differentiating into various specialized and distinct blood cell types. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML). The purpose of this study was to investigate the expression pattern of transcription factor mRNA in acute myeloid leukemia (AML) cells during in vitro differentiation. The 2 human leukemic cell lines HL-60 and NB4 had been used as model cell lines. Differentiation of HL-60 and NB4 cells was induced by all-trans retinoic acid (ATRA) for 4 days. Morphological changes were observed by May-Grunwald Giemsa stainings, the CD11b expression level was detected by flow cytometry. Transcription factor mRNA profiles (PU.1, C/EBPα, ε, γ, GATA-1, GATA-2) were determined by real time RT-PCR during in vitro HL-60 and NB4 differentiation; The expression level of transcription factor mRNA was relatively quantitatively analyzed by using 2(-ΔΔCT) and compared with control group. The results showed that the expression levels of PU.1 and C/EBP ε mRNA in NB4 differentiation group were 5.75 and 6.16, respectively, which were significantly higher than those in untreated group; while the expression level of C/EBPα, γ, GATA-1, GATA-2 mRNA in NB4 differentiation group were 62%, 31%, 63% and 8.7% respectively, which were significantly lower than those in untreated group; In HL-60 differentiation group, the expression levels of PU.1, C/EBPα, ε were 1.97, 1.95 and 2.35 respectively, which were significantly higher than those in untreated group; while the expression levels of C/EBPγ, GATA-1, GATA-2 in HL-60 differentiation group were 20%, 21% and 18% respectively, which were significantly lower than those in untreated group. It is concluded that dysregulation of transcription factors is a key contributing factor in the pathogenesis of acute myeloid leukemia.

Topics: Cell Differentiation; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; RNA, Messenger; Transcription Factors; Tretinoin

Sensitive to Apoptosis Gene (SAG), also known as RBX2 (RING box protein-2), is the RING component of SCF (SKP1, Cullin, and F-box protein) E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES) to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag(-/-) mES cells were much more sensitive to all-trans retinoic acid (RA)-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag(-/-) mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy). We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE), that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cyclopentanes; Drug Interactions; Embryonic Stem Cells; Enzyme Activation; Enzyme Inhibitors; Female; Gene Deletion; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, Myeloid, Acute; Male; Mice; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Tretinoin; Ubiquitin-Protein Ligases

We describe a patient with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) that clinically resembled acute promyelocytic leukemia (APL). The karyotype of his leukemic cells was 46, XY, del (3) (q?) and did not include a chromosomal translocation involving the retinoic acid receptor-α gene. However, retinoic acid syndrome developed, and partial remission was achieved after treatment with all-trans retinoic acid (ATRA) followed by chemotherapy. Our case might provide new insights into the mechanism of the growth inhibitory effect of ATRA on APL-like cells.

Topics: Aged; Antineoplastic Agents; Chromosome Deletion; Chromosomes, Human, Pair 3; Diagnosis, Differential; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Myelodysplastic Syndromes; Oncogene Proteins, Fusion; Tretinoin

Expression of N-myc downregulated gene 1 (NDRG1) is associated with growth arrest and differentiation of tumor cells. In hematopoietic cells, NDRG1 was identified in a screen for differentiation-related genes in human myelomonocytic leukemic U937 cells. In the present study, we found significantly higher NDRG1 mRNA levels in granulocytes of healthy donors than in primary acute myeloid leukemia (AML) cells. Another NDRG family member, NDRG2, was significantly higher expressed in normal macrophages compared to primary AML cells. Moreover, NDRG1 mRNA levels increased in two acute promyelocytic leukemia (APL) patients as well as in NB4 and HT93 APL cells upon all-trans retinoic acid (ATRA) therapy. In line with these observations, silencing of NDRG1 diminished neutrophil differentiation of leukemic cell lines. In conclusion, we found an association of low NDRG1 levels with an immature cell phenotype and provide evidence that NDRG1 is functionally involved in neutrophil maturation.

Topics: Antineoplastic Agents; Blotting, Western; Cell Cycle Proteins; Cell Differentiation; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Macrophages; Neutrophils; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Suppressor Proteins

Topics: Cell Differentiation; Dasatinib; Drug Synergism; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Protein Kinase Inhibitors; Pyrimidines; Thiazoles; Tretinoin

Onzin is a small, novel, and highly conserved protein with unique structure and tissue-restricted expression. The regulation of its expression and biological roles remain greatly elusive. Here, we provide the first demonstration that onzin expression is significantly downregulated during differentiation induction of acute myeloid leukemic (AML) cell lines and primary cells by all-trans retinoic acid (ATRA) and especially by phorbol 12-myristate 13-acetate (PMA). Applying chemical inhibitions, RNA interferences, and transfected expressions of dominant negative mutants or constitutive catalytic forms of the related kinases, we show that protein kinase C-epsilon (PKCepsilon)-extracellular signal-regulated protein kinase 2 (ERK2) signaling axis is required for PMA-induced downregulation of onzin expression. The ectopic expression of onzin partially inhibits PMA-induced monocytic differentiation of AML cells, whereas suppression of onzin by specific short hairpin RNAs enhances PMA-induced differentiation to a degree. Furthermore, onzin partially inhibits the transcriptional activity of hematopoiesis-related important transcription factor PU.1 via their interaction. Taken together, our results show that PMA downregulates onzin expression through PKCepsilon-ERK2 signaling pathway, which favors monocytic differentiation of leukemic cells.

Topics: Cell Differentiation; Cell Line, Tumor; Down-Regulation; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 1; Oncogene Proteins; Protein Kinase C-epsilon; Proteins; Proto-Oncogene Proteins; Signal Transduction; Tetradecanoylphorbol Acetate; Trans-Activators; Tretinoin

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Catechin; Cell Death; Cell Differentiation; Death-Associated Protein Kinases; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Gene Knockdown Techniques; Humans; Leukemia, Myeloid, Acute; Neutrophils; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Topics: Antigens, CD34; Antineoplastic Agents; Blotting, Western; Cell Differentiation; Chromatin Immunoprecipitation; Granulocytes; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Minor Histocompatibility Antigens; Neutrophils; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Although the potential role of Pim2 as a cooperative oncogene has been well described in lymphoma, its role in leukemia has remained largely unexplored. Here we show that high expression of Pim2 is observed in patients with acute promyelocytic leukemia (APL). To further characterize the cooperative role of Pim2 with promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha), we used a well-established PML-RARalpha (PRalpha) mouse model. Pim2 coexpression in PRalpha-positive hematopoietic progenitor cells (HPCs) induces leukemia in recipient mice after a short latency. Pim2-PRalpha cells were able to repopulate mice in serial transplantations and to induce disease in all recipients. Neither Pim2 nor PRalpha alone was sufficient to induce leukemia upon transplantation in this model. The disease induced by Pim2 overexpression in PRalpha cells contained a slightly higher fraction of immature myeloid cells, compared with the previously described APL disease induced by PRalpha. However, it also clearly resembled an APL-like phenotype and showed signs of differentiation upon all-trans retinoic acid (ATRA) treatment in vitro. These results support the hypothesis that Pim2, which is also a known target of Flt3-ITD (another gene that cooperates with PML-RARalpha), cooperates with PRalpha to induce APL-like disease.

Topics: Animals; Base Sequence; Bone Marrow Transplantation; DNA Primers; Female; fms-Like Tyrosine Kinase 3; Gene Expression; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Oncogene Proteins, Fusion; Oncogenes; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Tretinoin

Immunophenotyping by flow cytometry or immunohistochemistry is a clinical standard procedure for diagnosis, classification, and monitoring of hematologic malignancies. Antibody-based cell surface phenotyping is commonly limited to cell surface proteins for which specific antibodies are available and the number of parallel measurements is limited. The resulting limited knowledge about cell surface protein markers hampers early clinical diagnosis and subclassification of hematologic malignancies. Here, we describe the mass spectrometry based phenotyping of 2 all-trans retinoic acid treated acute myeloid leukemia model systems at an unprecedented level to a depth of more than 500 membrane proteins, including 137 bona fide cell surface exposed CD proteins. This extensive view of the leukemia surface proteome was achieved by developing and applying new implementations of the Cell Surface Capturing (CSC) technology. Bioinformatic and hierarchical cluster analysis showed that the applied strategy reliably revealed known differentiation-induced abundance changes of cell surface proteins in HL60 and NB4 cells and it also identified cell surface proteins with very little prior information. The extensive and quantitative analysis of the cell surface protein landscape from a systems biology perspective will be most useful in the clinic for the improved subclassification of hematologic malignancies and the identification of new drug targets.

Topics: Antigens, CD; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Flow Cytometry; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Membrane Proteins; Neoplasm Proteins; Phenotype; Proteome; Proteomics; Tandem Mass Spectrometry; Tretinoin

The high expression of folate receptor (FR) on cancer cells might be a potential target for cancer therapy. In this study, the FR-β expression and the modulation effect of all-trans retinoic acid (ATRA) in a number of cancer cell lines were analyzed. The gateway of ATRA activity on FR-β expression was further studied by a panel of retinoid activators and inhibitors. The results revealed that ATRA was capable of upregulating the expression of FR-β protein in KG-1 cells in a dosage-dependent manner, not in KG-1a, NB4, HL60, 293, L1210, JAR, and Hela cells. FR-β mRNA expression in KG-1 cells was higher when ATRA was present in culture medium at 10⁻⁶ mol/L for 5 days, and it went down to baseline when ATRA was removed from the medium, vice versa. The upregulation of FR-β expression in KG-1 cells by ATRA was not associated with cell proliferation and differentiation. In addition, activators of retinoid acid receptor (RAR)α and RARγ, CD336, and CD2781 also induced FR-β expression. The induction of FR-β expression by CD336 could be inhibited by RARγ antagonist CD2665; RARβ agonist CD-417 and CD-2314 as well as retinoid X receptor (RXR) agonist LG100364 could not induce FR-β expression. These results indicate that ATRA within a certain range of concentration could reversibly induce the expression of FR-β in a dosage- and cell type-dependent manner, and its action in KG-1 cells might be associated with the signal transduction of retinoid receptor RARα and RARγ, rather than RARβ and RXRs.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cervix Uteri; Choriocarcinoma; Female; Folate Receptor 2; Folate Receptors, GPI-Anchored; Humans; Kidney; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Uterine Neoplasms

Low oxygen tension is able to modulate the expression of several genes involved in physiological and pathological processes. A major regulator of gene expression is the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1), which also regulates angiogenesis-related genes, including the protein expression of angioregulatory cytokines. Angiogenesis has been shown to play a role in haematological disorders, and low oxygen tension might thereby influence leukaemogenesis and chemosensitivity in human acute myeloid leukaemia (AML).. We examined the effect of a hypoxic environment (1% O(2)) on in vitro-cultured, primary human AML cells with regard to HIF-1α expression, colony formation and cytokine release.. Our study demonstrated that hypoxic culture conditions increased HIF-1α expression in primary AML cells for a majority of the investigated patients when compared to culture at atmospheric (21%) oxygen tension. Hypoxia also increased the release of vascular endothelial growth factor (VEGF), osteopontin, as well as several CCL- (CCL3/4/5/7/8) and CXCL-chemokines (CXCL1 and proangiogenic CXCL8) by AML cells. The constitutive release of antiangiogenic CXCL9-11 was not altered by the low oxygen tension. The wide variation between patients as regards the release of the various cytokines persisted during hypoxia.. Culture of primary AML cells under low oxygen tension induces HIF-1α expression and increases the release of several cytokines, including proangiogenic mediators, compared to culture at ambient 21% O(2).

Topics: Adult; Aged; Aged, 80 and over; Angiogenesis Inducing Agents; Cell Differentiation; Cell Hypoxia; Cell Proliferation; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (ATRA), a natural ligand for the retinoic acid receptors (RARs), induces clinical remission in most acute promyelocytic leukemia (APL) patients through the induction of differentiation and/or eradication of leukemia-initiating cells. Here, we identify a novel natural ent-kaurene diterpenoid derived from Isodon pharicus leaves, called pharicin B, that can rapidly stabilize RAR-α protein in various acute myeloid leukemic (AML) cell lines and primary leukemic cells from AML patients, even in the presence of ATRA, which is known to induce the loss of RAR-α protein. Pharicin B also enhances ATRA-dependent the transcriptional activity of RAR-α protein in the promyelocytic leukemia-RARα-positive APL cell line NB4 cells. We also showed that pharicin B presents a synergistic or additive differentiation-enhancing effect when used in combination with ATRA in several AML cell lines and, especially, some primary leukemic cells from APL patients. In addition, pharicin B can overcome retinoid resistance in 2 of 3 NB4-derived ATRA-resistant subclones. These findings provide a good example for chemical biology-based investigations of pathophysiological and therapeutic significances of RAR-α and PML-RAR-α proteins. The effectiveness of the ATRA/pharicin B combination warrants further investigation on their use as a therapeutic strategy for AML patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cell Line, Tumor; Diterpenes, Kaurane; Drug Resistance, Neoplasm; Drug Synergism; Humans; Isodon; Leukemia, Myeloid, Acute; Protein Stability; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Our previous data demonstrated that folate receptor β (FR-β) targeted liposomal doxorubicin (FT-L-DOX) showed enhanced cytotoxicity relative to non-targeted liposomal doxorubicin (CON-L-DOX), and the effect was enhanced by selective FR-β upregulation by all-trans retinoic acid (ATRA) in AML blast cells. In this study, the enhanced cytotoxicity was investigated in the proliferating human AML clonogenic cells by combining FT-L-DOX with ATRA. Also, pharmacokinetic properties by pretreatment of ATRA were evaluated using FR-targeted liposomal calcein (FT-L-Calcein). Pharmacokinetic study showed that the area under the concentration curve (AUC) of FT-L-Calcein was decreased and total clearance was increased by pretreatment with ATRA. Meanwhile, the volume of distribution was significantly increased by pretreatment of ATRA. Moreover, calcein level in the liver, spleen and kidney was increased following intravenous administration of FT-L-Calcein by pretreatment of ATRA. In vitro cytotoxicity of FT-L-DOX was higher than that of CON-L-DOX and was increased by pretreatment with ATRA. Colony formation in AML cells was lower due to treatment with FT-L-DOX compared with CON-L-DOX and colony formation further decreased upon pretreatment with ATRA. Moreover, FT-L-DOX was more toxic to AML clonogenic cells than to AML blast cells. The results demonstrate that the efficiency of FR-mediated targeting of FT-L-DOX was preferentially enhanced by ATRA induced FR-β upregulation in AML clonogenic cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Colony-Forming Units Assay; Doxorubicin; Drug Delivery Systems; Female; Fluoresceins; Folate Receptor 2; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred ICR; Tissue Distribution; Tretinoin; Up-Regulation

Imbalances of histone acetyltransferase (HAT) and deacetylase activity (DAC) that result in deregulated gene expression are commonly observed in leukemias. These alterations provide the basis for novel therapeutic approaches that target the epigenetic mechanisms implicated in leukemogenesis. As the acetylation status of histones has been linked to transcriptional regulation of genes involved particularly in differentiation and apoptosis, DAC inhibitors (DACi) have attracted considerable attention for treatment of hematologic malignancies. DACi encompass a structurally diverse family of compounds that are being explored as single agents as well as in combination with chemotherapeutic drugs, small molecule inhibitors of signaling pathways and hypomethylating agents. While DACi have shown clear evidence of activity in acute myeloid leukemia, myelodysplastic syndromes and lymphoid malignancies, their precise role in treatment of these different entities remain to be elucidated. Successful development of these compounds as elements of novel targeted treatment strategies for leukemia will require that clinical studies be performed in conjunction with translational research including efforts to identify predictive biomarkers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Tretinoin

Topics: Animals; Cell Differentiation; Drosophila melanogaster; Drosophila Proteins; Hematopoietic Stem Cells; Humans; Iron; Iron Chelating Agents; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Models, Biological; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Oxidative Stress; Polycomb Repressive Complex 1; Reactive Oxygen Species; Signal Transduction; Tretinoin; Vitamin D

Transfusion-related acute lung injury (TRALI) is a clinical syndrome characterized by sudden onset of respiratory distress due to pulmonary edema during or following transfusion. Two proposed pathophysiologic mechanisms for TRALI were proposed: the antibody hypothesis and the two-event hypothesis. The two-event hypothesis postulates that a pathway to neutrophil activation and aggregation can occur without leukocyte antibodies. We report a case of TRALI occurring during remission induction course of acute myeloid leukemia in a 27-year-old woman who received All-transretinoic-acid (ATRA). We postulate that ATRA may have played a role in this life-threatening complication by priming neutrophil and enhancing their adherence and their activation in the pulmonary endothelium. TRALI improved with non-invasive ventilation support and use of high dose corticosteroids.

Topics: Adult; Anemia; Antineoplastic Agents; Female; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Leukocytosis; Remission Induction; Respiratory Distress Syndrome; Transfusion Reaction; Tretinoin

Acute myelogenous leukemia (AML) patients (24 consecutive patients, median age 71 years, 17 high-risk disease) were treated with all-trans retinoic acid, theophylline and valproic acid. Among 22 evaluable patients 9 responded with increased normal peripheral blood cell counts. The responses could be classified as hematological improvement according to response criteria for patients with myelodysplastic syndromes (MDS) for four patients only. The nine patients with increased normal cell counts had a median survival from start of therapy of 147 days compared with 48 days for the other patients. Four patients fulfilling the MDS criteria had a survival ranging from 112 to 644 days. The treatment was associated with decreased in vitro cytokine-dependent AML cell proliferation and increased blood levels of Endocan and angiopoietin-2 both for responders and non-responders. We conclude that the therapy causes disease stabilization for a subset of AML patients.

Topics: Aged; Aged, 80 and over; Blood Cell Count; Drug Therapy, Combination; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplasm Proteins; Proteoglycans; Risk Factors; Survival Analysis; Theophylline; Tretinoin; Valproic Acid

To screen and clone multi-drug resistance (MDR) related genes in MDR acute myeloid leukemia cells (HL-60/MDR).. HL-60/MDR was established using All-Trans Retinoic Acid. With the HL-60 cells as "tester" and HL60/MDR as "driver", the cDNA library of HL-60/MDR was established by suppression subtractive hybridization. Then 12 of the resulting subtracted cDNA clones were selected for DNA sequencing and homology analysis. The obtained expressed sequence tags (ESTs) were analyzed with the GenBank BLASTN program to identify sequence homologies to known genes.. The HL-60/MDR cells had different multi-drug resistance to six kinds of chemotherapeutic drugs. The 211 positive gene clones in differential cDNA library of HL-60/MDR cells were amplified with PCR and 46 gene clones exhibited differential expression (ratio >3). Twelve gene clones with significant differential expression (ratio >5) were screened out to homology analysis. Of these, 11 matched known genes and the rest 1 showed no significant homology to human or non-human known sequences. It was named as gene clone HA117.. This effort provides the partial list of genes differential expressed in HL-60/MDR cells and a novel gene HA117 was found to be related to MDR. Identification of these genes contributes to our understanding of MDR development, and potentially provides candidate target genes to overcome MDR.

Topics: Antineoplastic Agents; Cloning, Molecular; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Expressed Sequence Tags; Gene Library; Genes, Neoplasm; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Sequence Analysis, DNA; Tretinoin

Engagement of NKG2D by their ligands (NKG2D-L), as the human major histocompatibility complex class I-related molecules MIC-A and the UL16-binding proteins, on cytolytic lymphocytes leads to the enhancement of antitumour effector functions. These ligands are missing or expressed at very low levels on leukaemic cells; furthermore, they can be shed by tumour cells and inhibit cytolytic activity of lymphocytes. Herein, we show that in vivo administration of all-trans-retinoic acid (ATRA) or the histone deacetylase inhibitor sodium valproate (VPA) to patients affected with acute myeloid leukaemia (AML) M3 or M1 respectively, leads to the induction of transcription and expression of NKG2D-L at the surface of leukaemic cells. Apparently, no detectable shedding of the soluble form of these molecules was found in patients' sera. Conversely, AML blasts from patients treated with chemotherapy not including ATRA or VPA did not show any induction of NKG2D-L transcription. Furthermore, upon therapy with ATRA or VPA, leukaemic blasts become able to trigger lytic granule exocytosis by autologous CD8(+) T and natural killer lymphocytes, as shown by CD107a mobilization assay, followed by leukaemic cell lysis. These findings indicate that ATRA and VPA may contribute to the activation of cytolytic effector lymphocytes in vivo, possibly enhancing their anti-leukaemic effect.

Topics: Adult; Aged; Blast Crisis; Cytotoxicity, Immunologic; Female; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Ligands; Lymphocyte Activation; Male; Middle Aged; NK Cell Lectin-Like Receptor Subfamily K; Transcriptional Activation; Tretinoin; Valproic Acid

1,25-Dihydroxyvitamin D3 (1,25D) induces differentiation of myeloid leukemia cells, but resistant cells are also encountered. We studied the mechanistic basis for the resistance in a model system using enhancers of 1,25D, the antioxidant carnosic acid and a kinase inhibitor SB202190. Knock-down (KD) of JNK2p54 unexpectedly increased the intensity of differentiation induced by the 1,25D, carnosic acid and SB202190 (DCS) combination. This was associated with upregulation of activated JNK1p46, and the transcription factors regulated by the JNK pathway, c-Jun, ATF2 and JunB, as well as C/EBP beta. In contrast, KD of JNK1p46 reduced the intensity of DCS-induced differentiation, and partially abrogated activation of c-Jun/AP-1 transcription factors.

Topics: Abietanes; Antioxidants; Cell Differentiation; Cell Division; Drug Resistance, Neoplasm; Enzyme Inhibitors; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 9; Plant Extracts; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-jun; Pyridines; RNA, Small Interfering; Transcription Factor AP-1; Tretinoin

Topics: Calcitriol; Cell Differentiation; Cell Division; Drug Resistance, Neoplasm; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase 9; Monocytes; Receptors, Calcitriol; Retinoid X Receptors; Signal Transduction; Tretinoin

Priming with granulocyte macrophage colony-stimulating factor (GM-CSF) plus all-trans retinoic acid (ATRA) during induction chemotherapy may enhance response rates and survival in patients with acute myeloid leukemia (AML) due to the differentiation of human myeloblastic leukemia cells into granulocytes.. GM-CSF was administered to patients during induction chemotherapy and ATRA was ingested orally on days 1 to 14. Patients undergoing a regimen with GM-CSF and ATRA were evaluated as compared with a historical control group of subjects.. For patients enrolled in this study, the complete remission plus complete remission with incomplete platelet recovery rate was 61.5% as compared with 41.4% for the historical control group of subjects. The relapse rate was 38.5% and 44.8% for the study and control group of subjects, respectively. Two-year probabilities were 45.5% (study group) and 47.4% (control group) for disease-free survival and 38.5% (study group) and 36.2% (control group) for overall survival. The most frequent side effects included fever, headache, and skin lesions with pruritus and dyspnea with pulmonary congestion, similar to ATRA syndrome.. Priming with GM-CSF plus ATRA during anthracycline-based chemotherapy is feasible in terms of response rate, but the toxicity of the regimen is significant.

Topics: Adult; Antineoplastic Agents; Drug Therapy, Combination; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Pilot Projects; Prognosis; Remission Induction; Survival Rate; Treatment Outcome; Tretinoin; Young Adult

Our study explored the drug interaction of all-trans retinoic acid (ATRA) and RAD001 (everolimus), the inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in acute myelogenous leukemia (AML) NB4 and HL60 cells. RAD001 (10 nM) significantly enhanced the ATRA-induced growth arrest and differentiation of these cells, as measured by colony-forming assay and cell cycle analysis, and expression of CD11b cell surface antigen and nitroblue tetrazolium reduction, respectively. ATRA (0.1-1 microM) upregulated levels of RTP801, a negative regulator of mTORC1, and inhibited mTORC1 signaling as assessed by measurement of the levels of p-p70S6K and p-4E-BP1 in HL60 and NB4 cells. ATRA (0.1-1 microM) in combination with RAD001 (10 nM) strikingly downregulated the levels of p-70S6K and p-4E-BP1 without affecting the total amount of these proteins. Notably, RAD001 (10 nM) significantly augmented ATRA-induced expression of CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) and p27(kip1) and downregulated levels of c-Myc in these cells. Furthermore, RAD001 (5 mg/kg) enhanced the ability of ATRA (10 mg/kg) to inhibit the proliferation of HL60 cells growing as tumor xenografts in immune-deficient nude mice. Taken together, concomitant blockade of the RA and mTORC1 signaling may be a promising treatment strategy for individuals with AML.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Blotting, Western; CCAAT-Enhancer-Binding Proteins; CD11b Antigen; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Drug Synergism; Everolimus; Female; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Neoplasm Proteins; Nitroblue Tetrazolium; Proteins; Proto-Oncogene Proteins c-myc; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transplantation, Heterologous; Tretinoin; Tumor Stem Cell Assay; Up-Regulation

We saw a 34-year-old pregnant woman with acute promyelocytic leukemia, who developed acute respiratory failure from all-trans-retinoic acid (ATRA) syndrome. We applied noninvasive ventilation (NIV, continuous positive airway pressure plus pressure-support ventilation) to try to improve gas exchange, reduce the work of breathing, and prevent intubation. Initially we applied NIV continuously (24 hours a day), then gradually reduced the daily amount of time on NIV as her condition improved. She was discharged from the intensive care unit after 12 days. Three months after hospital discharge she gave vaginal birth to a healthy female baby. NIV was effective and safe for the mother and fetus, and NIV should be considered for respiratory failure in pregnant patients, especially if immunosuppressed.

Topics: Antineoplastic Agents; Female; Humans; Leukemia, Myeloid, Acute; Pregnancy; Pregnancy Complications, Neoplastic; Respiration, Artificial; Respiratory Insufficiency; Syndrome; Tretinoin

To identify patients who developed secondary clonal cytogenetic aberrations (CCA) following therapy for acute promyelocytic leukemia (APL), we retrospectively analyzed cytogenetic results from 123 patients diagnosed with APL between 1995 and 2007, who had ongoing cytogenetic analysis undertaken in our laboratory. During follow-up for APL we identified 12 patients (9.8%) who developed CCA, not detected at diagnosis of APL and unrelated to their original APL karyotype. All patients had received all-trans retinoic acid (ATRA) and chemotherapy and were in complete remission for APL when secondary CCA were identified. The median latency period between diagnosis of APL and emergence of secondary CCA was 27.5 months (range: 2-54 months). To date, four patients with CCA have been diagnosed with therapy-related myelodysplastic syndrome (t-MDS)/acute myeloid leukemia (t-AML), giving a median t-MDS/AML free survival of 78 months, with follow-up ranging between 20 and 136 months from APL diagnosis. Three patients have died: two patients died of t-AML and another developed relapsed APL with persistence of his secondary clone but no diagnosis of t-MDS/AML and died from transplant-related complications. Two patients are alive with t-MDS. Seven patients with CCA are alive with no morphological evidence of MDS at the time of their last known follow-up; thus median survival has not been reached. The appearance of these abnormalities in the absence of morphological evidence of MDS in the majority of patients is unusual, and highlights the importance of continued cytogenetic follow-up in these patients. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Chromosome Aberrations; Female; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Neoplasms, Second Primary; Remission Induction; Retrospective Studies; Survival Rate; Treatment Outcome; Tretinoin; Young Adult

Topics: Aged; Cell Nucleus; Disease-Free Survival; DNA-Binding Proteins; DNA, Complementary; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Middle Aged; Proto-Oncogene Proteins; Remission Induction; Retrospective Studies; Signal Transduction; Treatment Outcome; Tretinoin

All types of blood cell of the body are continuously produced by rare pluripotent self-renewing HSCs (haemopoietic stem cells) by a process known as haemopoiesis. This process provides a valuable model for examining how genetic programmes involved in cell differentiation are established, and also how cell-fate specification is altered in leukaemia. Here, we describe examples of how miRNAs (microRNAs) can influence myelopoiesis and how the identification of their target mRNAs has contributed to the understanding of the molecular networks involved in the alternative control between cell growth and differentiation. Ectopic expression and knockdown of specific miRNAs have provided powerful molecular tools able to control the switch between proliferation and differentiation, therefore providing new therapeutic tools for interfering with tumorigenesis.

Topics: Cell Differentiation; Cell Line; Granulocytes; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Models, Biological; Monocytes; Myeloid Cells; Tretinoin

Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia (AML) that is characterized by peculiar clinical and biologic features, including severe hemorrhagic diathesis, specific recurrent chromosomal aberration, and distinct morphologic features with predominant pathologic promyelocytes. A reciprocal translocation involving chromosomes 15 and 17, t(15;17)(q22;q21), is a characteristic feature of APL that represents approximately 5-8% of AML. The rearranged gene created by this translocation encodes a chimeric protein PML-RARA that is a transcriptional repressor. In contrast to other AML subtypes, APL is particularly sensitive to treatment with all trans-retinoic acid (ATRA) combined with chemotherapy, converting this once fatal leukemia to a highly curable disease. Nonetheless, therapy-related myelodysplastic syndrome-acute myelogenous leukemia (t-MDS/AML) has been reported as a rare complication of chemotherapy in APL. Of 30 APL cases described as t-MDS/AML in the literature, only 1 case relapsed as acute leukemia with t(3;21)(q26;q22). Here we describe a rare case of APL relapsing as secondary AML with t(3;21)(q26;q22) and clinically characterize this patient using the RUNX1 (previously AML1)-MDS1-EVI1 fusion transcript (with follow-up for 55 months), and review the relevant literature.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Chromosome Painting; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 3; Core Binding Factor Alpha 2 Subunit; DNA-Binding Proteins; Female; Humans; Karyotyping; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; MDS1 and EVI1 Complex Locus Protein; Middle Aged; Neoplasm Proteins; Neoplasms, Second Primary; Oncogene Proteins, Fusion; Proto-Oncogenes; Sequence Analysis, DNA; Transcription Factors; Translocation, Genetic; Tretinoin

Topics: Adenine; Adenine Nucleotides; Aminoglycosides; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Arabinonucleosides; Carbazoles; Clofarabine; Cytarabine; Dasatinib; Daunorubicin; fms-Like Tyrosine Kinase 3; Furans; Gemtuzumab; Humans; Leukemia, Myeloid, Acute; Naphthalimides; Organophosphonates; Prognosis; Protein Kinase Inhibitors; Pyrimidines; Sesquiterpenes; Staurosporine; Stem Cell Transplantation; Thiazoles; Tretinoin

Topics: Antineoplastic Agents; Azacitidine; Biomedical Research; Carbazoles; Carboplatin; Cytarabine; Daunorubicin; Decitabine; Enzyme Inhibitors; Etoposide; Flavonoids; Furans; Humans; Leukemia, Myeloid, Acute; Mitoxantrone; Piperidines; Quinolones; Staurosporine; Topotecan; Tretinoin

All-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukaemia (APL), inducing APL cells to differentiate into abnormal neutrophils. To investigate the possible relationship between the chromosome translocation t(15;17) found in APL and ATRA treatment, the human myeloid leukaemia cell lines HL60 and NB4, that are PML-RARalpha negative and positive, respectively, were treated with ATRA and immunophenotyped using a CD antibody microarray. For HL60 cells, ATRA induced major increases in descending order of CD38, CD11b, CD45RO, CD11c, CD54 and CD36 with repression of CD117 and CD44. For NB4 cells, ATRA induced major increases in descending order of CD11c, CD54, CD11a, CD11b, CD53, CD65, CD138, CD66c and T-cell receptor alpha/beta (TCRalpha/beta), with repression of CD38 and CD9. The induction of a number of these CD antigens is consistent with the known differentiation of these leukaemias to abnormal neutrophils. Approximately half of the antigens up-regulated by ATRA on NB4 cells were adhesion molecules, including CD11a, CD11b, CD11c, CD54, CD66c and CD138, consistent with the increased adhesiveness of leukaemia cells observed for APL patients treated with ATRA. On HL60 cells, ATRA induced expression of CD38, CD43 and CD45RO and repressed CD117, while the converse was true on NB4 cells that contain chimeric PML-RARalpha. For NB4 cells, ATRA induced some remarkable increases in CD antigens not seen for HL60: CD14 (16.6-fold), CD32 (27.8), CD53 (20.5), CD65 (139), CD66c (79.7), CD126 (15.1), and CD138 (57.6). The expression of these antigens may be regulated by PML-RARalpha in the presence of ATRA. Such CD antigens could be targets for synergistic treatment of APL with therapeutic antibodies following ATRA treatment.

Topics: Antigens, CD; Antineoplastic Agents; Gene Expression; HL-60 Cells; Humans; Immunophenotyping; Leukemia, Myeloid, Acute; Protein Array Analysis; Tretinoin

The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARalpha2 expression is dramatically reduced in primary AML blasts. Expression of the RARalpha1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARalpha1 and RARalpha2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARalpha2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.

Topics: Bone Marrow Cells; Cell Differentiation; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

Topics: Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Fatal Outcome; Female; Humans; Kidney Transplantation; Korea; Leukemia, Myeloid, Acute; Liver Transplantation; Male; Middle Aged; Treatment Outcome; Tretinoin

Topics: Antineoplastic Agents; Bone Marrow Cells; Cell Differentiation; Drug Synergism; Gefitinib; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Quinazolines; Tretinoin

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

Topics: Antineoplastic Agents; Cell Differentiation; Cells, Cultured; DNA Methylation; Gene Expression Regulation; Granulocytes; HL-60 Cells; Humans; Kruppel-Like Transcription Factors; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Promoter Regions, Genetic; RNA, Messenger; RNA, Neoplasm; Tretinoin; U937 Cells; Up-Regulation

Topics: Adult; Aged; Child; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Diagnosis, Differential; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Translocation, Genetic; Tretinoin

To evaluate the outcome in denovo AML patients treated with different remission induction and consolidation chemotherapy regimens in our population.. A retrospective study on acute myeloid leukaemia (AML) patients was carried out at Armed Forces Bone Marrow Transplant Centre Rawalpindi Pakistan between July 2001 and June 2006. During 5 years period 46 patients received treatment for AML at our centre. Twenty nine patients were males and 17 were females. Median age of patients was 21 years (range: 7-56 years). These 46 patients were categorized into two groups on the basis of type of leukaemia and chemotherapy given. In group-I 40 patients (group Ia: 23 patients of M1-M6, less M3 group Ib: 17 patients of AML M3) received anthracycline and cytarabin based chemotherapy. In group-II, six patients (AML- M3) received all trans retinoic acid (ATRA) based chemotherapy.. In group Ia, out of 23 patients, 14 patients (60.8%) achieved complete remission (CR) after remission induction chemotherapy, 10 patients remained in CR after 3rd and 4th consolidation. Eleven patients died and five patients relapsed during treatment and follow up. In this group overall CR, relapse rate (RR) and mortality was 30.4% (7/23), 21.7% (5/23) & 48% (11/23) respectively. In group Ib out of 17 patients, 9 patients (53%) achieved CR after remission induction. Eleven patients died during treatment while one patient relapsed in this group. Overall CR, RR & mortality was 29.4% (5/17), 6% (1/17) & 55% (11/17) respectively. In group II all patients achieved CR (100%) after 1st course of chemotherapy. Two of these patients unfortunately died of uncontrolled sepsis during 1st consolidation, while remaining 4 patients 66.6% are on maintenance chemotherapy and are still in CR.. Overall CR, RR and mortality in all groups was 35% (16/46), 13% (6/46) and 52% (24/46) respectively at a median follow-up of 36 + 8 months. Survival in AML-M3 patients treated with ATRA based chemotherapy is significantly superior than anthracycline based chemotherapy (66.6% vs. 29.4%). Infection and chemotherapy toxicity being major causes of mortality.

Topics: Adolescent; Adult; Anthracyclines; Antineoplastic Agents; Child; Cytarabine; Disease Progression; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Military Medicine; Military Personnel; Pakistan; Retrospective Studies; Time Factors; Treatment Outcome; Tretinoin

To analyze the prognosis and risk factors of acute nonlymphocytic leukemia (ANLL), 94 patients with acute nonlymphocytic leukemia were enrolled in this study, while survival rate and risk factors of prognosis were analyzed. The results indicated that the complete remission (CR) ratio was 51.1%. Overall survival and event-free survival rates of month 6, 12, 18, 24 were 68.6%, 55.8%, 53.8%, 46.4%, 21.3% and 57.9%, 38.6%, 33.3%, 31.6%, 0% respectively. The factors such as age<40 years, WBC<10.0x10(9)/L before chemotherapy, WBC in the period of bone marrow suppression<1.0x10(9)/L, chemotherapy within 1 month after occurrence of leukemia, blood transfusion before chemotherapy of APL had favourable influence on remission and survival rates of ANLL patients. CR1, the time to get CR, length of CR and relapse significantly correlated with prognosis (p<0.05). It is concluded that the individualized therapy concerning the risk factors should be applied to ANLL patients for improving the remission, survival rate and prognosis.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Female; Harringtonines; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Prognosis; Tretinoin; Young Adult

From November 1, 1995 to July 31, 2004, 49 children with de novo acute myeloid leukemia (AML) were treated at our institution. One patient who was treated by a different protocol was excluded. In total, 48 patients with de novo AML were enrolled in this study. Forty-two patients with AML other than acute promyelocytic leukemia (non-APL) were treated consecutively with 2 novel protocols: Mackay Memorial Hospital (MMH)-AML-96, designed as a pilot phase, and Taiwan Pediatric Oncology Group (TPOG)-AML-97A, on the basis of MMH-AML-96 with minor modifications. Six patients with APL were treated consecutively with 2 protocols, TPOG-APL-97 and APL-2001. As of July 31, 2006, the remission rates were 79%, 92%, and 98% after 1, 2, and 3 courses of induction therapy, respectively. The 5-year overall survival was 64%+/-6.9% (SE), and the 5-year event-free survival was 60%+/-7.1%; for non-APL AML, the rates were 62%+/-7.5% and 59%+/-7.6%; for APL, 83+/-15.2 and 67+/-19.3%. Among the factors analyzed, a complete remission achieved after 1 course of induction therapy, lactate dehydrogenase <500 IU/L at diagnosis, patients without invasive fungal infection during chemotherapy, and male sex were associated with a favorable outcome.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Drug Administration Schedule; Female; Humans; Infant; Leukemia, Myeloid, Acute; Male; Survival Rate; Survivors; Taiwan; Tretinoin

In this issue of the Journal, Soucek et al. challenge the assumption that increased functional granulocytic maturation of HL-60, an ATRA-responsive acute myeloid leukemia cell line devoid of the APL-specific PML-RARalpha fusion protein, results in more rapid or more sustained cell death. In this model cell line, the authors demonstrate that TGFbeta1, a well-known haemopoietic growth factor, enhances retinoid-dependent cyto-differentiation and growth arrest while inhibiting apoptosis. Concomitantly, treatment of HL-60 cells with the combination of TGFbeta1 and the retinoid partially suppresses ATRA-dependent induction of TRAIL. This is a death receptor ligand of the TNF family implicated in the paracrine mechanism underlying the apoptotic action of ATRA in APL blasts The protein activates the death-receptor-dependent or extrinsic apoptotic pathway, which is associated with caspase-8 activation. Down-regulation of TRAIL is correlated to an increase in the levels of the anti-apoptotic c-FLIP(L) and Mcl-1 proteins that are likely to be involved in the suppression of caspase-8 activation and apoptosis.

Topics: Acute Disease; Apoptosis; Apoptosis Regulatory Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Caspases; Cell Differentiation; Cell Proliferation; Granulocytes; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Membrane Glycoproteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Retinoic acid-induced expression of the CD38 ectoenzyme receptor in HL-60 human myeloblastic leukemia cells is regulated by RARalpha and RXR, and enhanced or prevented cell differentiation depending on the level of expression per cell. RARalpha activation caused CD38 expression, as did RXR activation but not as effectively. Inhibition of MAPK signaling through MEK inhibition diminished the induced expression by both RARs and RXRs. Expression of CD38 enhanced retinoic acid-induced myeloid differentiation and G0 cell cycle arrest, but at higher expression levels, induced differentiation was blocked and retinoic acid induced a loss of cell viability instead. In the case of 1,25-dihydroxyvitamin D3, induced monocytic differentiation was also enhanced by CD38 and not enhanced by higher expression levels, but without induced loss of viability. Expression levels of CD38 thus regulated the cellular response to retinoic acid, either propelling cell differentiation or loss of viability. The cellular effects of CD38 thus depend on its expression level.

Topics: ADP-ribosyl Cyclase 1; Cell Differentiation; Cell Survival; Fluorescent Antibody Technique; Gene Expression Regulation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Signal Transduction; Time Factors; Transfection; Tretinoin

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Knockout; Neoplasm Proteins; Neutrophils; Octamer Transcription Factor-1; Oncogene Proteins, Fusion; Proto-Oncogene Proteins; Trans-Activators; Translocation, Genetic; Tretinoin

Topics: Aminophylline; Antineoplastic Combined Chemotherapy Protocols; DNA-Binding Proteins; Drug Therapy, Combination; Female; Genes, Tumor Suppressor; Humans; Leukemia, Myeloid, Acute; Li-Fraumeni Syndrome; Middle Aged; Mutation; Nuclear Proteins; Treatment Outcome; Tretinoin; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Valproic Acid

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.

Topics: Adaptor Proteins, Signal Transducing; Blotting, Western; Cell Differentiation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Immunoprecipitation; Leukemia, Myeloid, Acute; Mutation; Paxillin; Phosphoproteins; Phosphorylation; Receptor, Macrophage Colony-Stimulating Factor; Receptors, Antigen, T-Cell; Resting Phase, Cell Cycle; Signal Transduction; Superoxides; Transfection; Tretinoin; Up-Regulation

During terminal cell differentiation, nuclear chromatin becomes condensed and the repertoire of epigentic heterochromatin proteins responsible for chromatin condensation is dramatically changed. In order to identify the chromatin regulatory factors associated with incomplete cell differentiation and impaired chromatin condensation in hematological malignancies, we examined expression levels of major heterochromatin proteins in normal blood cells and cells derived from a number of chronic and acute myeloid leukemia patients exhibiting different degrees of differentiation.. We used immunoblotting and immunofluorescence to examine the levels and localization of epigenetic heterochromatin factors in isolated cell nuclei and fractionated peripheral blood cells.. While the major epigenetic heterochromatin factor, histone H3 methylated at lysine 9, is present in all cell types, its main counterparts, nonhistone proteins, heterochromatin proteins 1 (HP1) alpha, beta, and gamma, are dramatically reduced in peripheral blood leukocytes of normal donors and chronic myeloid leukemia patients, but are substantially increased in the blood of accelerated phase and blast crisis patients. In the terminally differentiated cells, nuclear chromatin accumulates a nucleocytoplasmic serpin, monocyte and neutrophil elastase inhibitor (MNEI). HP1 and MNEI levels inversely correlate in a number of normal and leukemia myeloid cells and show strikingly opposite coordinated changes during differentiation of U937 cell line induced by retinoic acid.. Our results suggest that repression of HP1 and accumulation of MNEI are linked to terminal cell differentiation and that their levels may be monitored in blood cell populations to detect transitions in cell differentiation associated with leukemia progression and treatment.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Blast Crisis; Cell Differentiation; Chromosomal Proteins, Non-Histone; Epigenesis, Genetic; Gene Expression Regulation, Leukemic; Heterochromatin; Histones; HL-60 Cells; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukocytes; Neoplasm Proteins; Proteins; Serpins; Tretinoin; U937 Cells

Topics: Aged, 80 and over; Aminophylline; Blood Platelets; Drug Therapy, Combination; Female; Humans; Leukemia, Myeloid, Acute; Platelet Count; Tretinoin; Valproic Acid

The folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML), owing particularly to its selective up-regulation in the leukemic cells by all-trans retinoic acid (ATRA). Here we show, using KG-1 and MV4-11 AML cells and recombinant 293 cells, that the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VPA), and FK228 potentiated ATRA induction of FR-beta gene transcription and FR-beta mRNA/protein expression. ATRA and/or TSA did not induce de novo FR synthesis in any of a variety of FR-negative cell lines tested. TSA did not alter the effect of ATRA on the expression of retinoic acid receptor (RAR) alpha, beta, or gamma. Chromatin immunoprecipitation assays indicate that HDAC inhibitors act on the FR-beta gene by enhancing RAR-associated histone acetylation to increase the association of Sp1 with the basal FR-beta promoter. Under these conditions, the expression level of Sp1 is unaltered. A decreased availability of putative repressor AP-1 proteins may also indirectly contribute to the effect of HDAC inhibitors. Finally, FR-beta selectively mediated growth inhibition by (6S) dideazatetrahydrofolate in a manner that was greatly potentiated in AML cells by ATRA and HDAC inhibition. Therefore, the combination of ATRA and innocuous HDAC inhibitors may be expected to facilitate selective FR-beta-targeted therapies in AML.

Topics: Acetylation; Animals; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carrier Proteins; CHO Cells; Cricetinae; Drug Synergism; Enzyme Inhibitors; Folate Receptors, GPI-Anchored; Folic Acid Antagonists; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Promoter Regions, Genetic; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Retinoic Acid; RNA, Messenger; Substrate Specificity; Tetrahydrofolates; Transcription Factor AP-1; Tretinoin; Up-Regulation; Valproic Acid

We reported previously that treatment of human myeloblastic leukemia ML-1 cells with all-trans retinoic acid (ATRA) in combination with GM-CSF enhances the granulocytic differentiation, which is induced only slightly by ATRA alone. To investigate the mechanism underlying this differentiation and the synergistic effect of ATRA and GM-CSF, we used cDNA microarray to examine gene expression profiles of ML-1 cells treated with ATRA and/or GM-CSF. We identified 22 up-regulated genes in ML-1 cells treated with both reagents and examined the expression of these genes in cells treated with ATRA and/or GM-CSF by Northern blot analysis. Comparison of cells treated with both reagents and cells treated with ATRA or GM-CSF alone revealed that expression of nine of the 19 genes was induced synergistically by combined treatment with ATRA and GM-CSF. Expression of most of these genes was increased only slightly by ATRA alone, and this induction was enhanced by the addition of GM-CSF. These results indicate that GM-CSF enhances ATRA-induced gene expression. Moreover, studies with inhibitors of signaling molecules suggested that activation of JAK2 is associated with the synergistic induction of several genes by ATRA and GM-CSF. JAK2 inhibitor suppressed induction of NBT-reducing activity in ML-1 cells treated with both reagents. It is likely that the enhancer effect of GM-CSF on ATRA-induced gene expression leads to the differentiation induced synergistically by ATRA combined with GM-CSF. Further studies of the mechanism underlying this effect may identify better approaches for the treatment of RA-insensitive leukemia.

Topics: Cell Differentiation; Cell Line, Tumor; Gene Expression; Gene Expression Profiling; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kinetics; Leukemia, Myeloid, Acute; Oligonucleotide Array Sequence Analysis; Sensitivity and Specificity; Signal Transduction; Tretinoin; Up-Regulation

Epigenetic alterations of chromatin due to aberrant histone deacetylase (HDAC) activity and transcriptional silencing of all-trans retinoic acid (ATRA) pathway are events linked to the pathogenesis of acute myeloid leukemia (AML) that can be targeted by specific treatments. A pilot study was carried out in eight refractory or high-risk AML patients not eligible for intensive therapy to assess the biological and therapeutic activities of the HDAC inhibitor valproic acid (VPA) used to remodel chromatin, followed by the addition of ATRA, to activate gene transcription and differentiation in leukemic cells. Hyperacetylation of histones H3 and H4 was detectable at therapeutic VPA serum levels (>or=50 microg/mL) in blood mononuclear cells from seven of eight patients. This correlated with myelomonocytic differentiation of leukemic cells as revealed by morphologic, cytochemical, immunophenotypic, and gene expression analyses. Differentiation of the leukemic clone was proven by fluorescence in situ hybridization analysis showing the cytogenetic lesion +8 or 7q- in differentiating cells. Hematologic improvement, according to established criteria for myelodysplastic syndromes, was observed in two cases. Stable disease and disease progression were observed in five and one cases, respectively. In conclusion, VPA-ATRA treatment is well tolerated and induces phenotypic changes of AML blasts through chromatin remodeling. Further studies are needed to evaluate whether VPA-ATRA treatment by reprogramming differentiation of the leukemic clone might improve the response to chemotherapy in leukemia patients.

Topics: Antineoplastic Agents; Bone Marrow Cells; Cell Differentiation; Enzyme Inhibitors; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Myeloid, Acute; Treatment Outcome; Tretinoin; Valproic Acid

Leukemia is a type of cancer that starts in the bone marrow. The anticancer drugs used in the treatment of patients suffering from the disease have many side effects due to their toxicity. This fact has prompted researchers to search for other agents instead of, or in combination with, these anticancer drugs. Differentiating agents (DAs) including Na-Butyrate (NaBu), trans-retinoic acid (TRA), dibutyryl- cAMP (Bu-cAMP) and many others have been used for this purpose.. In this investigation, we studied 120 patients with acute myeloid leukemia (AML),presenting to the Oncology Institute of Tanta, Egypt. We studied the effect of some differentiating agents (DAs)mainly Na-Butyrate (Na-Bu., 1mM), trans-retinoic acid (TRA, l micro M) and dibutyryl-cAMP (Bu-cAMP, lmM) on the morphology of leukemia cells propagated ex-vivo for 3 and 6 days. We also studied the level of interferon- gamma and its release in the conditioned media of the leukemic cells compared to normal leukocytes.. The results revealed that DAs enhanced the conversion of the immature granulocytes into mature ones clearly at 6 days of treatment when we used the agents in combination. The results also showed that statistically significant elevation (p<0.001) of interferon- gamma level was found to be in the conditioned media of the treated leukemic cells (3 and 6 days) using the previously mentioned agents alone or in combination; that could reach almost the level in the cultured media of normal leukocytes.. In conclusion, this work could highlight the possibility of using DAs as a novel complementary therapy in the management of acute myeloid leukemia (AML) via the activation of the immune surveillance of patients suffering from AML through raising the interferon- gamma level. Further work is recommended to use DAs in clinical trials with and without conventional anticancer drugs for the management of patient with AML.

Topics: Antineoplastic Agents; Bucladesine; Butyrates; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Shape; Culture Media, Conditioned; Drug Combinations; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured

The efficacy of All-Trans Retinoic Acid (Atra) and Arsenic Trioxide (As(2)O(3)) in the treatment of Acute Promyelocytic Leukemia (APL) is well known. Further, these drugs inhibit cell growth and induce apoptosis in several cell lines, but few data are reported on leukemic blasts. The aim of this study was to evaluate the biological effects on non-M3 Acute Myeloid Leukemia (AML) cells. Blasts of six patients, after exposition to Atra and As(2)O(3) were tested for growth inhibition, induction of apoptosis and change in cell cycle distribution, evaluating cell viability, percentage of apoptotic cells and of blasts positive for Ki-67 and BrdU. In the present study we demonstrated that either Atra or As(2)O(3) inhibit leukemic cells proliferation by induction of apoptosis. The effects are time and dose dependent. We did not observe additive or synergistic effects with the combination of the drugs. Further, our results showed that Atra and As(2)O(3) have effects on cell cycle distribution reducing S-phase and proliferating cells. These results should be taken in to account preparing future laboratory and clinical experimental protocols that associate these drugs with antineoplastic agents with different cell cycle specificity.

Topics: Apoptosis; Arsenic Trioxide; Arsenicals; Bromodeoxyuridine; Cell Cycle; Cell Proliferation; Dose-Response Relationship, Drug; Drug Interactions; Humans; Ki-67 Antigen; Kinetics; Leukemia, Myeloid, Acute; Oxides; S Phase; Tretinoin; Tumor Cells, Cultured

Acute myeloblastic leukemia (AML) may be classified in a number of ways. Using the French American British classification, the M3 form of the disease or acute promyelocytic leukemia (APL) has been found to be sensitive in vitro and in vivo to the retinoid all trans retinoic acid (ATRA). The mechanism for this is by restoration of normal gene expression through the release of histone deacetylase complexes (HDACs). In contrast to APL, other forms of AML are either nonresponsive or show blunted responses to ATRA. We evaluated if the inhibitor of HDAC activity, valproic acid (VPA), could mimic or enhance retinoid sensitivity in the AML cell line, OCI/AML-2, and clinical samples derived from patients with AML. An Affymetrix GeneChip experiment demonstrated that VPA modulated the expression of numerous genes in OCI/AML-2 cells that were not affected by ATRA including p21, a retinoid responsive gene in APL. VPA induced p21 expression in OCI/AML-2 cells and the majority of the AML samples tested; this was associated with cell cycle arrest and apoptosis not seen with ATRA alone. The addition of ATRA to VPA accentuated many of these responses, supporting the potential beneficial combination of these drugs in the treatment of AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Leukemia, Myeloid, Acute; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Structure-Activity Relationship; Tretinoin; Valproic Acid

Valproic acid (VPA) has been demonstrated to be able to inhibit histone deacetylase activity and to synergize with all-trans retinoic acid (ATRA) in inducing the differentiation of acute myeloid leukemia (AML) cells. A pilot study of the VPA/ATRA combination was performed in 11 elderly patients with de novo AML (median age, 82 years). Complete marrow response was observed in 3 patients, including 1 complete remission. Two additional patients had hematologic improvement.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Humans; Leukemia, Myeloid, Acute; Pilot Projects; Time Factors; Treatment Outcome; Tretinoin; Valproic Acid

We describe a case of acute myeloid leukemia (AML) bearing the translocation t(11;17)(q23;q21). The morphological phenotype represented a monoblastic leukemia, AML French-American-British (FAB) M5a. Further analysis of the translocation revealed an involvement of the mixed-lineage leukemia (MLL) gene and a region closely proximal to the retinoic acid (RA) receptor alpha (RARA) gene. AMLs involving both a rearranged MLL and the 17q21 region, in which the RARA gene is located, have only been described in some individual cases. The functional role of this translocation is still unknown. Rearrangements of the MLL (11q23) gene in AML are usually related to the morphological phenotype FAB M5. In general, they are associated with an adverse prognosis. In acute promyelocytic leukemia, the translocation (15;17)(q22;q11-21) involving the RARA leads to a maturation arrest that can be overcome by RA, often inducing remission. In other forms of AML, however, the effects of RA are limited and diverse. To study whether RA might have a therapeutical potential in our case, we performed an in vitro analysis of RA effects on AML cells. We found that RA leads to enhanced cell death and up-regulation of CD38 and CD117. However, no hints of RA-induced in vitro differentiation were visible. Our data indicate that in AML cells bearing the t(11;17)(q23;q21), a differentiation arrest that is overcome by RA is not present. On the contrary, RA induces alterations in cellular regulation that are similar to the RA-induced changes observed in early hematogenic progenitors; thus, a possible therapeutical benefit of RA in such cases remains open.

Topics: ADP-ribosyl Cyclase 1; Antineoplastic Agents; Cell Death; Cell Differentiation; Child; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Drug Screening Assays, Antitumor; Female; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Histone-Lysine N-Methyltransferase; Humans; Leukemia, Myeloid, Acute; Myeloid-Lymphoid Leukemia Protein; Proto-Oncogene Proteins c-kit; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

The LYL1 gene encodes a basic helix-loop-helix transcription factor involved in T-cell acute lymphoblastic leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of LYL1 was at higher levels in the majority cases of acute myeloblastic leukemia (AML) or myelodysplastic syndrome when compared to normal bone marrow. Our study also showed that LYL1 was highly expressed in most AML cell lines and in CD34+ AML cells. To determine whether LYL1 had an affect on the phenotype and behavior of myeloid cells, we introduced full-length LYL1 cDNA into K562 cells using electroporation and U937 cells with retroviral infection. Both of the derivative cell lines with overexpression of LYL1 had an increased growth rate and clonogenecity. Forced expression of LYL1 in K562 cells enhanced spontaneous and hemin-induced erythroid differentiation but blocked spontaneous as well as PMA-induced megakaryocytic differentiation. Overexpression of LYL1 in U937 cells blocked all-trans retinoic acid-induced monocytic differentiation. The LYL1-transfected U937 cells were also more resistant to the cytotoxic drug cytarabine. These results demonstrate that LYL1 may play a role in early hematopoiesis and may be a potential oncogenic factor in AML.

Topics: Antineoplastic Agents; Antineoplastic Agents, Alkylating; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow; Cell Transformation, Neoplastic; Cytarabine; DNA-Binding Proteins; DNA, Complementary; Drug Resistance, Neoplasm; Electroporation; Gene Expression Profiling; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Neoplasm Proteins; Phenotype; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

Acute febrile neutrophilic dermatosis (Sweet syndrome) has been reported in a few adults receiving all-trans retinoic acid for acute promyelocytic leukemia. The authors report a case of Sweet syndrome associated with the administration of all-trans retinoic acid for acute promyelocytic leukemia in a pediatric patient.

Topics: Antineoplastic Agents; Child; Female; Humans; Leukemia, Myeloid, Acute; Skin; Sweet Syndrome; Treatment Outcome; Tretinoin

Acute myeloid leukemia (AML) predominantly affects older adults, a population with a poor prognosis, due to age, comorbidities and forms of disease. We present a retrospective study of 45 patients older than 60 years of age, with AML, who were diagnosed and/or treated in our clinic in the year 2001. Our study refers to 32 men, 63-80 years of age and 13 women, 62-85 years of age. Fourteen of them were diagnosed as de novo leukemia while 31 developed secondary leukemia, due to myelodysplasia, chronic myeloid leukemia and essential thrombocytemia. A therapeutic protocol that included 2 courses of induction chemotherapy with idarubicin 8mg/m2 for 3 days, aracytin 100 mg/m2 for 5 days and etoposide 75 mg/m2 for 5 days, followed by 2 courses of consolidation chemotherapy with aracytin 800 mg/m2/d for 4 days, was administered. In patients with acute promyelocytic leukemia we additionally administered all trans retinoic acid. Those with erythroleukemia also received erythropoietin, 10,000 IU 3 times a week. All patients received supportive therapy with blood products and G-CSF during blood marrow aplasia. Four patients refused therapy and three patients received only blood product support because of poor performance status. Nine out of the 38 patients who received chemotherapy (23.7%) achieved a complete remission after treatment, while, 13 out of 38 (34.2%) only a partial one (overall remission rate: 57.9 %). Ten patients relapsed in <6 months and 12 patients relapsed in >6 months. Patients who received only supportive treatment died 2-5 months after initial diagnosis. During therapy, 16 patients (42.1%) died due to: infection, cerebrovascular or gastrointestinal bleeding and acute myocardial infarction. In conclusion, it appears that a high percentage of the elderly patients with AML, despite the unfavourable prognosis, responded to chemotherapy (57.9%) and achieved longer survival durations compared to patients who refused therapy or received supportive treatment alone. Unfortunately, a large number of them exhibited serious complications during treatment, with a mortal outcome. Close follow-up and supportive care highly contributed to an improvement of treatment outcome in elderly patients with acute myeloid leukemia.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Etoposide; Female; Granulocyte Colony-Stimulating Factor; Humans; Idarubicin; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Recurrence; Remission Induction; Retrospective Studies; Time Factors; Treatment Outcome; Tretinoin

Folate receptor (FR) type beta is a promising target for therapeutic intervention in acute myelogenous leukemia (AML) owing particularly to its specific up-regulation in AML cells by all-trans retinoic acid (ATRA). Here we identify functional elements in the FR-beta gene and examine the molecular mechanism of transcriptional induction of FR-beta by ATRA. The basal promoter activity of FR-beta resulted from synergistic interaction between Sp1 and ets binding sites (EBSs) and repression by upstream AP-1-like elements, whose action required EBSs. A minimal promoter containing the Sp1 and ets elements was ATRA-responsive. The repressor elements bound Fos family proteins; association of the proteins with the repressor elements correlated negatively with FR-beta expression in peripheral blood neutrophils and monocytes and also in KG-1 (AML) cells grown in the absence or in the presence of ATRA. Furthermore, down-regulation of FR-beta in KG-1 cells treated with O-tetradecanoylphorbol 13-acetate (TPA) was accompanied by increased AP-1 binding to the repressor elements. From chromatin immunoprecipitation (ChIP) assays, the nuclear retinoic acid receptor alpha (RARalpha) associated with the Sp1 region, and RARs beta and gamma associated with the AP-1 and Sp1 regions; treatment of KG-1 cells with ATRA did not alter Sp1 binding but increased the association of RARalpha and decreased the association of RARs beta and gamma. ATRA also decreased RAR expression levels. The results suggest that the FR-beta gene is a target for multiple coordinate actions of nuclear receptors for ATRA directly and indirectly acting on a transcriptional complex containing activating Sp1/ets and inhibitory AP-1 proteins. The multiple mechanisms favor the prediction that ATRA will induce FR-beta expression in a broad spectrum of AML cells. Further, optimal FR-beta induction may be expected when all 3 RAR subtypes bind agonist.

Topics: Blood Cells; Carrier Proteins; Folate Receptors, GPI-Anchored; Genes, Regulator; Humans; Leukemia, Myeloid, Acute; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Receptors, Cell Surface; Receptors, Retinoic Acid; Sp1 Transcription Factor; Transcription Factor AP-1; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34 + CD34 - phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38 +, some are CD38 - . AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo. Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 - cell lines, HL60 and CB-1, as well as in normal CD34 + CD34 - hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34 + leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34 + -AML (M0 and M1) patients, RA caused an increase in CD38 + that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 - cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34 + cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 - cells are the targets of leukemic transformation, transition of these cellsinto CD38 + phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Antigens, CD; Antigens, CD34; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Line; Cell Line, Tumor; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Immunophenotyping; Leukemia; Leukemia, Myeloid, Acute; Membrane Glycoproteins; Myeloid Cells; Phenotype; Time Factors; Tretinoin

In the present study, the effects of 9-cis retinoic acid (RA) and 13-cis RA on acute myeloblastic leukaemia (AML) cell growth and the induction of apoptosis as well as its relationship with bcl-2 and p53 were compared with those of all-trans RA (ATRA). The study was performed with the subclones of the retinoid-sensitive OCI/AML-2 cell line. The most prominent inhibitory effect on clonogenic cell growth and morphological apoptosis was shown by 9-cis RA. In addition, Western blotting revealed the most obvious translocation of p53 from cytosol to nucleus in the case of 9-cis RA, which was the only retinoid able to change the conformation of p53 from mutational to wild type, as demonstrated by flow cytometry. There was no difference between the retinoids in the downregulation of bcl-2 as analysed by Western blotting and flow cytometry. The RA receptor (RAR)-alpha antagonist had no effect on apoptosis in any of the three retinoids studied using the annexin V method. In conclusion, this study shows that 9-cis RA was a more potent agent than ATRA or 13-cis RA in inducing growth arrest and apoptosis in the OCI/AML-2 subclones. The effect was associated with the downregulation of bcl-2 and was hardly mediated through the RAR-alpha receptor, but might be related to the activation of p53.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Count; Cell Division; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescence; Humans; Isotretinoin; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Topics: Adult; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mercaptopurine; Methotrexate; Myelodysplastic Syndromes; Neoplasms, Second Primary; Prednisone; Remission Induction; Tretinoin; Vincristine

Topics: Antineoplastic Combined Chemotherapy Protocols; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Myelodysplastic Syndromes; Tretinoin

All-trans retinoic acid (ATRA) combined with granulocyte macrophage colony-stimulating factor (GM-CSF) synergistically increases superoxide-generating activity in human myeloblastic leukemia ML-1 cells. ATRA is known to increase the expression of some NADPH components; however, little is known about the effect of GM-CSF on the expression of these components. We examined the expression of NADPH oxidase components in ML-1 cells treated with ATRA, GM-CSF, or a combination of ATRA and GM-CSF. Expression of p47phox and gp91phox proteins increased markedly after treatment with both reagents. p47phox expression was increased by ATRA alone, and the expression was increased synergistically by the combination of ATRA with GM-CSF. gp91phox was increased by ATRA or GM-CSF alone. The expression of p47phox and gp91phox mRNA underwent similar changes to those seen in protein level. These results indicate that GM-CSF induces expression of gp91phox and enhances ATRA-induced p47phox expression. We speculate that the remarkable induction of gp91phox and p47phox protein is associated with an increase in superoxide-generating activity due to the synergistic effect of ATRA plus GM-CSF.

Topics: Base Sequence; DNA Primers; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxides; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

P-glycoprotein (P-gp)/multi-drug resistance 1 (MDR1) gene is recognized to be, at least in part, responsible for the refractoriness to chemotherapy of leukemia. The transcriptional mechanism of MDR1 gene is largely unknown. However, recent reports have clarified that early growth response 1 gene (Egr1) positively regulates MDR1 transcription, while Wilms' tumor suppressor gene (WT1) does negative regulation of MDR1 gene expression in 12-O-tetradecanoylphorbol-13-acetate treated K562 cells. In addition, Egr1 and WT1 are structurally related transcription factors and bind to quite similar DNA sequences. Our study of mRNA expression profile of Egr1, WT1 and MDR1 in fresh AML samples demonstrated that there are disease-specific patterns. Egr1 mRNA was frequently and strongly expressed in monocytic leukemia cells, especially in AML M4 cells. WT1 mRNA was undetectable in t(8;21) AML cells. mRNA expression of MDR1 was frequent in AML M1 and t(8;21) AML cells, in which the expression level was highest in AML M1 and was low in monocytic leukemia (M4 and M5). Then, expression level of MDR1 was inversely correlated with Egr1. By liquid culture of leukemia cell lines and fresh AML cells with the addition of all-trans retinoic acid (ATRA), modulation of P-gp/MDR1 and Egr1 was observed and the pattern of modulation was divided into four groups: (1) blastic AML type, in which distinct expression of P-gp/MDR1 and CD34 was not influenced by ATRA; (2) t(8;21)AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 was kept high; (3) AML M3 type, in which P-gp/MDR1 expression was reduced with granulocytic differentiation by ATRA; (4) monocytic AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 expression decreased, and strong Egr1 expression was downregulated just prior to the augmentation of P-gp/MDR1 expression. WT1 expression was not influenced by the addition of ATRA in each group. Previous reports have suggested that P-gp/MDR1 plays an important role in resistance to chemotherapy, and is recognized as one of the stem cell marker. However, P-gp/MDR1 expression augmented by ATRA, which was observed in monocytic AML, was recognized as a functional molecule of mature monocyte/macrophage, because CD34 expression decreased and CD13 expression increased by ATRA. Finally, expression of P-gp/MDR1 in monocytic leukemia, which was functionally confirmed by Rh123 efflux study, was thought to be closely related to the characteristic modulatio

Topics: Antigens, CD34; ATP Binding Cassette Transporter, Subfamily B, Member 1; CD13 Antigens; Cell Differentiation; Cell Line; Cell Separation; DNA Primers; DNA-Binding Proteins; Down-Regulation; Drug Resistance, Multiple; Early Growth Response Protein 1; Humans; Immediate-Early Proteins; Immunophenotyping; Karyotyping; Leukemia, Myeloid, Acute; Monocytes; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Tretinoin; Tumor Cells, Cultured; WT1 Proteins

Acute myeloid leukemia (AML) is a heterogeneous leukemia characterized by the blockage of myeloid differentiation at different stages, which define distinct AML subtypes. We have recently reported that the ligation of CD44 with 2 activating monoclonal antibodies (mAbs), A3D8 and H90, triggers terminal differentiation of leukemic blasts in AML-M1/2 to AML-M5 subtypes, which are the most frequent ones. However, fresh AML blasts have short in vitro lifespans. Therefore, to find relevant in vitro cellular models for further studying the mechanisms involved in CD44-induced differentiation, we investigated whether CD44 ligation with A3D8 and H90 mAbs can induce terminal differentiation of THP-1, NB4, and HL60 cells, each interesting models of AML-M5 (monoblastic subtype), AML-M3 (promyelocytic subtype), and AML-M2 (myeloblastic subtype), respectively. We also study whether CD44 ligation induces a loss of proliferative capacity, an important feature of late-stage myeloid differentiation. In the second part of our study, we investigated whether A3D8 and H90 anti-CD44 mAbs can induce the differentiation and inhibit the proliferation of KG1a cells, which are very immature AML-M0 blasts. Using functional, antigenic, and cytologic criteria, we presently show that A3D8 and/or H90 induce terminal differentiation of THP-1, HL60, and NB4 cell lines and strongly inhibit their proliferation. Interestingly, cell-specific effects of H90 and A3D8 are observed. We also observe that incubation with A3D8 for 3 to 6 days induces an apoptotic cell death that is moderate in the case of THP-1 and HL60 cells and massive in the case of NB4 cells. Finally, our results demonstrate for the first time that it is possible to reverse the leukemic blockage of KG1a cells by using both an anti-CD44 mAb and retinoic acid. This result may provide a new experimental basis for a differentiative therapy in AML-M0 patients.

Topics: Antibodies, Monoclonal; Apoptosis; Cell Differentiation; Cell Division; Granulocytes; HL-60 Cells; Humans; Hyaluronan Receptors; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured

It has been shown that the novel synthetic triterpenoid CDDO inhibits proliferation and induces differentiation and apoptosis in myeloid leukemia cells. In the current study the effects of the C-28 methyl ester of CDDO, CDDO-Me, were analyzed on cell growth and apoptosis of leukemic cell lines and primary acute myelogenous leukemia (AML). CDDO-Me decreased the viability of leukemic cell lines, including multidrug resistant (MDR)-1-overexpressing, p53(null) HL-60-Dox and of primary AML cells, and it was 3- to 5-fold more active than CDDO. CDDO-Me induced a loss of mitochondrial membrane potential, induction of caspase-3 cleavage, increase in annexin V binding and DNA fragmentation, suggesting the induction of apoptosis. CDDO-Me induced pro-apoptotic Bax protein that preceded caspase activation. Furthermore, CDDO-Me inhibited the activation of ERK1/2, as determined by the inhibition of mitochondrial ERK1/2 phosphorylation, and it blocked Bcl-2 phosphorylation, rendering Bcl-2 less anti-apoptotic. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and monocytic differentiation in primary cells. Of significance, colony formation of AML progenitors was significantly inhibited in a dose-dependent fashion, whereas normal CD34(+) progenitor cells were less affected. Combinations with ATRA or the RXR-specific ligand LG100268 enhanced the effects of CDDO-Me on cell viability and terminal differentiation of myeloid leukemic cell lines. In conclusion, CDDO-Me is an MDR-1- and a p53-independent compound that exerts strong antiproliferative, apoptotic, and differentiating effects in myeloid leukemic cell lines and in primary AML samples when given in submicromolar concentrations. Differential effects of CDDO-Me on leukemic and normal progenitor cells suggest that CDDO-Me has potential as a novel compound in the treatment of hematologic malignancies.

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; Blast Crisis; Caspase 3; Caspases; Cell Differentiation; Cell Survival; Cytarabine; DNA Fragmentation; Drug Interactions; Flow Cytometry; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Membrane Potentials; Mitogen-Activated Protein Kinases; Oleanolic Acid; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoids; Tretinoin; Tumor Cells, Cultured

The use of all-trans retinoic acid (ATRA) in combination with chemotherapy has markedly improved the prognosis for patients with acute promyelocytic leukemia (APL); the higher complete remission (CR) and survival rates now reported in this disease almost approach those obtained for other highly curable hematologic malignancies. Of 77 patients with APL who were consecutively treated at a single institution and who achieved CR after induction and consolidation therapy, 5 (6.5%) acquired therapy-related myelodysplasia (tMDS), acute myelogenous leukemia (AML), or both (tMDS-AML). Of these, 3 of 46 (6.5%) patients received front-line chemotherapy with or without ATRA and acquired tMDS-AML while in first remission of APL. Two underwent repeated chemotherapy cycles with ATRA because of APL relapse and acquired tMDS-AML while in the second or third remission of APL. In 2 patients, clinical and biologic characteristics of tMDS-AML were as expected for postalkylating forms (long latency, MDS phase preceding AML, karyotypic aberrations involving chromosomes 5 or 7), even though one of them had not previously received alkylating drugs. Three of the 5 patients died shortly after tMDS-AML diagnosis, one is alive with tMDS, and one is alive and in CR after allogeneic bone marrow transplantation. The occurrence of tMDS-AML after successful therapy for APL is an emerging problem. The availability of prognostic score systems at initial diagnosis and monitoring of residual disease by polymerase chain reaction might allow better tailoring of treatment intensity in APL to spare unnecessary toxicity and to minimize the risk for tMDS-AML in patients who are presumably cured.

Topics: Adolescent; Adult; Antineoplastic Agents; Cytogenetic Analysis; Female; Follow-Up Studies; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Myelodysplastic Syndromes; Neoplasms, Second Primary; Treatment Outcome; Tretinoin

To present a special case with the karyotype and molecular marker of acute myeloid leukemia (AML)-M2 who was induced to complete remission by all-trans retinoic acid (ATRA) alone.. A recently hospitalized young female patient with acute leukemia was initially diagnosed as M3 subtype based on morphological French-American-British (FAB) classification. Karyotype analysis using standard G and R banding techniques and RT-PCR were applied to further define the diagnosis. After primarily cultured bone marrow cells from the iliac aspiration were tested for in vitro induced differentiation, the patient was treated with oral all-trans retinoic acid alone, 60 mg per day until complete remission was achieved. Peripheral blood and bone marrow changes were monitored over the whole treatment course.. The characteristic chromosomal aberration for M3, the t(15;17) reciprocal translocation, was not found while a t(8;21) translocation was verified. Furthermore, an amplified product of the AML-1/ETO fusion gene instead of the PML/RAR alpha fusion gene was detected by RT-PCR and the diagnosis was corrected from M3 to M2. Primary cultured bone marrow cells can be fully induced to terminal differentiation after 4 days exposure to ATRA. A hematological complete remission was achieved after 40 days treatment with ATRA as a single therapeutic agent, suggesting an alternative pathway mediating ATRA-induced myeloid differentiation.. A leukemia patient with a subtype other than M3, such as M2 in this case, may also be induced to complete remission by the mechanism of ATRA-induced terminal differentiation. This implies that there may be a pathway other than PML/RAR alpha fusion gene product which mediates ATRA-induced myeloid maturation in leukemia cells.

Topics: Adolescent; Antineoplastic Agents; Core Binding Factor Alpha 2 Subunit; Female; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Reverse Transcriptase Polymerase Chain Reaction; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Tretinoin

Up-regulation of folate receptor (FR) type-beta in acute myelogenous leukemia (AML) by all-trans retinoic acid (ATRA) and its restricted normal tissue distribution makes it a potential target for therapeutic intervention. The FR-beta in peripheral blood granulocytes was unable to bind folate and appeared to have a variant GPI membrane anchor, evident from its insensitivity to phosphatidylinositol-specific phospholipase C but not nitrous acid. Granulocyte FR-beta lacked mutations, and neither deglycosylation nor detergent solubilization restored folate binding. The posttranslational modification causing its nonfunctionality was evidently absent in FR-beta from AML cells from patient marrow, which bound folate. From flow cytometric analysis of 78 AML bone marrow specimens of different subtypes, 68% expressed FR-beta, most of which were also CD34+. In model cell lines that are FR - (KG-1a, L1210, and Chinese hamster ovary [CHO]) or FR + (KG-1, L1210 JF, and recombinant CHO-FR-beta), selective FR-mediated binding and cytotoxicity was obtained using folate-coated liposomes encapsulating fluorescent calcein (f-L-calcein) and doxorubicin (f-L-DOX), respectively, which could be blocked by 1 mM free folic acid. In the FR-beta-expressing KG-1 human AML cells, treatment with ATRA further increased this specificity. In mouse ascites leukemia models generated using L1210JF or KG-1 cells, increased median survival times were obtained with f-L-DOX treatment compared to nontargeted L-DOX. In the KG-1 model, ATRA treatment increased the cure rate with f-L-DOX from 10% to 60%. The above combined data from our 2 laboratories further support the feasibility and potential usefulness of selective ATRA-facilitated liposomal drug delivery in FR-beta + AMLs.

Topics: Animals; Antigens, CD34; Antineoplastic Combined Chemotherapy Protocols; Carrier Proteins; Cell Death; Disease Models, Animal; Doxorubicin; Drug Delivery Systems; Drug Evaluation, Preclinical; Folate Receptors, GPI-Anchored; Folic Acid; Gene Expression Regulation; Humans; Leukemia, Myeloid, Acute; Liposomes; Mice; Receptors, Cell Surface; Survival Rate; Therapeutic Equivalency; Treatment Outcome; Tretinoin; Tumor Cells, Cultured

Histone deacetylase (HDAC)-dependent transcriptional repression of the retinoic acid (RA)-signaling pathway underlies the differentiation block of acute promyelocytic leukemia. RA treatment relieves transcriptional repression and triggers differentiation of acute promyelocytic leukemia blasts, leading to disease remission. We report that transcriptional repression of RA signaling is a common mechanism in acute myeloid leukemias (AMLs). HDAC inhibitors restored RA-dependent transcriptional activation and triggered terminal differentiation of primary blasts from 23 AML patients. Accordingly, we show that AML1/ETO, the commonest AML-associated fusion protein, is an HDAC-dependent repressor of RA signaling. These findings relate alteration of the RA pathway to myeloid leukemogenesis and underscore the potential of transcriptional/differentiation therapy in AML.

Topics: Acetylation; Antineoplastic Agents; Cell Differentiation; Core Binding Factor Alpha 2 Subunit; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Oncogene Proteins, Fusion; RUNX1 Translocation Partner 1 Protein; Signal Transduction; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

AML with trilineage myelodysplasia (AML/TMDS) is recognized as having of poor prognosis due to its resistance to chemotherapy in comparison with de novo AML. An AML/TMDS patient who failed to respond to ordinary induction therapy achieved complete remission with combination therapy consisting of all-trans retinoic acid (ATRA) and low-dose Ara-C. No serious toxicity was observed. ATRA combined with low-dose Ara-C could be an alternative treatment for this kind of patient.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Female; Humans; Leukemia, Myeloid, Acute; Middle Aged; Neural Tube Defects; Tretinoin

To describe the retinoic acid syndrome, a complication of therapy with all-trans retinoic acid (ATRA) in acute promyelocitic leukemia (APL).. Retrospective study of five patients with a morphologic diagnosis of APL by the French-American-British (FAB) classification that were treated for remission induction with ATRA and developed the ATRA syndrome.. Three patients in newly diagnosed APL and two in leukemia relapse were analyzed. All patients received with ATRA 45 mgrs/m2/day, and three of them also received chemotherapy. Patients developed fever, respiratory distress, pulmonary infiltrates, weight gain and edemas. The onset of this symptom complex occurred from 1 to 11 days after starting treatment and was preceded by an increased in peripheral blood leukocytes. Infections or congestive heart failure were ruled out. The clinical course progressed while patients being treated with wide spectrum antibiotics. Four patients were treated with high doses corticosteroid therapy (dexametasone 10 mgrs intravenously every 12 hours), in three of them full recovery was attained and one died. One patient that did not received steroids died.. The use of all-trans retinoic acid to induce hematologic remission in APL patients is associated in same patients with the development of ATRA syndrome, a life threatening complication. Symptoms begin in the first days of treatment. If this syndrome is suspected, early treatment with high dose steroids should be initiated.

Topics: Adolescent; Antineoplastic Agents; Female; Humans; Leukemia, Myeloid, Acute; Lung Diseases, Interstitial; Male; Middle Aged; Pulmonary Edema; Respiratory Insufficiency; Retrospective Studies; Syndrome; Tretinoin

When bcl-2 is immunoprecipitated from (32)P-labeled cell extracts of all-trans retinoic acid (ATRA)-treated acute myeloblastic leukemia (AML) blasts, a phosphorylated protein of approximately 30 kd is coprecipitated. This protein has been identified as ribosomal protein S3a. The biologic effects of S3a include favoring apoptosis and enhancing the malignant phenotype. We sought to determine whether S3a, like bcl-2, influenced the response of cells to chemotherapeutic drugs and ATRA. Cell lines were studied in which S3a was genetically increased or disrupted; increased S3a was regularly associated with increased plating efficiency and increased sensitivity to either cytosine arabinoside (ara-C) or doxorubicin (DNR). S3a did not affect the sensitivity of cells to paclitaxel. Pulse exposures to either (3)HTdR or ara-C showed a greater percentage of clonogenic cells in the S phase of the cell cycle in cells with increased S3a than in controls. Cells with increased S3a responded to ATRA by increased ara-C or DNR sensitivity, whereas cells with reduced S3a protein were either protected by ATRA or not affected. We studied cryopreserved blast cells from patients with AML or chronic myelomonocytic leukemia (CMML). S3a protein levels were heterogeneous in these populations. In 32 cryopreserved blast populations, S3a levels were significantly correlated with both bcl-2 and with cell growth in culture. As in cell lines, high S3a in cryopreserved blasts was associated with ATRA-induced sensitization to ara-C. No significant association was seen between S3a levels and response to treatment.

Topics: Animals; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line; Cryopreservation; Cytarabine; DNA Replication; DNA, Neoplasm; Doxorubicin; Drug Resistance, Neoplasm; Gene Targeting; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Chronic; Neoplasm Proteins; Neoplastic Stem Cells; Paclitaxel; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Rats; Ribosomal Proteins; Tissue Preservation; Treatment Outcome; Tretinoin

All-trans retinoic acid (ATRA) induces growth arrest and apoptosis in acute myeloblastic leukemia (AML) cells. Since cellular redox state regulates these events, we were interested in studying whether it has any role in the responsiveness of AML cells to ATRA.. Two human AML cell lines, the ATRA-sensitive OU-AML-3, and the ATRA-resistant OU-AML-7, were used as models. Clonogenic cell culture assay, annexin V method, and measurement of mitochondrial membrane potential were used for the determination of cell growth and apoptosis. Peroxide formation was analyzed by flow cytometry, glutathione and g-glutamylcysteine synthetase (g-GCS) activity was determined spectrophotometrically, and the expression of manganese superoxide dismutase (MnSOD) by Western blotting.. ATRA inhibited clonogenic cell growth and induced apoptosis particularly in OU-AML-3 cells. The OU-AML-7 cells had a higher basal level of glutathione and g-GCS activity than the OU-AML-3 cells. ATRA enhanced the generation of peroxides after 24h exposure, which was more prominent in the sensitive than the resistant cell line and was not preventable by N-acetyl-L-cysteine. ATRA also increased the activity of g-GCS, which was associated with increased intracellular glutathione in the resistant cell line, while the glutathione level was maintained in the sensitive cell line. During ATRA exposure, MnSOD was induced in the sensitive cell line, but not until after 72 h. Buthionine sulfoximine significantly increased the inhibitory effect of ATRA on colony formation in both cell lines, but only marginally enhanced the effect of ATRA on the induction of apoptosis.. The balance between oxidative and antioxidative actions of ATRA, as well as the basal redox state of the cells seem to have a definite influence on the responsiveness of AML cells to ATRA.

Topics: Apoptosis; Cell Division; Clone Cells; gamma-Glutamyl Hydrolase; Glutathione; Humans; Leukemia, Myeloid, Acute; Oxidants; Oxidation-Reduction; Peroxides; Reactive Oxygen Species; Superoxide Dismutase; Tretinoin; Tumor Cells, Cultured

Acute promyelocytic leukemia (APL) is characterized by a block to myeloid differentiation caused by expression of the fusion oncoprotein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha). The purpose of this study was to identify genes that are regulated in a PML-RARalpha-dependent fashion by retinoic acid (RA), because such genes may be integrally involved in APL pathogenesis and/or myeloid differentiation. A cDNA microarray approach was used to identify genes induced in response to RA in TF1 myeloid leukemia cells expressing PML-RARalpha (TF1-PR cells). The B94 gene (TNFAIP2; Unigene Hs.101382), originally identified as a tumor necrosis factor alpha-inducible gene in endothelial cells, was one of several genes found to be induced by RA specifically in TF1-PR cells, but not in TF1-neo (control) cells. The induction of B94 was most pronounced in cells expressing the PML-RARalpha short isoform and was negligible in cells that expressed a mutant PML-RARalpha protein containing a deletion of the PML coiled-coil domain. B94 induction by RA occurred within 1 h, did not require new protein synthesis, and was inhibited by actinomycin D, suggesting rapid transcriptional activation. B94 was also induced by RA in NB4, UF1, and HL-60 cells, but not in other hematopoietic cell lines tested, suggesting that its up-regulation by RA may be specific to cells that express PML-RARalpha or are at the late myeloblast or promyelocyte stage of myeloid development. A screen of bone marrow cells from normal donors or patients with acute myelogenous leukemia showed that B94 was highly expressed in normal marrow and in marrow from patients with acute myelogenous leukemia French-American-British subtypes M0-M2, but was repressed in marrow cells from APL patients. Treatment of APL blasts in vitro with all-trans-RA resulted in up-regulation of B94 mRNA. These results suggest that B94 plays a role in myeloid development and support the hypothesis that B94 is a target gene of PML-RARalpha in APL.

Topics: Bone Marrow Cells; Cytokines; Female; Fetus; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Neoplasm Proteins; Oncogene Proteins, Fusion; Tretinoin; Tumor Necrosis Factor-alpha

We retrospectively analyzed treatments and outcomes for 83 acute myelogenous leukemia (AML) patients aged 60 years or more (median age 71) admitted to our hospital between August 1984 and January 1998. Complete remission was achieved in 36% of 78 patients who received anti-leukemic therapy, and median overall survival was 227 days. In addition to abnormal karyotypes involving chromosome 5 or 7, administration of less than 120 mg/m2/course of daunorubicin (DNR) during the initial treatment phase was an unfavorable prognostic factor for both CR and survival. Only 41% of all patients received 120 mg/m2/course of DNR or more, and had a significantly higher CR rate (56%) and longer survival, with a median of 389 days. It was suggested that intensive chemotherapy was effective for selected elderly AML patients who were relatively younger and had good performance status, although the number of such patients was limited in our study.

Topics: Age Factors; Aged; Aged, 80 and over; Chromosomes, Human, Pair 5; Chromosomes, Human, Pair 7; Cytarabine; Daunorubicin; Drug Therapy, Combination; Gene Deletion; Humans; Leukemia, Myeloid, Acute; Middle Aged; Mitoxantrone; Remission Induction; Retrospective Studies; Treatment Outcome; Tretinoin

We previously reported that all-trans retinoic acid (ATRA) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically induced granulocytic differentiation in human myeloblastic leukemia ML-1 cells. The combination of these agents also suppressed DNA-synthesis. In the present study, we investigated the suppression of cyclin dependent kinase (CDK) activities resulting in G1 arrest in differentiated ML-1 cells. We show that treatment of ML-1 cells with ATRA plus GMCSF results in G1 arrest and suppression of CDK activities. Protein levels of the G1 CDKs were essentially unchanged during this time. However, we observed an increase in CDK2-bound p27 and CDK4-bound p18, and a decrease in CDK6-bound cyclin D3. These results suggest that complex regulation of CDKs play a key role in G1 arrest of ML-1 after treatment with ATRA and GM-CSF. We also showed that an increase in CDK2-bound p27 and CDK4-bound p18 are caused by treatment with ATRA and a decrease in CDK6-bound cyclin D3 is induced synergistically by treatment with both reagents. Furthermore, we propose that the changes in binding of p18 and cyclin D3 to CDKs are due to changes at the protein expression level and that the increase in p27 binding to CDK2 is due to a novel mechanism.

Topics: Antineoplastic Agents; Carrier Proteins; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinases; Enzyme Inhibitors; G1 Phase; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Microfilament Proteins; Muscle Proteins; Neoplasm Proteins; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Efficacy of differentiating agents requires that their specific cellular targets are still expressed and functional in the leukemic cells. One hypothesis to target sensitive cells is to select leukemic clones which harbor disrupted transcription factors. CBFalpha and CBFbeta are core-binding proteins which have been identified as transcription regulators of hematopoietic genes and shown to be altered in numerous leukemias. In M2 AML, the t(8;21) translocation, CBFalpha (AML1) is altered and produced as the AML1-ETO fusion protein. The fusion protein blocks transcription and differentiation mediated by G-CSF. Interestingly, AML1-ETO leukemic cell lines are sensitive to numerous cytokines in vitro and can be induced to differentiate in the presence of G-CSF and PMA.. As in the APL differentiation model, primary culture provides a useful tool for therapeutic screening of differentiation inducers, we analysed the in vitro sensitivity of 10 fresh M2 AML t(8;21) leukemic samples to G-CSF and the functionality of G-CSF intracellular pathways. In vitro data were compared with in vivo data from four patients treated with rhG-CSF at the dosage of 5 microg/kg/day i.v. for two to three weeks before the initiation of AML induction chemotherapy and immunophenotypic analysis performed weekly to monitor in vivo differentiation.. In vitro, an increase in CD34+ cells expressing differentiation antigens (CD11b, CD13 or CD15) was noted along with a decrease of immature CD34+/differentiation antigen negative cells. After two weeks of a daily rhG-CSF administration in vivo, a significant, albeit transient, decrease of blast count was achieved, concomitant with an increase in differentiated leukemic cells suggesting that in vivo differentiation occurs. Fresh t(8;21) leukemic cells possess functional G-CSF signaling pathways as normal activity and kinetics of STAT1 and STAT3 binding was observed. Furthermore, differentiation induction leads to a subsequent degradation of the AML1-ETO oncoprotein.. The data presented here supports the claim that G-CSF can induce in vitro and in vivo differentiation of M2 AML t(8;21) cells.

Topics: Antigens, CD; Apoptosis; Blood Cells; Bone Marrow Cells; Cell Differentiation; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Core Binding Factor Alpha 2 Subunit; DNA-Binding Proteins; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptors, Granulocyte Colony-Stimulating Factor; Recombinant Proteins; RUNX1 Translocation Partner 1 Protein; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transcription Factors; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

Topics: Humans; Leukemia, Myeloid, Acute; Lung Diseases; Tretinoin

The efficacy of all-trans retinoic acid (ATRA) in patients with acute promyelocytic leukemia (APL) has been well documented. However, ATRA is not as effective against other types of acute myelogenous leukemia (AML) or myelodysplastic syndromes. We present a patient with AML (FAB: M2) associated with a t(2;17;4)(p13;q21;p16) chromosomal defect in which the 17q21 breakpoint was not within the retinoic acid receptor alpha locus which is typically rearranged in APL. This patient was successfully treated with ATRA and granulocyte colony-stimulating factor and improvement of hematological parameters lasted for 19 months without the use of cytotoxic agents.

Topics: Antineoplastic Agents; Chromosome Aberrations; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 4; Drug Therapy, Combination; Female; Follow-Up Studies; Granulocyte Colony-Stimulating Factor; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Leukemia, Myeloid, Acute; Leukocyte Count; Middle Aged; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

The present study investigated whether all-trans retinoic acid (ATRA)-induced apoptosis in acute myeloblastic leukaemia (AML) is related to changes in mitochondrial function. Two human AML cell lines, OU-AML-3 and OU-AML-7, known to be inducible to time-dependent apoptosis of varying degrees by ATRA, were used. Apoptosis induced by ATRA was shown to be a slow event. It was detected by the DNA electrophoretic method and cytofluorimetrical annexin V assay after 48 h exposure, and by morphology and polyADPribose polymerase (PARP) cleavage after 72 h exposure of AML cells to ATRA. The efflux of mitochondrial cytochrome c to cytosol was notable in Western blotting after 48 h exposure of the cells to ATRA and was observed before the drop in the mitochondrial membrane potential, which only took place after 72 h exposure, when measured by flow cytometry and a JC-1 probe. The apoptotic events in mitochondria were more evident in the OU-AML-3 than the OU-AML-7 cell line. This might relate to the different bcl-2 contents of the cell lines: the basic bcl-2 levels of the OU-AML-7 cell line were almost twofold compared to that of the OU-AML-3 cell line, as analysed by the ELISA method. However, both of the cell lines showed progressive down-regulation of bcl-2, which began after 12-24 h exposure of the cells to ATRA as determined by ELISA, Western blotting and flow cytometry. The present results show that mitochondria have a role in ATRA-induced apoptosis in AML cells and down-regulation of bcl-2 is related to it. In view of the previously published studies, the present results underline the fact that the timing of apoptotic events, such as fragmentation of DNA, externalization of phosphatidylserine, cytochrome c efflux, change in mitochondrial membrane potential and cleavage of PARP, are, to a notable extent, cell type and inducer-dependent.

Topics: Apoptosis; Cell Division; Cytochrome c Group; Down-Regulation; Gene Expression; Genes, bcl-2; Humans; Leukemia, Myeloid, Acute; Mitochondria; Tretinoin; Tumor Cells, Cultured

Differentiation therapy using retinoic acids (RAs) or 1alpha25-dihydroxyvitamin D3 (D3) is an attractive alternative to chemotherapy in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). However, with the exception of RA therapy for acute promyelocytic leukaemia (APL), RAs and D3 are not potent enough at doses that can be tolerated by patients. We demonstrate that clofibric acid (CA) enhances the response of HL60 cells to all-trans RA and D3. Our findings and those of others in the field lead us to suggest that combination therapy using all-trans RA and CA should be considered as potential therapy for AML and MDS.

Topics: Aged; Antineoplastic Agents; Clofibric Acid; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Middle Aged; Myelodysplastic Syndromes; Tretinoin

Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2-type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D-responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD-responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation.

Topics: Acute Disease; Calcitriol; Cell Differentiation; Cell Lineage; Cell Nucleus; Dimerization; DNA; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Monocytes; Neoplasm Proteins; Neoplastic Stem Cells; Promoter Regions, Genetic; Protein Multimerization; Receptors, Calcitriol; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

To determine the clinical significance of the lung resistance protein (LRP) in acute myeloid leukemia (AML), we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo AML. LRP expression of leukemic blasts was determined by immunocytochemistry by means of monoclonal antibody LRP-56. LRP expression at diagnosis was detected in 31 out of 86 (36%) patients and correlated with white blood cell count (p = 0.01). The complete remission rate of induction chemotherapy was 72% for all treated patients (n = 82). The complete remission rate was 81% for patients without LRP expression but only 55% for patients with LRP expression (p = 0.01). Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively. At a median follow-up of 16 months, median overall survival was 17 months for LRP-negative patients but only 8 months for LRP-positive patients (p = 0.006). Disease-free survival was 9 months for LRP-negative patients and 6 months for LRP-positive patients (p = 0.078). Thus LRP predicts for poor outcome indicating that the LRP gene is a clinically relevant drug resistance gene in AML.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Disease-Free Survival; Drug Resistance, Multiple; Etoposide; Female; Humans; Idarubicin; Immunohistochemistry; Karyotyping; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplasm Proteins; Predictive Value of Tests; Prognosis; Survival Rate; Tretinoin; Vault Ribonucleoprotein Particles

A 64-year-old man who had taken acute myelogenous leukemia (AML-M2) in 1989 have relapsed with t(12; 17) (p13; q11.2-21) chromosomal abnormality and presenting marked infiltration to the skin in 1994. Blasts were seen on his peripheral blood smear (15%) and bone marrow examination showed increased leukemic cells (56%), with maturation. Leukemic cells expressed CD13 and CD33 antigen but not HLA-DR. Although leukemic cells had not promyelocytic feature morphologically, detection of PML/RAR alpha infusion signal of peripheral leukemic cells were positive for 8% (1% for control) by fluorescence in situ hybridization method. Because he did not response to standard combination chemotherapy and because we considered the possibility that t(12; 17) (p13; q11.2-21) observed in this case are t(15; 17) variant, we tried all trans retinoic acid (ATRA) to him. Interestingly, ATRA was very effective for skin lesion but hematologically it had no effect at all, and he died because of bacterial pneumonia.

Topics: Antineoplastic Agents; Chromosome Aberrations; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 17; Humans; Leukemia, Myeloid, Acute; Leukemic Infiltration; Male; Middle Aged; Skin; Tretinoin

Among 379 patients with AML with FAB type M1, 2 and M4-7 diagnosed between 1978 and 1997 in our institution, 19 (5%) had hypofibrinogenemia (HF), ie a fibrinogen level <180 mg/dl. Compared to patients with normal fibrinogen (n = 360) patients with HF had significantly elevated markers of activation of coagulation (TAT, F1.2, FPA) and fibrinolysis (D-dimer, FDP) indicating that disseminated intravascular coagulation/hyperfibrinolysis was the cause of hypofibrinogenemia. Patients with HF had significantly longer prothrombin times, thrombin clotting and reptilase times. Factor X and VIII were significantly lower than in patients without HF. With the exception of M7, HF occurred in all FAB subtypes, but was most common in M5 (12.1%). Patients with HF did not differ from those with normal fibrinogen with regard to age, sex, leukocyte count and other hematological parameters. During induction chemotherapy fibrinogen normalized rapidly (median 5 days) and there was no increased incidence of early hemorrhagic death. The overall and disease-free survival was similar to that of patients without HF.

Topics: Adult; Antineoplastic Agents; Blood Coagulation Disorders; Disease-Free Survival; Female; Fibrinogen; Humans; Incidence; Karyotyping; Leukemia, Myeloid, Acute; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins, Fusion; Prognosis; Tretinoin

Serine phosphorylation of bcl-2 has been reported after treatment of cells with protein kinase C, okadaic acid, taxol, and other chemotherapeutic agents that attack microtubules. We report here that bcl-2 is phosphorylated on serine in acute myeloblastic leukemia (AML) blasts exposed to all trans retinoic acid (ATRA). Two-dimension gels (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) disclosed a novel acidic isoform of bcl-2 in ATRA-treated blast cells from a continuous line and from two AML patients; when the cell lysates were digested with lambda-phosphatase, bcl-2 reverted to the control position, indicating that it was phosphorylated. Metabolic labeling experiments using 32Pi showed that, while control bcl-2 was labeled, incorporation was greatly increased when cells were treated with ATRA. A comparison of bcl-2 from blasts treated with ATRA or taxol showed that bcl-2 was phosphorylated on serine in cells treated with either agent; however, both qualitative and quantitative differences were seen. Qualitatively, the phosphorylated isoform from taxol-treated cells was slightly larger than the native isoform and could be distinguished on 10% to 20% SDS-polyacrylamide gradient gels, while the phosphorylated bcl-2 after ATRA ran as a single band on gradient gels at the same position as control bcl-2. Quantitatively, all bcl-2 from ATRA-treated cells was in the phosphorylated isoform, while after taxol, both phosphorylated and native bcl-2 was present; incorporation of 32Pi into bcl-2 was stimulated to greater extent in ATRA-treated compared with taxol-treated cells. We used immunoprecipitation experiments to ask if bcl-2 phosphorylated after ATRA or taxol had altered capacity to dimerize with bax. No change in dimerization was demonstrated. We conclude that: bcl-2 is phosphorylated on serine after treatment of AML blasts with ATRA; bcl-2 phosphorylation after ATRA is different from that seen after taxol; bcl-2 phosphorylated after either agent retains capacity to dimerize with bax. The ATRA or taxol-induced phosphorylation of bcl-2 can also be seen in blast cells obtained from AML patients.

Topics: Dimerization; Electrophoresis, Gel, Two-Dimensional; Humans; Immunosorbent Techniques; Leukemia, Myeloid, Acute; Paclitaxel; Phosphoamino Acids; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

To evaluate the prognostic significance of CD7 expression in de novo acute myeloid leukemia (AML), we studied 63 patients with AML who had been admitted to our hospital between September 1989 and January 1996. Even of the patients were later eliminated from the study (9 due to insufficient surface marker analyses, and 2 due to early death). The remaining 52 patients (median age: 42.5 years) were evaluated for morphologic subtype, immunophenotypic classification, complete remission (CR), disease-free survival (DFS) and overall survival (OS). All 52 patients were grouped by the French-American-British classification system: 10 as M1, 16 as M2, 11 as M3, 8 as M4, 5 as M5, and 2 as M6. Ten of the patients expressed CD7 on their leukemia cells (positive rate > or = 25) and were classified as CD7(+)AML, with morphological subtypes as follows: 3 as M1, 6 as M2, and 1 as M3. Thirty-three of the 42 patients with CD7 + AML (78.6%) and 6 of the 10 patients with CD7 + AML (40%) achieved CR. DFS and OS rates for the patients with CD7(+)AML were 22.1% and 35.4%, respectively; those for the CD7(+)AML patients were 53.3% and 44.4%, respectively. No significant differences in gender hematological findings, clinical manifestations such as hepatosplenomegaly, lymphadenopathy, or incidence of central nervous system involvement, CR rate, and DFS distinguished patients with CD7(+)AML from those with CD7(+)AML. These suggest that CD7 expression is unlikely to be a prognostic factor in AML.

Topics: Adolescent; Adult; Aged; Antigens, CD7; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Child, Preschool; Cytarabine; Daunorubicin; Female; Humans; Leukemia, Myeloid, Acute; Middle Aged; Prednisolone; Prognosis; Survival Rate; Thioguanine; Tretinoin

The present study examined differentiation-inducing activity by various tumor-necrosis-factor(TNF) mutants against the human leukemic cell lines HL-60 and U-937. Mutant TNF 471, from which 7 N-terminal amino acids of native TNF were deleted and Pro8, Ser9 and Asp10 were replaced by Arg, Lys and Arg, possessed the highest activity among the TNF mutants, and its activity was 120-fold that of native TNF. The various biological activities of TNF were signaled through 2 distinct receptors, p55 and p75. Although cytotoxicity was reported to involve mainly p55, this differentiation-inducing activity was not well understood. The fact that the affinity of TNF 471 was higher to p55 and lower to p75 than that of native TNF by a binding competition assay suggested that the differentiation-inducing activity was also signaled through p55. To verify this hypothesis, the human myelogenous leukemic cell line, KG-1, which scarcely expresses either receptor and does not differentiate with TNF, was transduced with the p55 or p75 gene. Subsequently p55 transfectants manifested a greater ability to differentiate; however, p75 transfectants did not differ from parental cells or from mock-transfectants. Further, the differentiation of p55 transfectants induced by TNF was reduced by the inhibitor of protein-kinase-C (PKC), staurosporine. These results indicate that the differentiation-inducing activity was signaled through the TNF receptor, p55, via PKC and that the excellent ability of TNF 471 to induce differentiation was related to its high affinity for p55.

Topics: Antigens, CD; Antineoplastic Agents; Cell Differentiation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mutation; Protein Kinase C; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Signal Transduction; Staurosporine; Transduction, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells.

Topics: Cell Differentiation; Cell Division; Gene Expression Regulation, Leukemic; Genes, Homeobox; Granulocyte Colony-Stimulating Factor; HL-60 Cells; Homeodomain Proteins; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Neoplasm Proteins; Neoplastic Stem Cells; Oligonucleotides, Antisense; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

To investigate whether apoptosis occurs in vivo in patients with acute leukemia during induction chemotherapy and its significance.. Apoptotic effect on peripheral blood of 20 patients with different types of acute leukemia prior to and during routine chemotherapy was studied by cell morphology, DNA electrophoresis and FCM-DNA analysis.. After chemotherapy, with falls in WBC and blast count, no apoptotic cell appeared on smears of peripheral blood, nor did ladder pattern of DNA on electrophoresis and subdiploid peak on FCM.. Although anticancer drugs have been shown to induce apoptosis in vitro, circulating apoptotic cells are hardly detected probably due to their rapid clearance from the circulation. It is more difficult to study apoptosis in vivo than in vitro.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Daunorubicin; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prednisone; Tretinoin; Vincristine

Two homologous high-affinity receptors for the chemoattractant interleukin-8, IL-8RA and IL-8RB, and one for the chemoattractant C5a (C5aR) have been cloned. These membrane proteins are members of the rhodopsin superfamily of G-protein coupled seven-transmembrane segment receptors. New monoclonal antibodies (mAb) directed against the deduced N-terminal sequences of the IL-8RA (mAb SE2) and IL-8RB (mAb HC2) were generated to determine the IL-8R expression on human blood leukocytes and two human myeloid cell lines. The C5aR expression was detected using the mAb W17/1. Approximately 107,000 C5aR, 55,000 IL-8RA, and 25,000 IL-8RB molecules per cell could be detected on human granulocytes by flow cytometric analysis. On peripheral blood monocytes, 42,000 C5aR molecules/cell and 3000 IL-8RB molecules/cell were expressed. However, we were unable to quantitate IL-8RA expression, which was detectable but below 2500 molecules per cell and thus outside the standard range for the quantitation of receptor molecules by flow cytometry. On AML-193 cells, only the IL-8RB was constitutively expressed, whereas on HL-60 cells, we could not detect expression of any of the three receptors. Vitamin D3 (250 ng/ml, 7 days), which has been shown to induce differentiation of AML-193 and HL-60 cells into the monocytic phenotype, led to an up-regulation of IL-8RB and C5aR in both cell lines in the absence of any expression of IL-8RA. In contrast, all-trans retinoic acid (0.1 microM, 7 days), which induces differentiation into the granulocytic phenotype, led to an up-regulation of IL-8RB in AML-193 cells and to an expression of IL-8RB and C5aR in HL-60 cells. Again, neither cell line expressed IL-8RA. These findings suggest that regulation of IL-8RA expression differs from that of its IL-8RB homolog and may be a late event in leukocyte maturation.

Topics: Antigens, CD; Cell Differentiation; Chemotactic Factors; Cholecalciferol; Complement C5a; GTP-Binding Proteins; HL-60 Cells; Humans; Interleukin-8; Leukemia, Myeloid, Acute; Leukocytes; Protein Binding; Receptor, Anaphylatoxin C5a; Receptors, Complement; Receptors, Interleukin; Receptors, Interleukin-8A; Tretinoin; Virulence Factors, Bordetella

Topics: Antineoplastic Combined Chemotherapy Protocols; Colony-Stimulating Factors; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Humans; Immunotherapy; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Tretinoin

The acute promyelocytic leukemia cell line, NB4, can be induced to differentiate to mature granulocytes by retinoic acid treatment. A novel retinoic acid-inducible cDNA clone, designated RI58, was isolated from a cDNA library constructed from retinoic acid-treated NB4 cells by differential hybridization. RI58 cDNA encodes a protein of 58kDa which has a similarity in its amino acids sequence to interferon (IFN)-inducible proteins. In addition, RI58 was induced by recombinant human IFN-alpha (rhIFN-alpha) in NB4 cells. RI58 was detectable within 4 hours post-stimulation with rhIFN-alpha, while it took as long as 1day after retinoic acid stimulation. Culture supernatant from retinoic acid-treated NB4 cells also induced RI58 expression similarly as rhIFN-alpha. This activity in culture supernatant was inhibited by anti-leukocyte IFN antiserum which showed specific reactivity to rhIFN-alpha. These results indicate that RI58 is induced by retinoic acid stimulation through autocrinally secreted IFN-alpha from NB4 cells. In the retinoic acid-treated NB4 cells, the expression of RI58 was increased along the process of differentiation. On the other hand, it was expressed constitutively in untreated non-hematopoietic cell lines and mature hematopoietic cell lines.

Topics: Amino Acid Sequence; Autocrine Communication; Base Sequence; Cell Differentiation; Cell Line; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation, Neoplastic; Humans; Interferons; Leukemia, Myeloid, Acute; Molecular Sequence Data; Neoplasm Proteins; Sequence Homology, Amino Acid; Tretinoin; Tumor Cells, Cultured

Using flow cytometry, we have investigated the effects of 0.5 microM all-trans-retinoic acid (ATRA) on bcl-2 expression in the blast cells of 25 acute myeloblastic leukemia (AML) patients and the HL-60 cell line after incubation for 6 days. We observed a significant decrease of bcl-2 expression after treatment with ATRA in 12 of 25 AML samples and the HL-60 cells. The mean fluorescence intensity (MFI) ratio for the bcl-2 levels of the ATRA responders (n = 12) was reduced to 7.9 +/- 4.8 following incubation with ATRA compared with 10.9 +/- 6.5 (mean +/- SD) for control samples incubated without ATRA (p = 0.011). There was no significant difference between the baseline bcl-2 MFI ratio in the ATRA responders (11.14 +/- 7, n = 12) and the non responders (14.18 +/- 11.3, n = 13; p = 0.432). The down-regulation of bcl-2 expression by ATRA was not significantly associated with CD34-negative or -positive AML. There was no correlation between AML subtypes and regulation of bcl-2 expression by ATRA. Complete remission and overall survival were not significantly improved in bcl-2 down-regulated cases. Our data confirm that ATRA can down-regulate the bcl-2 expression in AML blasts. Because many chemotherapeutic agents also operate through the activation of programmed cell death and bcl-2 levels are positively associated with resistance to apoptosis, ATRA can be used in combination chemotherapy to increase the chemosensitivity of some patients with AML.

Topics: Antineoplastic Agents; Down-Regulation; Flow Cytometry; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-bcl-2; Tretinoin

Defensins are microbicidal peptides and the principal constituents of neutrophil primary granules. They are presumed to play a prominent role in innate host defenses. We examined defensin mRNA levels during drug-induced differentiation of the promyelocytic leukemia cell line, HL-60. Transcription was restricted to promyelocyte, myelocyte, and very early metamyelocyte stages of the granulocytic pathway. Complete downregulation occurred during late granulocytic maturation or early during phorbol ester-promoted differentiation along the monocyte/macrophage lineage. Retinoic acid (RA) was the strongest inducer of defensin mRNA accumulation, even at doses too low to effect morphologic changes; the initial (first 48 hours), gradual increase resulted from transcriptional activation and was enhanced by granulocyte colony-stimulating factor. In contrast, addition of hybrid polar compounds led to a transient, drug-specific downregulation within the same time period, apparently by means of selectively induced, biphasic degradation of transcripts. Subsequent increase in transcript levels was faster and more pronounced with hexamethylene bisacetamide, relative to dimethyl sulfoxide (DMSO). DMSO-promoted effects were strikingly different in serum-free medium or in the presence of the tyrosine kinase inhibitor, genistein. Under these conditions, and although differentiation was unaffected, early defensin mRNA downregulation was final. The effect did not occur with RA and expression of other myeloid-specific genes was also unchanged. Addition of selected cytokines caused a similar "dip," only at earlier times and uncoupled from differentiation. Tumor necrosis factor-alpha markedly induced defensin levels after 2 days in previously untreated HL-60 cells, but inhibited expression in RA-differentiated cells. These results begin to detail a complex regulation of defensin mRNA synthesis with both spatial and temporal control elements, and a unique modulation by chemical agents, cytokines, and serum-factors.

Topics: Acetamides; Biomarkers; Blood Proteins; Cell Differentiation; Cycloheximide; Cytokines; Dactinomycin; Defensins; Dimethyl Sulfoxide; Dimethylformamide; DNA, Complementary; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Genistein; Granulocyte Colony-Stimulating Factor; Granulocytes; HL-60 Cells; Humans; Interferon-gamma; Isoflavones; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lymphocytes; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Recombinant Proteins; RNA Processing, Post-Transcriptional; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The blast stem cells of acute myeloblastic leukemia become more sensitive in culture to the chemotherapeutic agents cytosine arabinoside (Ara-C) and daunorubicin (DNR) when exposed to all-trans retinoic acid (ATRA) after drug. We have proposed that down regulation of bcl-2 by ATRA is part of the mechanism of sensitization. The hypothesis is based on reduced expression of bcl-2 mRNA, as seen in Northern blots, after ATRA. Nuclear run on experiments, however, failed to account completely for the effect at the transcriptional level. Accordingly, we looked for post-transcriptional effects of ATRA on bcl-2, using metabolic labelling of the protein to measure stability. We found that the half-life of bcl-2 protein is markedly shortened after treatment with ATRA. Hydrocortisone (HC) protects cells against the toxic effects of Ara-C or DNR when given before drug. HC does not alter bcl-2 expression at the level of mRNA; however, metabolic labelling shows that newly synthesized bcl-2 protein is stabilized in blast cells treated with HC. Response to Ara-C by growth factor responsive blast cells is influenced by the factor in the cultures; cells are more sensitive in cultures with G-CSF and less sensitive when GM-CSF is present. We compared two blast cell lines, OCI/AML-5, primarily responsive to GM-CSF, and OCI/AML-10, primarily responsive to G-CSF. Growth factor did not influence the stability of bcl-2 protein in either line. In contrast, Western blots showed that the amount of bcl-2 protein was greater in cultures with GM-CSF or GM-CSF in combination with G-CSF than in cultures with G-CSF or no added factor. This pattern was seen regardless of the mitogenic response to G-CSF or GM-CSF. We interpret our findings as indicating that bcl-2 protein is transcriptionally activated; that the stability of the protein is decreased after ATRA and increased after HC; that the amount of bcl-2 protein is greater in cultures with GM-CSF than in cultures with G-CSF, regardless of which factor gives the greater mitogenic response. We propose that these post-transcriptional modifications of transcriptionally activated bcl-2 account, in part, for the regulation of drug sensitivity by ATRA, HC and growth factors.

Topics: Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Blotting, Northern; Blotting, Western; Cytarabine; Daunorubicin; Down-Regulation; Gene Expression Regulation, Leukemic; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrocortisone; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Remission Induction; RNA, Messenger; Transcription, Genetic; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Human permanent leukemia cell lines represent powerful research tools in a multitude of investigations. The two new continuous leukemia cell lines MUTZ-2 and MUTZ-3 were derived from the peripheral blood of patients with acute myeloid leukemia (AML) FAB M2 and AML FAB M4. MUTZ-2 and MUTZ-3 cells have morphological and immunophenotypical features of myeloid and monocytic cells, respectively. While MUTZ-2 is negative, MUTZ-3 cells express the monocytic surface marker CD14, albeit weakly. The monocytic nature of MUTZ-3 cells is underlined by the expression of the monocyte-specific esterase (MSE), myeloperoxidase (MPO) and tartrateresistant acid phosphatase (TRAP) enzymes; MUTZ-2 is negative for MSE and TRAP, but expresses MPO. For sustained cell growth, both cell lines require constitutively the addition of cytokines to the culture medium and retain an absolute dependence on conditioned medium or recombinant growth factors for proliferation and survival. Incubation with single recombinant cytokines from a broad spectrum of growth factors established that the strongest proliferation response of MUTZ-2 cells was elicited by FLT-3 ligand, granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), interferon-gamma (IFN-gamma) and stem cell factor (SCF), whereas granulocyte-macrophage CSF (GM-CSF), M-CSF, interleukin-3 (IL-3) and SCF were the most effective growth factors in inducing proliferation of MUTZ-3. Both cell lines were proliferatively responsive to several further cytokines, however, to a lesser extent. Exposure to phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or the physiological all-trans retinoic acid (ATRA) had growth-inhibitory and differentiation-inducing effects on both cell lines. Using a clonogenic cell recovery assay, both cell lines were found to be sensitive to the chemotherapeutic drugs cytosine arabinoside (Ara-C) and daunorubicin (DNR), MUTZ-2 cells being more sensitive to both Ara-C and DNR treatment than MUTZ-3 cells. Chromosomal trisomies 8 and 10 were found in MUTZ-2 cells without any additional structural abnormalities. MUTZ-3 carries the rare, but recurrent AML-associated translocation (12;22)(p13;q11-q12) reflecting the karyotype of the original tumor. The main characteristics of these cell lines remained the same during about 1 year of continuous culture as well as after freezing and thawing. In summary, we established and characterized two new leukemia cell lines with myeloid or monocytic features which ar

Topics: Acid Phosphatase; Adult; Antineoplastic Agents; Base Sequence; Cell Differentiation; Cell Division; Chromosome Aberrations; Cytokines; Esterases; Hematopoietic Cell Growth Factors; Humans; Leukemia, Monocytic, Acute; Leukemia, Myeloid, Acute; Male; Middle Aged; Molecular Sequence Data; Peroxidase; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

HL-60 myeloid leukaemia cells are ineffective as stimulators of allogeneic lymphocytes in mixed leucocyte culture (MLC). These cells can be induced to differentiate along the monocytic or granulocytic pathways with or without acquisition of major histocompatibility complex (MHC) class II antigen by various agents. Surprisingly, treatment of HL-60 cells with 10 nM all-trans retinoic acid (RA) for 7 days (HL-60-R7) resulted in a marked increase in MLC stimulation although the cells lacked detectable MHC class II antigen expression at the initiation of the MLC. In contrast, treatment with interferon-gamma (IFN-gamma), with or without RA, induced MHC class II antigen expression but failed to enhance MLC stimulation. Lymphocytes responding to HL-60-R7 were predominantly CD8+ and/or CD16+ and displayed enhanced cytolytic capacity for HL-60 and HL-60-R7 cells as well as natural killer (NK)-sensitive K562 cells. Nevertheless, monoclonal antibodies (mAb) to MHC class II antigens substantially inhibited the MLC and some CD4+ lymphocytes in the responding population were required, although this requirement could be replaced by the addition of interleukin-2 (IL-2). HL-60-R7 (and HL-60) cells were shown to acquire detectable MHC class II antigen expression during the first 3 days of the MLC. Thus a low level of activation by MHC class II+ stimulator cells appears to be required for the response. Analysis of the role of cytokines with costimulatory activity for T cells and/or NK cells indicated that tumour necrosis factor-alpha (TNF-alpha) was important in the proliferative response, while interleukins-1, -6 and -12 and stem cell factor did not seem to be involved. Cell interaction molecules lymphocyte function-associated antigen-1 (LFA-1) (CD11a), intracellular adhesion molecule-1 (ICAM-1) (CD54), ICAM-3 (CD50) and B7.2 (CD86) were up-regulated on HL-60-R7. Blocking mAb to LFA-1 and B7.2 potently inhibited the proliferative response indicating a key role for these molecules in the enhanced immunostimulation by HL-60-R7 cells. The results may have implications for the mechanism of the therapeutic effect of RA in acute promyelocytic leukaemia and may also provide valuable information in regard to the immunogenicity of tumour cells in general.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Division; Cell Survival; Cytokines; Cytotoxicity, Immunologic; HL-60 Cells; HLA-D Antigens; Humans; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Tretinoin

High levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia (AML) cells have been associated with low complete remission rates and poor survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased by the down-regulation of bcl-2 expression by antisense oligonucleotides. All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic cell line, with both effects being accompanied by a decrease in bcl-2 expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have investigated the effects of ATRA (1 microM) on bcl-2 expression in the blast cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h, respectively. Using Kolmogorov-Smirnov statistical analysis where a D value of > 0.12 was statistically significant, we found that in 8/25 AML samples and the K562 cells there was a significant decrease in bcl-2 protein expression after incubation with ATRA (D value range 0.14-0.44). The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA responders (n = 8) was reduced to 35.5 +/- 6.9 following incubation with ATRA compared to 47.6 +/- 8.2 (mean +/- SEM) for control samples incubated in the absence of ATRA (P = 0.014). There was no significant difference between the baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the ATRA responders (48.9 +/- 5.7, n = 8) and the non-responders (41.3 +/- 3.9, n = 17) (mean +/- SEM) (P = 0.28). The down-regulation of bcl-2 expression by ATRA was particularly associated with CD34-negative AML and of the eight AML patients' cells that responded to ATRA by down-regulating bcl-2, seven were CD34 negative (P < 0.05). Our data suggest that the addition of ATRA to combination chemotherapy would increase the chemosensitivity of some patients with AML, particularly CD34-negative AML, due to down-regulation of bcl-2 expression.

Topics: Antigens, CD34; Down-Regulation; Genes, bcl-2; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Tretinoin

Paclitaxel dose responses in culture have been investigated alone and in association with cytosine arabinoside (ARA-C) and all-trans retinoic acid (ATRA), with the objective of identifying a role for paclitaxel in the treatment of acute myeloblastic leukaemia (AML). Initial studies were done to determine if paclitaxel dose responses of AML blast cell precursors were altered by regulatory compounds known to modify the dose responses of ARA-C. In contrast to ARA-C, paclitaxel dose responses were independent of cell culture method, the growth factors G-CSF and GM-CSF, and the ligands all-trans retinoic acid (ATRA) and hydrocortisone. Most blast cell populations were sensitive to paclitaxel; compared with normal marrow progenitors the dose responses were markedly heterogenous with some more, and others less, sensitive. Remission marrow progenitor paclitaxel responses resembled those of AML blasts in heterogeneity. The cell culture model tested the effect of pacliataxel and ATRA on the ARA-C dose responses of OCI/ AML-5; paclitaxel exposure was either before or after ARA-C to test for an effect of schedule; ATRA was added to the MEC cultures after paclitaxel and ARA-C. Repeat experiments were done to test three dose levels each of paclitaxel and ATRA. When paclitaxel was given after ARA-C, synergism was found for all but one of the dose combinations tested; only three examples of synergy were seen when paclitaxel preceded ARA-C. The studies justify trials combining ARA-C, paclitaxel and ATRA using a schedule suggested by the cell culture findings.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Cytarabine; Dose-Response Relationship, Drug; Drug Synergism; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Paclitaxel; Tretinoin; Tumor Cells, Cultured

Leukemic cells spontaneously secrete cytokines involved in the proliferation of the clone; in this study we evaluated the effects of all-trans retinoic acid (ATRA) on the in vitro autocrine production of cytokines by acute myeloid leukemia cells. Thirty acute nonlymphoid leukemia cases (ANLL) (10 APL and 20 ANLL of other cytotypes than APL) were studied; the in vitro secretions of IL-1 alpha, IL-3, IL-4, IL-6, IL-10, G-CSF, GM-CSF, TNF-alpha were tested with and without ATRA addition. After 5 d exposure to ATRA 10(-6) M APL-treated samples showed a significant reduction of IL-6 (p = 0.008) and GM-CSF (p = 0.03) and a significant increase of IL-1 alpha (p = 0.01) production, if compared to untreated APL samples. No difference was seen in IL-3, IL-10 and IL-4 productions; G-CSF production resulted absent in all but 3 APL cases, in which addition of ATRA determined increase in the production. Interestingly, the 3 G-CSF-producing cases did not obtain clinical remission with ATRA; GM-CSF and IL-6 were spontaneously produced by all the cases, and 7 of 10 APL patients subsequently obtained complete remission after induction. TNF-alpha was produced only in 1 case. No statistical difference was seen in all the productions obtained from other than promyelocytic acute leukemic cells, both with and without ATRA addition. However, it is noteworthy that the production of IL-6 was more than twice as high in ANLL non-APL than in APL cases. In conclusion, these data could thus suggest possible complementary mechanisms of the exhaustion of the leukemic clone upon treatment with ATRA.

Topics: Bone Marrow; Cell Differentiation; Cytokines; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Prognosis; Tretinoin; Tumor Cells, Cultured

Retinoic acid and hydrocortisone (HC) have been shown to regulate the drug sensitivity of the blast cells of acute myeloblastic leukemia (AML). We asked if the proto-oncogene bcl-2 played a role in this regulation. As target cells we used the continuous lines, OCI/AML-1, OCI/AML-2 or OCI/AML-5; expression of bcl-2 can be detected by Northern analysis of RNA from OCI/AML-2 or OCI/AML-5 cells; bcl-2 expression can be found in OCI/AML-1 cells only by using RT-PCR. Exposure of OCI/AML-2 or OCI/AML-5 cells to retinoic acid (all-trans retinoic acid, ATRA) led to a down-regulation of bcl-2 expression that was first seen after 2 h of exposure and was complete after a day. The down-regulation could be prevented by exposing the cells to ara-C either before or after ATRA; decrease in bcl-2 protein was moderate and only obvious after 36 h of ATRA treatment. Nuclear run-on experiments provided evidence that bcl-2 down-regulation was occurring at transcriptional and post-translational levels. Since bcl-2 is considered to have anti-oxidant activity, we tested the sensitivity of the three cell lines to H2O2; we found that OCI/AML-1, the line with very low bcl-2 expression, was a 100-fold more H2O2-sensitive than OCI/AML-2 or OCI/AML-5, where bcl-2 expression can be detected readily. We then asked if H2O2 sensitivity could be regulated. We found that exposure of cells to HC before H2O2 was protective while ATRA after peroxide treatment increased killing; this is the same pattern of regulation observed when AML blasts are exposed to HC before, or ATRA after ara-C. Finally, we asked whether N-acetylcysteine (NAC), a known radical scavenger would protect cells against ara-C killing. Significant protection was observed when NAC was given before drug, but not if given after drug. NAC protection against ara-C killing was seen for OCI/AML-1 and 2 cells, but not for OCI/AML-5 cells. We interpret the results as follows: ara-C kills cells in two ways: first, directly, by incorporation into DNA and chain termination; second, indirectly, by inducing the production of toxic radicals. Bcl-2 reduces the oxidant activity of such radicals, and is protective. ATRA regulates ara-C toxicity by its action on bcl-2. Left unexplained are the action of HC, which does not affect bcl-2 expression and the mechanism by which ara-C prevents down-regulation of bcl-2 by ATRA.

Topics: Acetylcysteine; Antioxidants; Base Sequence; Blotting, Northern; Cytarabine; Down-Regulation; Drug Interactions; Drug Screening Assays, Antitumor; Free Radicals; Gene Expression Regulation, Leukemic; Humans; Hydrocortisone; Hydrogen Peroxide; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphocytes; Molecular Sequence Data; Polymerase Chain Reaction; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

A 67-year-old man presented with acute myelogenous leukemia (M2). Peripheral blood examination revealed a leukocyte count of 1,700/mu l with 1% myeloblasts, and bone marrow aspiration showed 42.6% myeloblasts with Auer bodies. Culture of his marrow cells at diagnosis showed that granulocyte colony-stimulating factor (G-CSF) promoted cell proliferation, while all-trans retinoic acid (ATRA) inhibited the proliferative effect of G-CSF and induced differentiation. Combination therapy with G-CSF, ATRA, and low-dose cytotoxic drugs achieved complete remission without severe marrow suppression.

Topics: Aged; Antineoplastic Agents; Arabinonucleotides; Bone Marrow; Cyclophosphamide; Cytidine Monophosphate; Drug Therapy, Combination; Granulocyte Colony-Stimulating Factor; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Male; Tretinoin

In this study, we evaluated the in vitro growth of normal hematopoietic progenitors (CFU-GM, BFU-E, CFU-GEMM, CFU-meg) stimulated by optimal sources of colony stimulating activity in the absence or presence of 10(-6) M all-trans retinoic acid (ATRA). ATRA alone did not show any colony-stimulating ability when added in culture to partially purified bone marrow populations. On the other hand, it significantly increased the number of CFU-GM (p = 0.003) and both the number (p = 0.009) and size (p = 0.002) of CFU-meg in the presence of appropriate colony-stimulating activity. Since ATRA had only modest stimulatory effects on purified CD34+ cells, the megakaryocyte colony-stimulating activity of ATRA was mainly due to an increased production of endogenous cytokines by bone marrow accessory cells. In parallel experiments, the in vitro growth of the different hematopoietic progenitors was evaluated in 28 patients affected by acute non-lymphoid leukemia (ANLL), mainly acute promyelocytic leukemia (APL). Bone marrow cells were harvested after remission induction obtained: (i) in ten APL patients treated with ATRA followed by one chemotherapy cycle (CHT) (3/7: Daunorubicin+Ara-C): group A ('ATRA/CHT'); (ii) eight APL patients treated with one CHT cycle alone (3/7 as above): group B ('APL-CHT'); (iii) in ten ANLL-non-APL patients after one CHT cycle (3/7 as above): group C ('ANLL-CHT'). The number of the different hematopoietic progenitors, and in particular CFU-GM and CFU-meg, was significantly higher in APL patients treated with ATRA plus CHT (group A) compared to APL (group B) or ANLL-non-APL (group C) patients treated with CHT alone (CFU-GM: p = 0.01; CFU-meg: p = 0.03). Our data demonstrate that ATRA is able to potentiate both normal and APL megakaryocytopoiesis and suggest that the in vivo administration of ATRA could be beneficial in other pathological conditions, where the megakaryocyte progenitor cell compartment is impaired.

Topics: Antigens, CD; Antigens, CD34; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Bone Marrow Cells; Colony-Forming Units Assay; Cytarabine; Daunorubicin; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Megakaryocytes; Tretinoin

Damage to DNA can be assessed using a technique for labeling nicks in DNA by incubating paraformaldehyde-fixed cells in a mixture of biotin-labeled dUTP, dATP with dNTP and DNA polymerase I. The addition of labeled nucleotides can then be identified by fluorescence by their reaction with streptavidin. We have used this method to examine damage to the DNA of OCI/AML-2 cells caused by cytosine arabinoside (ara-C) and the effects of hydrocortisone and retinoic acid on this damage (regulated drug sensitivity). Concurrent measurements of clonogenic cells were used to allow a comparison of damage as shown by labeled nicks in DNA with loss of colony-forming capacity. Both methods gave comparable ara-C dose-response curves, for cells incubated with the drug for 24 h. Both methods showed that exposure of OCI/AML-2 cells to hydrocortisone before ara-C greatly reduced the toxicity of the drug; and that retinoic acid given after ara-C increased both its lethal effects on colony formation and the extent of DNA damage as assessed by labeled nicks. Clonogenic assays required for colony formation are not readily adapted to the study of development and repair of damage. The labeled nick assay is suitable for such kinetic studies. OCI/AML-2 cells were exposed in suspension to either hydrocortisone before ara-C or retinoic acid after ara-C. At 24 h intervals thereafter, cells were harvested, assayed by both methods, and recultured after dilution to the original cell concentration. In cultures exposed only to ara-C (controls), the number of cells with labeled nicks increased during the first 24 h and cells with damaged DNA could be detected for 48-72 h, depending on the ara-C dose in spite of the dilution at each passage. OCI/AML-2 cells exposed to hydrocortisone before drug showed fewer nick-labeled cells than controls at the first observation and damaged cells rapidly disappeared from the population with increasing time. For cells treated with retinoic acid after ara-C, the nick-labeled cell population was greater than controls and remained greater throughout subsequent observations. We propose that in the control cultures, sublethal damage either became lethal with time and was seen as increased numbers of cells with damaged DNA, or alternatively, sublethal damage was repaired. From this point of view we consider that hydrocortisone promotes repair of sublethal damage while retinoic acid inhibits repair.

Topics: Cytarabine; DNA Damage; DNA Repair; DNA, Neoplasm; Drug Screening Assays, Antitumor; Flow Cytometry; Fluorescence; Genetic Techniques; Humans; Hydrocortisone; Kinetics; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

The experiments reported here continue the study of regulated drug sensitivity by extending the observations to anthracyclines. Previous work has shown that hydrocortisone (HC) protects AML blast stem cells from the lethal effects of cytosine arabinoside (ara-C) while retinoic acid (ATRA) increases ara-C sensitivity; further mechanisms of regulation of ara-C sensitivity might include increase or decrease in repair of sublethal damage. Anthracycline dose-response curves are characterized by an initial shoulder, followed by exponential decrease in survival with increasing dose. The shoulder portion of such curves may indicate the accumulation of sublethal damage. We used two assays to look for evidence of regulation of anthracycline sensitivity by HC or ATRA; the clonogenic assay for blast stem cells detects drug effects on this crucial population, but only after several days on incubation, during which time repair might occur. Measurements of nicks in DNA show damage in the bulk population of cells, but these can be detected very soon after exposure to drug. Both methods showed the HC protected cells in two continuous cell lines (OCI/AML-2 and OCI/AML-5) while ATRA made the cells more sensitive. Blast cells freshly-obtained from six AML patients were also tested. Both assays showed HC protection and ATRA sensitization in three populations. The clonogenic assay detected both effects in cells from a fourth patient; the nicked DNA assay confirmed both effects in a fifth patient, where the results of the clonogenic assay did not reach statistical significance. Neither ATRA nor HC influenced the sensitivity of blasts from a sixth patient; but these cells were highly resistant to drug. Kinetic studies showed that damage persisted longer after treatment with anthracyclines than with ara-C. OCI/AML-2 cells treated with HC before drug accumulated fewer cells with nicked DNA after daunorubicin (DNR). Cells exposed to ATRA after DNR showed increased toxicity in kinetic experiments. We conclude that sensitivity to anthracyclines may be regulated by ligands for steroid receptors. Furthermore, since growth factors do not regulate anthracyclines' sensitivity, different mechanisms may be operative for the action of ligands for cell surface receptors. Finally, we suggest that retinoic acid might be considered for inclusion in standard anthracycline/ara-C regimens for the treatment of AML.

Topics: Adult; Aged; Cell Survival; Cytarabine; Daunorubicin; DNA Damage; DNA Repair; DNA, Neoplasm; Dose-Response Relationship, Drug; Female; Genetic Techniques; Humans; Hydrocortisone; Kinetics; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

Bone marrow transplantation procedure has emerged as an effective treatment for hematological malignancies. However, recurrence of leukemia is still the major cause of treatment failure. Subsequent treatment in this category of patients, generally considered incurable, has not been yet standardized. At our institution, 13 patients, 7 with acute non lymphoid leukemia (ANLL) and 6 with acute lymphoid leukemia (ALL), were treated at relapse after bone marrow transplantation either autologous or allogeneic (AuBMT 8, ABMT 4) performed in complete remission (CR). The interval between BMT and relapse was less than 9 months in 6 patients (2 ABMT and 4 AuBMT) and more than 9 months in 7 patients. Early relapsed patients showed no response to treatment and died at a median of 5.5 months (range 1-13) after relapse. Late relapse after BMT was characterized by a high percentage of response (5 CR and 1 PR), particularly after intensive chemotherapy and by a longer survival (median 14 months; range 2-36). Chemotherapy after transplantation should be carefully evaluated in patients relapsed after BMT in order to select a population that can achieve long term disease free survival.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Cytarabine; Female; Humans; Idarubicin; Italy; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Male; Middle Aged; Neoplasm, Residual; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prednisone; Remission Induction; Retrospective Studies; Salvage Therapy; Survival Analysis; Treatment Outcome; Tretinoin; Vincristine

Topics: Adult; Humans; Leukemia, Myeloid, Acute; Male; Myositis; Tretinoin

A patient is described with undifferentiated acute myeloblastic leukemia refractory to two courses of daunorubicin and cytosine arabinoside. Because some the myeloblasts developed morphologic features of promyelocytes, the patient was treated with all-trans-retinoic acid (ATRA) in an attempt to promote maturation. Cytogenetic studies and sensitive molecular analysis did not reveal any abnormality classically associated with acute promyelocytic leukemia. Serial bone marrow biopsies demonstrated myeloid maturation, and the patient uneventfully went into a sustained complete remission. A review of the literature confirms this to be an apparently hitherto undescribed response to ATRA that may have therapeutic implications in similar patients.

Topics: Aged; Base Sequence; Biopsy; Bone Marrow; Cytarabine; Daunorubicin; Female; Humans; Leukemia, Myeloid, Acute; Molecular Sequence Data; Remission Induction; Tretinoin

Topics: Adult; Cell Differentiation; Drug Combinations; Humans; Interferon-alpha; Leukemia, Myeloid, Acute; Male; Tretinoin

The effects of retinoic acid (RA) on the proliferation of acute myeloblastic leukemia (AML) cells were studied. AML samples were divided into three groups. Namely, RA stimulated blast colony formation by AML samples in group A and inhibited that by the samples in group B, regardless of added growth factors. For the samples in group C, RA inhibited the colonies formed by granulocyte colony-stimulating factor (G-CSF) but stimulated those by granulocyte macrophage CSF (GM-CSF). To investigate the mechanism involved, the effects of RA on growth factor receptors on AML cells were examined by flow cytometry using fluorolabeled ligands. For the samples in groups A and B, RA affected neither G-CSF receptor (GR) nor GM-CSF receptor (GMR). For the samples in group C, exposure to 10(-7) M RA for 1 day clearly increased GMR, but did not affect GR. This finding supports the hypothesis that the increase of GMR is one of the causes of the stimulative effects of RA on cells cultured with GM-CSF in group C.

Topics: Cell Division; Gene Expression; Humans; Leukemia, Myeloid, Acute; Receptors, Granulocyte Colony-Stimulating Factor; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; RNA, Messenger; RNA, Neoplasm; Tretinoin

It has been shown previously that multilineage human hematopoiesis is maintained within human fetal bone marrow (BM) fragments implanted into severe combined immunodeficient (SCID) mice. We describe here an application of this animal model, the SCID-hu mouse, to the study of human myeloid leukemias. BM cells from 8 patients with various types of myeloid leukemias were injected directly into human bone grafts in the SCID-hu mouse. Cells from 7 patients grew in the human marrow without spreading to the mouse marrow. Cells from 6 of these patients were successfully transferred in vivo to secondary SCID-hu recipients. The surface phenotype and the cytologic features of the leukemia cells were conserved during passage in vivo. Thus, human myeloid leukemia cells could be reproducibly propagated in the human marrow environment in SCID-hu mice. The differentiation of promyelocytic leukemia cells in the SCID-hu mice was induced by all-trans retinoic acid, suggesting that the biologic features of the leukemia cells were maintained as well. Finally, evidence for a leukemic progenitor cell population in one case of acute myelogenous leukemia was provided with this system. This model may provide a useful tool for studying the biology of human myeloid leukemia as well as for evaluating new therapeutic modalities for myeloid leukemias.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Bone Marrow; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; Lewis X Antigen; Mice; Mice, SCID; Neoplasm Transplantation; Sialic Acid Binding Ig-like Lectin 3; Transplantation, Heterologous; Tretinoin

50 cases were treated with Myelodysplastic Syndrome (MDS) by combined TCM-WM therapy. They were classified into RA 17 cases, RAS 6, RAEB 19, CMML 1 and RAEBT 7. The patients were divided into two groups, one with RA and RAS receiving treatment of hemopoietic and immune drugs plus Chinese medicinal herbs, the other with RAEB, CMML and RAEBT receiving treatment of LD Ara-c and LD Hom chemotherapy plus medicinal herbs. The effective rates were 47.83% and 62.96% respectively, the total effective rate being 56%. 6 cases (RAEB 4, RA 1, RAS 1) were treated with all-trans retinoic acid used as an inducer of differentiation, 2 of them were effective. 11 patients with MDS who had transformed into acute leukemia were treated by LD Ara-c and combined TCM-WM chemotherapy, the remission rate was 54.55% and the survival period was 9-27 months after remission. In some cases low dose chemotherapy resulted in hemocytopenia, bone marrow inhibition, infection, mild nausea and anorexia.

Topics: Adolescent; Adult; Aged; Cell Transformation, Neoplastic; Cytarabine; Drugs, Chinese Herbal; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Pyridoxine; Stanozolol; Tretinoin

Retinoic acid exhibits effects on the proliferation and differentiation of many hematopoietic cells. Cellular responsiveness to retinoic acid (RA) is conferred through two distinct classes of nuclear receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs). The RARs bind to both 9-cis- and all-trans-RAs, but 9-cis-RA alone directly binds and activates RXR. This suggested that 9-cis-RA could have expanded hematopoietic activities as compared with all-trans-RA. We compared the abilities of 9-cis- and all-trans-RAs to induce differentiation and inhibit proliferation of three acute myelogenous leukemia (AML) cell lines and fresh leukemic cells from 28 patients and found that: (1) 9-cis-RA in general was more potent than all-trans-RA in suppressing the clonal growth of two AML cell lines and 17 AML samples from patients, including four from individuals with acute promyelocytic leukemia (APL). Eleven leukemic samples, including three from patients with chronic myelogenous or chronic myelomonocytic leukemia, were relatively refractory to both retinoids. (2) The range of activities of both retinoids was similar except that the clonal growth of samples from three AML patients were inhibited by 9-cis-RA, but not by all-trans-RA. (3) Both retinoids inhibited the clonal proliferation of leukemia cells without necessarily inducing their differentiation; in fact, the only fresh AML cells that were able to undergo differentiation were from patients with APL and one individual with M2 AML. (4) Both retinoids enhanced myeloid and erythroid clonal growth from normal individuals, and 9-cis-RA showed slightly more stimulation of the myeloid clonal growth than did the all-trans-RA. Our study suggests that 9-cis-RA is worthy of further study for the treatment of selected individuals with AML.

Topics: Cell Differentiation; Cell Division; Erythroid Precursor Cells; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

Thirteen refractory/resistant AML patients no suitable for additional aggressive chemotherapy, were treated with a combination including all-trans retinoic acid (45 mg/m2 sine die) and low doses of Ara-C (20 mg/m2 subcutaneously, twice in a day, days 1-10, every 28 days). Ten patients were evaluable; 8 of them achieved a complete remission, two patients with an important tumor burden, failed to achieve a response. One complete remission patient relapsed after 7 months but is still receiving the same therapy and is now in partial remission. We believe this combination effective as inducer of complete remission in those AML patients which cannot tolerate additional heavy treatments. The role of tumor burden in affecting response to therapy remains to be still evaluated.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Cytarabine; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Remission Induction; Tretinoin

The effects of recombinant human G-CSF (rhG-CSF) and retinoic acid (RA) were studied on the proliferation and differentiation of HL-60 cells and human acute myeloid leukemic cells. Synergistic effect on granulocyte differentiation was observed when HL-60 cells and primary acute promyelocytic leukemic cells were cocultured with RA plus rhG-CSF. rhG-CSF combined with RA increased more significantly the percentage of mature cells than RA alone and greatly increased NBT reduction activity (P < 0.001). These results suggested that proliferated effect of rhG-CSF on leukemic cells may be important for inducing differentiation of myeloid leukemic cells. But this effect might expose the patients to the risk of acute myeloblastic leukemia if G-CSF was used alone. However, RA could not only rule out the latter situation but retain former merit as well. The authors suggest that the combined use of G-CSF with RA is probably a new approach to the treatment of leukemia.

Topics: Cell Differentiation; Drug Synergism; Granulocyte Colony-Stimulating Factor; Granulocytes; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

To study the regulation of expression of the myc protooncogene, cells from normal individuals and patients with acute myelogenous leukemia (AML), and chronic phase and blastic crisis of chronic myeloid leukemia (CML) cells were put in overnight culture in the presence or absence of fetal calf serum. Myc expression in normal marrow cells and chronic phase CML cells fell after culture in vitro. In contrast, myc expression was maintained or increased in a majority of the AML and blastic crisis CML specimens. These data demonstrate that the regulation of myc expression is disordered in many AML and blastic crisis specimens but not in chronic phase CML cells.

Topics: Base Sequence; Blast Crisis; Blotting, Southern; Bone Marrow Cells; Cell Count; Gene Expression Regulation, Leukemic; Genes, myc; Genes, ras; Humans; Interferon alpha-2; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Molecular Sequence Data; Mutation; Recombinant Proteins; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Combined Modality Therapy; Granulocyte Colony-Stimulating Factor; Humans; Immunologic Factors; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Middle Aged; Remission Induction; Tretinoin

Ten patients with active acute myelogenous leukemia (AML) received either 13 cis retinoic acid (RA) + alpha interferon (IFN) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 3 days. Cell cycle measurements were performed before and at the conclusion of administration of the bioactive agent(s). The proliferative rate of the leukemia cells in vivo decreased in four of five patients receiving RA+IFN whereas in one patient proliferation accelerated. The proliferative rate of AML cells accelerated in three of the five patients who received rhGM-CSF and slowed in two patients. These data show that while the proliferative rate of AML cells can be altered in vivo, the effect produced by bioactive agents may be the opposite of the desired effect. Furthermore, the studies described here demonstrate the usefulness of marrow biopsies for measuring the percent S-phase cells and the importance of measuring the duration of S phase so that the effects of bioactive agents on the cell cycle time of the leukemia cells can be determined.

Topics: Biopsy; Bone Marrow; Cell Cycle; Cell Division; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-alpha; Kinetics; Leukemia, Myeloid, Acute; Tretinoin

Topics: Aged; Female; Humans; Leukemia, Myeloid, Acute; Multiple Organ Failure; Prednisone; Respiratory Distress Syndrome; Tretinoin

Topics: Animals; Bone Marrow; Cell Differentiation; Cell Division; Growth Inhibitors; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Leukemia, Experimental; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mice; Tretinoin

The myelodysplastic syndrome (MDPS) provides an opportunity for identifying host factors (genetic, endocrine, immune) involved in initiation and progression of preleukemia into frank acute myeloid leukemia. The aim of this study was to identify bone marrow (BM) cellular and humoral dysfunctions central to the development of MDPS and useful in therapeutic follow-up studies. Our preclinical studies have shown that (1) the characteristic stromal cell composition of the normal BM microenvironment was impaired in MDPS and in AML in 67 and 86% of the cases, respectively; (2) the 1 alpha,25(OH)2D3 concentration in BM plasma was abnormal in 50% of MDPS and 30% of AML; and (3) an inverse correlation existed in MDPS between the 1 alpha,25(OH)2D3 concentration and the frequency of F-CFU, (r = 0.41, p < 0.02), suggestive of a regulatory interaction between this secosteroid hormone and BM stromal cells. The analysis of clonal extinction of BM blast cells in response to all trans retinoic acid (RA), 1 alpha,25(OH)2D3, and colony stimulating factors (PHA-LCM), either alone or in various combinations, revealed individual patterns of responses in the cases of MDPS or AML. The results indicate the necessity for preclinical studies to select patients for combined differentiation therapy. Our ongoing clinical trials suggest that RA (Roaccutan, 20 mg/day continuously) as induction therapy, followed at weeks 6 to 8 by prednisone (40 mg/day for 15 days) and 1 alpha,25(OH)2D3 (Rocaltrol, 3 x 0.25 micrograms/day for 3 months) may induce a long-lasting hematological remission in MDPS.

Topics: Bone Marrow; Calcitriol; Cell Differentiation; Cells, Cultured; Drug Therapy, Combination; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Prednisone; Tretinoin

The uptake of colloidal gold particles by human monocytes was studied by electron microscopy, with special emphasis on changes in this uptake during the differentiation and maturation of these cells. The way in which leukemic cells of childhood acute non-lymphocytic leukemia (ANLL) can function in this gold uptake was also examined. In monocytes, microendocytosis was temperature-dependent; colloidal gold uptake increased as temperatures rose from 4 degrees C to 37 degrees C. It appeared that gold particles first adhered to the cell surface membrane, were then incorporated into the cytoplasmic vesicles, and then were transported into the granules. Original HL-60 cells and retinoic acid (RA)-treated HL-60 cells, which were differentiating and maturing along the granulocyte lineage, did not ingest colloidal gold particles, but 1,25(OH)2D3-treated HL-60 cells showed colloidal gold uptake during their differentiation and maturation along the monocyte lineage: 68.6% of the cells contained gold particles. Gold uptake was demonstrated in 27.3% of original U937 cells; the percentage increased to 70.3% when they were induced to mature by RA. In 15 specimens of childhood ANLL, none of the M1, M2 or M3 cells showed colloidal gold uptake, whereas 76-97% of M4 and M5 cells showed this uptake. These findings indicate that colloidal gold uptake is a marker of monocyte differentiation and maturation and can provide additional information for ANLL cytology.

Topics: Adolescent; Biomarkers; Calcitriol; Cell Differentiation; Child; Child, Preschool; Endocytosis; Female; Gold; Humans; Infant; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; Lymphoma, Large B-Cell, Diffuse; Male; Microscopy, Electron; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

Recent work has shown that acute promyelocytic leukemia (APL) cells have a characteristic translocation involving the retinoic acid receptor on chromosome 17 and the myl protein on chromosome 15. Patients with APL respond to the administration of all-trans-retinoic acid. A cell line with t15;17 (NB4) has recently been reported; this line responds to all-trans-retinoic acid with differentiation. There is also a recent report showing that all-trans-retinoic acid is more active than cis-retinoic acid in inducing differentiation in freshly obtained APL cells. All-trans-retinoic and cis-retinoic acid are compared for their effects on growth in culture of freshly obtained AML cells, cell lines without t15;17, and NB4 cells. While all of these AML populations responded to both forms of retinoic acid, NB4 cells only were much more sensitive to all-trans-retinoic acid compared to cis-retinoic acid. The difference was seen when the NB4 cells were exposed in suspension and not when colony-formation in methylcellulose was used as an end point. Both forms of retinoic acid increased the sensitivity of blast cells to cytosine arabinoside; for NB4 cells, the sensitization was much greater when all-trans-retinoic acid was used rather than cis-retinoic acid. We conclude that the increased effects of all-trans-retinoic acid are specific for APL cells, and that a major effect of retinoic acid is on blast stem cell self-renewal.

Topics: Cell Survival; Cytarabine; Drug Administration Schedule; In Vitro Techniques; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Stereoisomerism; Tretinoin; Tumor Cells, Cultured

This paper describes the use of the bromodeoxyuridine/propidium iodide method to assess the effects of bioactive and cytotoxic agents on the kinetic characteristics of acute myelogenous leukemia cells. By careful selection of gates, the following parameters can be measured simultaneously using only 50,000 cells: the proportion of cells in S-phase, the distribution of cells within the S-phase compartment, the relative rate of DNA synthesis, the relative distribution of S-phase times, the proportion of S0 cells, and the proportion of cells in G1 and G2/M. This method was used to demonstrate that while retinoic acid, alpha-interferon, and cytosine arabinoside may all "inhibit" DNA synthesis, the actual effects of these agents differ. Retinoic acid appears to arrest cells in G1 without affecting the rate of DNA synthesis, while alpha-interferon and cytosine arabinoside "inhibit" DNA synthesis by reducing the rate of synthesis per se.

Topics: Animals; Bone Marrow; Bromodeoxyuridine; Cell Cycle; Cell Nucleus; Cell Survival; Cells, Cultured; Chickens; Cytarabine; DNA Replication; DNA, Neoplasm; G1 Phase; G2 Phase; Interferon alpha-2; Interferon-alpha; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Mitosis; Propidium; Recombinant Proteins; S Phase; Tretinoin; Tumor Cells, Cultured

Although mRNA for the retinoic acid receptor alpha (RAR-alpha) is expressed in many different myeloid leukemias, most of these leukemia cells exhibit little if any phenotypic response when exposed to retinoic acid (RA). To determine whether such RA resistance is related to altered RA receptor structure or function, we performed a detailed analysis of nuclear RA receptors in RA-resistant K-562 cells. These cells exhibit RA receptors of the same approximate molecular weight and similar kd as those exhibited by the RA-sensitive HL-60 leukemia cell line, but the number of RA receptors in the RA-resistant K-562 cells (80 per cell) is significantly lower than that exhibited by RA-sensitive HL-60 cells (550 per cell). Retroviral-mediated transduction of RAR-alpha cDNA into K-562 significantly increased the number of RA receptors to 2,000 per cell. These RAR-alpha-transduced K-562 cells, when incubated with RA, exhibit diminished cell proliferation associated with decreased c-myc expression and an accumulation of cells in G0/G1. In addition, these RA-treated cells exhibit downregulation of the CD15 surface antigen and a slight increase in hemoglobin production but manifest no other evidence of significant erythroid, megakaryocytic, or myeloid differentiation. These results indicate that an elevated number of nuclear RA receptors can be involved in altering proliferation but not necessarily the differentiation of certain RA-treated myeloid leukemia cells.

Topics: Antigens, CD; Antigens, Differentiation; Blotting, Northern; Carrier Proteins; Cell Differentiation; Cell Division; Cell Nucleus; DNA, Neoplasm; Gene Expression; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Lewis X Antigen; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins c-bcr; Proto-Oncogene Proteins c-myc; Receptors, Retinoic Acid; Transfection; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid, hexamethylene bisacetamide and 5-azacytidine are inducers of granulocytic differentiation of HL-60 human myeloid leukaemic cells, which eventually leads to inhibition of cell proliferation. The effect of graded concentrations of all-trans-retinoic acid (RA) (1 nM-1 microM), hexamethylene bisacetamide (HMBA) (0.5-4 mM) and/or 5-azacytidine (5azaC) (1 nM-1 mM), alone and in combination with each other on colony formation and growth of HL-60 cells was studied in agar capillary clonogenic micro assays in order to identify new potential therapeutic regimens for elderly patients with acute myeloid leukaemia. ED90 concentrations, inducing 90% inhibition of colony formation for RA, HMBA and 5azaC, were 128 nM, 2.7 mM and 40 microM, respectively. The drug interactions between these differentiating agents were analysed by Berenbaum's general algebraic solution. The combinations: RA + HMBA, 5azaC + HMBA and RA + 5azaC were significantly synergistic in inhibiting HL-60 colony formation. Their interaction indices were 0.62, 0.83, and 0.97, respectively, at a specific effect level of 15%. The addition of 1 mM HMBA to 100 nM 5azaC- and 1 nM RA-treated cultures significantly increased the colony-formation inhibition from only 2.6% and 7.0% to 46.4%, and 43.1%, respectively. Also, HMBA showed marked synergism with RA and 5azaC in inhibiting colony growth. The interaction indices (I) of HMBA + RA and HMBA + 5azaC were 0.013 and 0.009, respectively, at the same specific level of 15%. Moreover, the triple combination of RA + HMBA + 5azaC showed synergism in inhibiting both the colony formation (I = 0.7) and colony growth (I = 0.4) at the same specific level of 15%. Since RA, HMBA and 5azaC were effective when administered alone in phase I clinical trials of myeloid leukaemic patients, their synergistic combinations could provide shorter and less toxic courses of treatment in elderly myeloid leukaemic patients. I is less than 1, = 1 or greater than 1 in synergistic, additive or antagonistic interactions, respectively.

Topics: Acetamides; Aged; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Cell Differentiation; Cell Division; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

This paper describes the properties of a continuous cell line derived from the blast cells of a patient with acute myeloblastic leukemia (AML), secondary to the treatment of Hodgkin's disease. The line grows slowly without stimulation but responds to interleukin-3 (IL-3), GM-CSF and mast cell growth factor (MGF), a ligand for the receptor encoded by the c-kit oncogene. When OCI/AML-4 cells are exposed to MGF with IL-3 or GM-CSF, additive or synergistic effects are seen. Combinations of MGF and G-CSF, IL-6 or CSF-1 give less growth than MGF alone. OCI/AML-4 cells are sensitive to retinoic acid; a dose related decrease in clonogenic cells is observed when OCI/AML-4 cells are exposed to retinoic acid in suspension culture. OCI/AML-4 cells are sensitive to cytosine arabinoside (ara-C), but the ara-C dose-response curve can be changed by altering the regulatory milieu in suspension culture. The cells are more ara-C sensitive in MGF or G-CSF than in IL-3 or GM-CSF. Following a 24 h exposure to retinoic acid, the ara-C sensitivity increases; in contrast, after a similar exposure to hydrocortisone, the cells become less ara-C sensitive. These changes in ara-C sensitivity occur in cells that are actively making DNA, as indicated by the reduction in colony formation after exposure to tritiated thymidine. Since OCI/AML-4 cells respond to many of the regulators that affect the growth of freshly obtained AML blast cells, it is proposed that this cell line may be useful for the study of regulation on AML in general and the interaction between different regulators in particular.

Topics: Antigens, CD; Blotting, Northern; Cell Division; Cell Survival; Cytarabine; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Hydrocortisone; In Vitro Techniques; Interleukin-3; Interleukin-6; Leukemia, Myeloid, Acute; Macrophage Colony-Stimulating Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; Receptor, Macrophage Colony-Stimulating Factor; Stem Cell Factor; Tretinoin; Tumor Cells, Cultured

We analysed the effects of recombinant human G-CSF (rhG-CSF) and retinoic acid (RA) on proliferation and differentiation of HL-60 cells and human acute myeloid leukemic (AML) cells. A synergistic effect on granulocyte differentiation was observed when HL-60 cells and primary cultured acute promyelocyte leukemic cells were cocultured with 10(-8)mol/L RA plus 1:2000 or 1:1000 rhG-CSF. The rhG-CSF plus RA treated cells demonstrated significant increase in the percentage of mature cells. Morphological changes and nitroblue tetrazolium (NBT) reduction activity evidenced more increase than RA treatment alone (P less than 0.001). The results suggest that RA not only inhibits the proliferative action of G-CSF, but also retains and enhances the action of G-CSF to induce differentiation. Therefore, we believe that the combined use of G-CSF with RA may improve the treatment of leukemia.

Topics: Cell Differentiation; Cell Division; Granulocyte Colony-Stimulating Factor; Granulocytes; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Acute myeloid leukaemia (AML) is characterized by the inability of myeloid cells to reach terminal maturation. We examined to what degree granulocytic maturation could be achieved by stimulating AML blast cells in an in vitro serum-free system with a combination of granulocyte-colony stimulating factor (G-CSF) and all-trans-retinoic acid (RA). Specimens from 41 AML patients were cultured for 7 d and then examined for cytochemistry (myeloperoxidase, Sudan Black, naphthyl-ASD-chloracetate esterase, periodic acid Schiff) an nitroblue tetrazolium reduction. The expression of CD11a, CD11b, CD11c, CD15, CD18, CD10, CD24 and B13-3 membrane antigens was also evaluated. Morphological and cytochemical studies were also performed after AML colony culture and culture of normal bone marrow cells (NBM). The comparative analysis of the panel of parameters was indicative of granulocytic maturation although to different degrees. The cells from 25/41 cases showed morphologic maturation (May-Grünwald-Giemsa). A positive correlation was evident between morphological maturation and CD11b expression (11/22 patients) as well as that of CD11c and CD15 (6 patients). Napthyl ASD chloroacetate esterase and PAS stainings also correlated with morphology (in 10/22 and 10/24 patients respectively). Nevertheless, the pattern of granulocytic maturation was remarkably variable among the 41 cases examined. The cells from only a few patients acquired the full spectrum of granulocytic markers. The comparison with normal bone marrow blasts indicates that serum-free culture conditions can, per se, limit granulocytic maturation, but it also confirms the intrinsic inability of AML cells to attain complete maturation in response to two potent granulocytic inducers.

Topics: Adult; Antigens, CD; Antigens, Neoplasm; Cell Differentiation; Granulocyte Colony-Stimulating Factor; Granulocytes; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Tretinoin; Tumor Cells, Cultured

The activity of cytidine deaminase markedly increases during the differentiation of HL-60 cells induced by dimethylsulfoxide or 1,25-dihydroxy vitamin D3, but does not increase when the inducer is retinoic acid. Here it is demonstrated that retinoic acid inhibits the increase in cytidine deaminase activity elicited by the other two inducers. This inhibitory effect of retinoic acid (i) was not the result of a direct action on the enzymatic activity; (ii) was correlated with the differentiating effect of retinoic acid, as indicated by the similar time-course and dose-dependence of both effects, and by additional studies with various retinoids and with an HL-60 variant resistant to retinoic acid-induced differentiation; (iii) required the continued presence of the drug for more than 24 h, and could not be reversed after 48 h; (iv) was manifest, after a lag-time of 24 h, at whatever time retinoic acid was added during the 5 days of treatment of the cells with the differentiation inducers; and (v) was prevented by the addition of the protein synthesis inhibitor cycloheximide. These data indicate that retinoic acid negatively regulates the expression of cytidine deaminase in HL-60 cells, and suggest that this effect is mediated by a protein, the synthesis of which should be controlled by the nuclear receptor of retinoic acid.

Topics: Calcitriol; Cell Differentiation; Cycloheximide; Cytidine Deaminase; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Retinoids; Structure-Activity Relationship; Time Factors; Tretinoin; Tumor Cells, Cultured

A biological hypothesis which is based upon the response of AML blast cells to retinoic acid alone and in combinations with other differentiating agents in primary culture, is proposed for the FAB classification of Acute Myeloid Leukaemias. The present biological hypothesis accounts for the biological and clinical observations in AML.

Topics: Cell Differentiation; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Models, Biological; Tretinoin

The responses to retinoic acid (RA) of acute myeloblastic leukemia (AML) blasts and normal hemopoietic progenitors was examined under defined growth factor conditions. For the leukemic cells marked patient to patient variation was seen; blast colony formation by cells from some patients was stimulated by RA without growth factors or in the presence of recombinant granulocyte colony-simulating factor (rG-CSF), recombinant granulocyte-macrophage-CSF rGM-CSF and recombinant interleukin-3 (rIL-3); for other populations inhibition was observed under the same conditions. Some blast cells were stimulated by RA in the presence of rGM-CSF and rIL-3 and inhibited when cultured with RA and rG-CSF. Supernatants prepared from blasts cultured with RA and growth factors did not show activities that were not readily explained by the carry-over of growth factors; this result did not provide evidence that RA and growth factors interact to produce factors. Titrations of RA showed that activity was first observed at concentrations of 10(-9) M and was maximum at concentrations of 10(-7) M. Different effects of RA in combination with rG-CSF compared with rGM-CSF or IL-3 were not seen when the cells were tested in suspension culture rather than in methylcellulose, a finding that may be interpreted to mean that the interaction between RA and factors affects terminally-dividing blast cells. Three normal bone marrow samples were cultured with RA and growth factors. Colony formation was stimulated by RA in the presence of rGM-CSF or rIL-3 but inhibited by RA with rG-CSF. Thus a differential effect of RA in combination with growth factors occurs in normal hemopoietic cells and persists in some AML populations.

Topics: Bone Marrow; Cells, Cultured; Colony-Forming Units Assay; Colony-Stimulating Factors; Culture Media; Dose-Response Relationship, Drug; Drug Interactions; Humans; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphocytes; Methylcellulose; Tretinoin

The human AML-193 cell line requires exogenous granulocyte-monocyte colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) for growth in liquid or semisolid medium. However, these CSFs do not stimulate the differentiation of the cell line. We show that addition of all-trans retinoic acid (RA) or 1,25 dihydroxyvitamin D3 (D3) induces AML-193 cells to differentiate into the granulocytic or monocytic lineage, respectively. On the other hand, addition of either G- or M-CSF alone exerts virtually no differentiative effect. Terminal granulocytic or monocytic differentiation was observed when AML-193 cells were treated with RA and G-CSF, or D3 and M-CSF, respectively, as evaluated by cell morphology, analysis of surface antigens, and phagocytic functions. These positive interactions indicate that the differentiating activity of G- and M-CSF on leukemic cells may be unmasked by preliminary treatment with RA and D3, respectively, ie, the physiologic inducers override the leukemic differentiation blockade and CFSs exert their differentiative activity on the unblocked leukemic cells. These preliminary observations on a single cell line may pave the way for the designing of clinical protocols combining physiologic inducer(s) and hematopoietic growth factor(s) in the treatment of acute leukemia.

Topics: Antibodies, Monoclonal; Antigens, CD; Calcitriol; Cell Differentiation; Cell Line; Clone Cells; Cytokines; Drug Interactions; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kinetics; Leukemia, Myeloid, Acute; Macrophage Colony-Stimulating Factor; Recombinant Proteins; Tretinoin

Addition of mycophenolic acid to cultures of HL-60 cells results in a decreased cellular level of guanine nucleotides and the induction of cell differentiation. During the early stages of this induction, steady-state levels of cellular IMP dehydrogenase (IMPDH), messenger RNA (mRNA), and protein are increased, perhaps because of cellular compensation for the inhibition of IMPDH activity. The subsequent decrease in IMPH mRNA and protein levels after several days of treatment suggests a change in the control of IMPDH expression. In contrast to the pattern of increased IMPDH expression observed in the mycophenolic acid-treated cells, treatment of HL-60 cells with two other inducers of differentiation, namely retinoic acid and phorbol 12-myristate 13-acetate, resulted in stable or decreased levels of cellular IMPDH mRNA and protein. However, the kinetics of this expression were different. These results suggest that a number of factors influence the regulation of IMPDH expression during the induction of HL-60 cell differentiation, including the nature of the inducer. A decrease in the cellular IMPDH activity was observed for all of the inducers, suggesting that this decreased activity may be a determining factor in the acquisition of a mature phenotype in the HL-60 cells.

Topics: Blotting, Northern; Blotting, Western; Cell Differentiation; Cell Division; Humans; IMP Dehydrogenase; In Vitro Techniques; Ketone Oxidoreductases; Leukemia, Myeloid, Acute; Mycophenolic Acid; Ribonucleotides; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured

The characteristic lesion in acute myeloid leukemia (AML) is the failure of myeloid cells to differentiate normally, leading to the accumulation of immature blast cells (BC) in the bone marrow. We determined whether BC and leukemia colony-forming cells (L-CFC) from AML patients could differentiate in vitro after short-term culture with interferon-gamma (IFN gamma), 1,25 dihydroxyvitamin D3 (D3), retinoic acid (RA), tumor necrosis factor-alpha (TNF alpha), and granulocyte-monocyte colony-stimulating factor (GM-CSF). Expression of myeloid differentiation antigens CD15, CD14, CD33, and p124 was determined on the BC by immunofluorescence and on the L-CFC by monoclonal antibody (MoAb) and complement (C')-mediated cytotoxicity followed by cloning in methylcellulose. We found that 26 of 39 (67%) cases demonstrated changes in the expression of myeloid differentiation antigens on the BC, and 6 of 7 (86%) cases showed an altered L-CFC myeloid antigen phenotype after short-term culture with differentiating agents. Alterations in myeloid antigen expression in the L-CFC population correlated with a reduction in L-CFC cloning potential. In the BC, alterations of myeloid differentiation antigens occurred in a manner consistent with those observed during normal myelopoiesis. For example, CD14 antigen expression (a late-stage monocyte antigen) increased on BC from 12 of 39 (31%) cases, and p124 (an antigen expressed both by myeloid progenitor cells and by a subset of monocytes) increased on 15 of 39 (38%) cases. Changes in the expression of CD33 antigens (expressed normally by myeloid progenitor cells and by mature monocytes) on the BC were variable, with 7 of 29 cases (24%) showing a decrease and 7 of 29 cases (24%) showing an increase. When comparisons were made between pairs of differentiation agents that caused the altered expression of an antigen on either the BC or L-CFC of a patient, the majority of changes were in the same direction (either both "increased" or both "decreased"). This suggests that the direction of antigen change is characteristic of the leukemia cell subpopulation for each patient and not of the stimulatory agent. This study demonstrates that cells from more than two thirds of AML cases examined responded to various differentiation agents in vitro as measured by changes in the expression of myeloid cell-associated surface antigens and by alterations in cloning potential of the L-CFC, a finding of potential clinical significance.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Calcitriol; Cell Differentiation; Cell Division; Colony-Stimulating Factors; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Tretinoin; Tumor Necrosis Factor-alpha

We studied differentiation inducing effects of retinoic acid (RA), 1 alpha, 25-dihydroxyvitamin D3 (D3) and interferons (IFNs), alone and in combination, on fresh myeloid leukemic cells from 8 patients. RA not only induced the differentiation of leukemic cells in 5/8 cases, but potentiated differentiation by IFNs either in granulocytic or monocytic pathways. In particular, interferon-alpha enhanced granulocytic differentiation and interferon-gamma induced mono-macrophage differentiation of promyelocytic leukemic cells in the presence of RA. Differentiation induced by D3, alone or in combination with IFNs, was limited in all cases. RA plus IFNs might be an effective combination for differentiation therapy for some types of myeloid leukemia.

Topics: Cell Differentiation; Cholecalciferol; Esterases; Humans; Interferon Type I; Interferon-gamma; Leukemia, Myeloid, Acute; Muramidase; Nitroblue Tetrazolium; Tretinoin; Tumor Cells, Cultured

HL-60 human nonlymphocytic leukemia cells undergo terminal differentiation along either the myeloid or monocytic pathway in a process previously shown to involve two sequential steps, early events leading to a precommitment state and late events leading to onset of terminal differentiation. The present report shows that bromodeoxyuridine induces the early events leading to precommitment. In this course bromodeoxyuridine causes the rapid down regulation of the c-myc protooncogene. The course is similar to other common inducers of HL-60 differentiation including retinoic acid, dimethyl sulfoxide, 1,25-dihydroxyvitamin D3, and sodium butyrate. HL-60 cells which were initially exponentially proliferating were exposed to 10 microM bromodeoxyuridine for 24 h, a period corresponding to one division cycle in these cells. When the cells were subsequently exposed to either retinoic acid or 1,25-dihydroxyvitamin D3, onset of G1/0 specific growth arrest and display of the differentiated phenotype occurred within 24 h. This is in contrast to the 48-h exposure needed for onset of terminal differentiation if either inducer is used singly during continuous exposure, as has been reported previously. Thus bromodeoxyuridine consummated the early events, including the rapid down regulation of c-myc message levels, which occur during the first division cycle of the induced cellular metabolic cascade leading to onset of terminal differentiation. The ability of bromodeoxyuridine to drive events in the metabolic cascade leading to onset of terminal differentiation was specific for early events, inasmuch as it was relatively ineffective at driving late events. Down regulation of c-myc was not in itself sufficient to result in subsequent terminal differentiation, since pulse exposure to bromodeoxyuridine followed by culture in inducer free medium resulted in little G1/0 specific growth arrest or phenotypic differentiation. Continuous exposure to bromodeoxyuridine, in contrast, resulted in significant G1/0 specific growth arrest but little phenotypic differentiation, indicating that the regulation of cell cycle transit and differentiation are separable.

Topics: Bromodeoxyuridine; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Down-Regulation; Humans; Leukemia, Myeloid, Acute; Oncogenes; Tretinoin; Tumor Cells, Cultured

Differentiation induction therapy provides an alternative for treatment of patients with acute myeloid leukaemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + low concentration of cytarabine (Ara-C) + dimethylformamide (DMF) on the differentiation of blasts from 24 AML patients was studied. Nonadherent mononuclear cells were cultured at a concentration of 5 x 10(5) cells/ml in 24-well tissue culture plates containing RPMI 1640 culture medium with 20% fetal calf serum, 10% autologous serum and 10% 5637-conditioned medium and incubated with 10(-6) M RA, 10(-6) M Ara-C and/or 100 mM DMF alone and in combination with each other for 6 days in primary culture at 37 degrees C in a humidified incubator under 5% CO2. The triple combination of 10(-6) M RA + 10(-6) M Ara-C + 100 mM DMF induced 90% of blasts from 22 out of 24 AML patients to differentiate. These highly effective results justify a clinical trial of this triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Transformation, Neoplastic; Cytarabine; Dimethylformamide; Humans; Leukemia, Myeloid, Acute; Tretinoin

Differentiation induction therapy provides an alternative therapeutic approach for patients with acute myeloid leukaemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + 6-thioguanine (6-Th) + hexamethylene bisacetamide (HMBA) on differentiation of blasts from 24 AML patients was studied. Nonadherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in 24-well tissue culture plates containing RPMI-1640 culture medium with 20% fetal calf serum and 10% 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 1.5 X 10(-6) M 6-thioguanine and/or 2 mM hexamethylene bisacetamide for six days at 37 degrees C in a humidified incubator under 5% CO2. Morphological, cytochemical and functional differentiation into mature cells were induced in blasts from 22 out of the 24 AML patients following exposure to the triple combination of 10(-6) M RA + 1.5 X 10(-6) M 6-Th + 2 mM HMBA in primary culture. These effective results justify a clinical trial of such triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.

Topics: Acetamides; Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Drug Screening Assays, Antitumor; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Thioguanine; Tretinoin; Tumor Cells, Cultured

Recent findings that retinoic acid (RA) induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line HL-60 in vitro and blast cell maturation in patients suffering from acute non-lymphocytic leukemia (ANLL) prompted an investigation on the ability of this agent to induce terminal maturation in blast cells from ANLL patients in vitro. We tested the ability of RA at 3 x 10(-6) M, 3 x 10(-7) M and 3 x 10(8-) M concentrations to induce differentiation in blastoid cells from 16 patients with ANLL using cytochemical and cytologic parameters, in addition to cytofluorometric methods. Leukemic cells in primary culture from all the patients underwent cytochemical and biochemical changes after treatment with RA. However, the extent of differentiation-positive cell clones (D+ clones) varied from patient to patient. Morphologic maturation was observed in a significant number of bone marrow samples. Leukocyte alkaline phosphatase and NBT reduction ability of cells, which are biochemical markers of granulocytic differentiation, were also significantly increased with a simultaneous decrease in DNA and RNA synthesis (which was estimated using a Phywe ICP-11 impulse flow cytometer).

Topics: Adolescent; Adult; Bone Marrow; Cell Differentiation; DNA; Female; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; RNA; Statistics as Topic; Tretinoin; Tumor Cells, Cultured

The in vitro induced differentiation of a number of human leukemia cell lines by chemical inducers not only provides a valuable model system for the study on the mechanism of hematopoietic cell proliferation and differentiation at both cellular and molecular levels, but also reveals a new prospect in the treatment of leukemia. In order to find out the possibility of applying inducing agents to the patients with various types of leukemia, the bone marrow cells in primary culture from 50 patients with leukemia were tested for their inducibility in response to the inducers. Only M3 leukemia bone marrow cells can be markedly induced by retinoic acid to the myeloid terminal cells with positive NBT reduction while the cells of other types respond with uncertainty. TPA is able to cause a macrophage-like differentiation in bone marrow cells of all types of leukemia except M1. However, the leukemic cells of chronic myelogenous leukemia in lymphocytic blast crisis will lose response to TPA. The cultured bone marrow cells of acute lymphocytic leukemia respond neither to retinoic acid nor to TPA. Homoharringtonine, a chemotherapeutic drug used in the so-called HOAP regimen for acute nonlymphocytic leukemia, seems to possess the capability of inducing HL-60, the promyelocytic leukemia cell line, to NBT positive myeloid terminal cells, although the inducing effect is weaker than retinoic acid.

Topics: Adolescent; Adult; Aged; Bone Marrow; Child; Female; Harringtonines; Homoharringtonine; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) has been shown to increase differentiation in some leukemia cell lines (HL-60 and KG-1) but not others (K562). Similarly, RA has been reported to have variable effects on fresh blast cells. Recently, molecular clones have been obtained for the nuclear receptor for retinoic acid. The experiments described in this paper were designed to compare expression of the receptor to biological activity in myeloblastic leukemia cells. In four continuous AML cell lines, a positive correlation was found between retinoic acid receptor (RAR) expression by Northern analysis or RNA dot blot and the ability of RA to inhibit colony formation. Kinetic studies of the most sensitive cell line showed that inhibition of colony formation was associated with reduced blast cell self-renewal and differentiation-like events. RAR was detected in freshly obtained blast cells from 23 AML patients. Patient-to-patient variation was observed; however, a correlation was not found between RAR expression and response of the freshly obtained blast cells to RA.

Topics: Carrier Proteins; Cell Survival; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Neoplastic Stem Cells; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

From ten consecutive patients with acute myeloid leukemia leukemic cells were isolated and cultured with and without 10(-6), 10(-7) and 10(-8) M 1.25(OH)2D3 (vit D3), retinoic acid (RA) cytosine-arabinoside (ARA-C) and 1.0, 1.25 and 1.5% dimethyl sulfoxide (DMSO). Maturation was measured with a comprehensive panel of qualitative and quantitative parameters of maturation. Six of those ten leukemias showed significant (p less than 0.01) changes in at least three parameters after exposure to either one of the differentiation inducers. Vit D3 induced maturation in four leukemias, in three of them clearly in monocytic direction. ARA-C showed changes in one leukemia in only three parameters not pointing to either granulocytic or monocytic direction. Maturation in granulocytic direction was observed after exposure to RA in one leukemia. Maturation induction was observed in six out of ten freshly isolated leukemic cells with vit D3 being the most potent inducer of maturation in monocytic direction. The data about inducibility of maturation in freshly isolated human leukemic cells are reviewed and discussed.

Topics: Adult; Aged; Cell Differentiation; Cell Separation; Cholecalciferol; Cytarabine; Dimethyl Sulfoxide; Female; Granulocytes; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Monocytes; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

Cells from three patients showed maturation after incubation with retinoic acid (2 had M-3 AML and 1 had CML-B). Three additional patients showed spontaneous maturation (1 with M-2 and 2 with M-4 AML), and in them cell maturation was also achieved after incubation with retinoic acid and cytosine arabinoside (10 nM). These results confirm different maturation capability of leukaemic cells, as well as the possibility to induce cellular maturation with retinoic acid, especially in patients with acute promyelocytic leukaemia (M-3).

Topics: Adult; Aged; Cell Differentiation; Cells, Cultured; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Middle Aged; Tretinoin

Retinoic acid (RA) is a potent morphogen that has been shown to increase differentiation in some leukemic cell populations. RA has been used in treatment of some patients with acute myeloblastic leukemia (AML) and myelodysplastic syndromes. In previous experiments we had observed that RA may decrease the self-renewal of blast cells in established cell lines, and in our clinic RA has been tested as maintenance treatment in association with chemotherapeutic drugs. Accordingly, we asked if exposure of AML blast cells to RA affected their subsequent response to ara-C. We found that brief exposure to RA regularly increased the ara-C sensitivity of cells from two established AML cell lines. A similar, though less marked, effect was seen when the blast cells from one patient were tested directly; in a second instance, highly ara-C resistant blasts did not become sensitive when exposed to RA. Experiments using high specific activity tritiated thymidine did not disclose any changes in the proportion of AML cells in the DNA synthesis phase of the cycle at times when their responses to ara-C were changing. We interpret our findings as support for continuing efforts to integrate RA in the management of AML patients and suggest that the mechanism of ara-C sensitization may not depend on changes in the cell cycle.

Topics: Cell Survival; Cytarabine; DNA; Doxorubicin; Drug Interactions; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.

Topics: Acylation; Cell Differentiation; Cell Line; Electrophoresis, Polyacrylamide Gel; Humans; Hydrolysis; Leukemia, Myeloid, Acute; Nuclear Proteins; Subcellular Fractions; Tretinoin

In a series of experiments investigating the differentiation of human promyelocytic leukemia cells (HL60) in the presence of chemical inducers, we have transduced the bacterial neomycin-phosphotransferase (NPT) gene into the HL60 genome. We show here, that HL60 cells expressing the NPT gene under the control of different transcriptional promoters differentiate significantly slower than the parental HL60 cells. Competition experiments involving the use of excess amounts of specific NPT substrate (i.e. G418) revealed that the delay in the differentiation response of neo-resistant HL60 cells results from the action of the neomycin-phosphotransferase protein. We conclude that the activity of the NPT gene may prove a useful tool in elucidating the control of differentiation in HL60 cells.

Topics: Alcohol Oxidoreductases; Cell Differentiation; Genetic Vectors; Kanamycin Kinase; Leukemia, Myeloid, Acute; Phosphotransferases; Tretinoin; Tumor Cells, Cultured

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.

Topics: Adult; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Gangliosides; Granulocytes; Humans; Leukemia, Myeloid, Acute; Microscopy, Electron; Structure-Activity Relationship; Superoxides; Tretinoin

The uptake of all-trans-retinoic acid (RA) and two new retinoids [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylcarbamoyl )benzoic acid (Am80) and (E)-4-[3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]benzoic acid (Ch55)] by HL-60 human promyelocytic leukemia cells was investigated. For the investigation, [3H]RA and [3H]Am80 with high specific radioactivities (more than 50 Ci/mmol) were used. [3H]Am80 was prepared by hydrogenolysis of the corresponding chlorinated derivative of Am80 with tritium gas. The retinoids RA, Am80 and Ch55 were efficiently taken up by HL-60 cells, and induced differentiation of the cells into mature granulocytes. The specific bindings (uptake) of RA, Am80 and Ch55 (the bindings inhibited competitively by the other two retinoids) by HL-60 cells were due to a newly detected binding protein. The protein that bound specifically to RA appeared identical to that which bound specifically to Am80 by high-performance liquid chromatography (HPLC), and was named retinoid-specific binding protein (RSBP). One HL-60 cell was found to contain about 1500 molecules of RSBP distributed between the nuclear fraction and cytosolic fraction in proportions of about 4:1. The bindings of the three retinoids (RA, Am80 and Ch55) to RSBP (i.e., formation of retinoid-RSBP complexes) greatly enhanced the affinity of RSBP for the nuclei. The apparent molecular weight of RSBP was estimated to be 95,000 daltons by size exclusion HPLC. The association constants (Ka) of RSBP were calculated to be 2.4 X 10(10) M-1 for RA and 4.4 X 10(10) M-1 for Am80 from Scatchard plots. The bindings of RA, Am80 and Ch55 to RSBP were mutually competitive, indicating that the binding sites for RA, Am80 and Ch55 were identical. The very high affinities of these retinoids for RSBP (Ka's of the order of 10(10) M-1) correspond to the effective concentrations of these retinoids in HL-60 cell culture medium for induction of differentiation of the cells. The mutually competitive bindings of these retinoids strongly support the idea that RSBP is the true receptor of retinoids.

Topics: Benzoates; Carrier Proteins; Cell Differentiation; Cell Line; Chromatography, High Pressure Liquid; Humans; Leukemia, Myeloid, Acute; Molecular Weight; Receptors, Retinoic Acid; Tretinoin

A cAMP binding protein was detected in HL-60 cells using photoaffinity labeling with 8-azido [32P]cAMP. The binding protein was found in a 0.35 M NaCl nuclear protein extract from untreated HL-60 cells and from the HL-60 cells induced to mature with retinoic acid. While the quantity of the cAMP binding protein did not change following the induced differentiation, a second form of the subunit, altered in charge, was present at 3 and 5 days after retinoic acid treatment. The findings indicate that the regulatory subunit of the type II cAMP-dependent protein kinase could be involved in nuclear functions associated with human myeloid cell differentiation.

Topics: Azides; Carrier Proteins; Cell Nucleus; Cyclic AMP; Cyclic AMP Receptor Protein; Humans; Leukemia, Myeloid, Acute; Molecular Weight; Protein Kinases; Tretinoin

Promyelocytic leukemia HL-60 cells induced to differentiate along the granulocytic and monocytic pathways respond to stimulation with phorbol myristate acetate by producing superoxide radicals. The amount of superoxide radical generation can be monitored by spectrophotometric measurement of cytochrome c reduction. We have developed a microtiter assay that assesses differentiation of HL-60 cells on the basis of cytochrome c reduction. HL-60 cells were incubated with known standards or unknown samples, including crude fermentation broths, for 6 days; then cytochrome c reduction was quantified as a function of increasing absorbance at 550 nm on a microtiter plate reader. HL-60 cells induced to differentiate showed up to a 10-fold increase in absorbance over that of control cells. Differentiation was confirmed by morphological assessment and by flow cytometric analysis of the DNA cell-cycle distribution and the cell-surface transferrin receptor. Analysis of 198 crude fermentation broth samples confirmed the feasibility of using this assay for large-scale drug screening.

Topics: Cell Differentiation; Cytochrome c Group; Drug Evaluation, Preclinical; Humans; Leukemia, Myeloid, Acute; Nitroblue Tetrazolium; Spectrophotometry; Superoxides; Tretinoin; Tumor Cells, Cultured

Topics: Alkaloids; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Harringtonines; Humans; Leukemia, Myeloid, Acute; Microscopy, Electron; Tretinoin; Tumor Cells, Cultured

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Headache; Humans; Leukemia, Myeloid, Acute; Male; Stereoisomerism; Tretinoin

We previously reported that human promyelocytic leukemia HL-60 cells, when treated with various inducers in magnesium-deficient medium, became committed to differentiate but did not express the differentiation-related phenotypes (Okazaki et al., J. Cell. Physiol., 131:50-57, 1987). In the present study we demonstrated the existence of an intracellular differentiation-inducing activity (int-DIA) in differentiation-committed phenotype-nonexpressing HL-60 cells by using cybrid formation between untreated HL-60 cells and cytoplasts from HL-60 cells treated in magnesium-deficient medium with 100 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell extracts from similarly treated HL-60 cells also showed int-DIA, which when added (10 mg total protein/ml) to culture of untreated HL-60 cells, could increase the percentages of nitroblue tetrazolium (NBT)- and nonspecific esterase (NSE)-positive cells from 1% to 53%, and from 0 to 32%, respectively. They also induced differentiation of human monoblastic leukemia U-937 cells and of human myeloblastic leukemia KG-1 cells but not of erythroleukemia K-562 cells. These results suggested that the int-DIA had a common effect on differentiation induction in several human myeloid cell lines and may be involved in inducing cells to proceed from a commitment to a phenotype-expression step during human myeloid cell differentiation.

Topics: Calcitriol; Cell Differentiation; Cell Division; Cell Extracts; Humans; Hybrid Cells; Intracellular Membranes; Leukemia, Myeloid, Acute; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The human leukemic cell line HL-60 undergoes differentiation to granulocytic-like cells in response to dimethyl sulfoxide (DMSO) or retinoic acid (RA). This differentiation is accompanied by an arrest in cell proliferation. Studies have implicated alterations in the phospholipid fatty acid (FA) composition as a result of HL-60 differentiation. However, changes in FA's are also known to occur during the arrest of cellular proliferation. Using a highly efficient capillary gas-liquid chromatography technique, the phospholipid FA composition of HL-60 and of DMSO-resistant and RA-resistant HL-60 subclones was determined in proliferating cells, in density-arrested cells, and in terminally differentiated cells. The same specific modifications in some of the FAs of the three cell lines were observed when proliferation was inhibited by cell density; 16:0 and 18:2n-6 were decreased and 22:6n-3 increased. Moreover, 16 and 18 dimetylacetals were both increased when proliferation was decreased, indicating modifications in plasmalogen contents. Granulocytic differentiation of HL-60 cells and of its subclones with DMSO and/or RA provoked modifications in phospholipid FAs different from that found in density-arrested, undifferentiated cells such as decreases in monoenoic FAs of 16 and 18 carbons as well as an increase in arachidonic acid, the major polyunsaturated FA. The biological significance of these changes upon arrest of proliferation and differentiation are discussed. These results indicate that, when arrest of proliferation accompanies differentiation, these two phenomena can be responsible for different changes and, whenever possible, they have to be considered separately in order to know which modifications are effectively due to differentiation itself.

Topics: Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Fatty Acids; Humans; Leukemia, Myeloid, Acute; Phospholipids; Tretinoin

Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these, eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction, NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy, none of the patients showed any abnormalities in the coagulation parameters we measured, suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin, with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL.

Topics: Adolescent; Adult; Bone Marrow; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Disseminated Intravascular Coagulation; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Time Factors; Tretinoin

Topics: Adolescent; Adult; Alkaloids; Cell Transformation, Neoplastic; Child; Female; Harringtonines; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin; Tumor Cells, Cultured

Polymorphonuclear leukocytes contain proteases that are capable of degrading articular cartilage matrix in disease states such as rheumatoid arthritis and osteoarthritis. In this study, the HL-60 human promyelocytic leukemia cell line was examined for ability to degrade cartilage proteoglycans. The HL-60 cells contained proteoglycan-degrading enzymes, which may contribute to the joint inflammation sometimes seen in acute leukemia. However, the protease activity was much less than in mature neutrophils and was not enhanced by the induction of myeloid maturation with dimethyl sulfoxide or retinoic acid. The diminished enzyme activity of induced HL-60 cells compared to normal neutrophils is another functional deficiency of these cells.

Topics: Animals; Arthritis; Cartilage; Cattle; Cell Line; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Neutrophils; Peptide Hydrolases; Proteoglycans; Tretinoin

Two sublines of HL-60 cells differing markedly in their ability to undergo differentiation to granulocytes after treatment with retinoic acid (RA), dimethyl sulfoxide (DMSO) and the pyrimidine analog, 5-aza-2'-deoxycytidine (azadCyd) were studied. The sensitive subline (HL-60 S) responded well to 1 microM RA, 1% DMSO and 1 microM azadCyd, showing 89 +/- 5%, 46 +/- 5% and 29 +/- 6% mature nitroblue tetrazolium (NBT)-positive cells, respectively. However, the resistant subline (HL-60 R) showed only modest maturational effects (12 +/- 3%, 11 +/- 2% and 9 +/- 2%, respectively) after treatment with the same agents. Using the HL-60 R as a model for resistance to differentiation induction in the HL-60 cell line, studies were carried out to determine whether the combined use of RA, DMSO and azadCyd could reverse the resistance of these tumor cells to the induction of maturation expressed by the individual agents. When these agents were given in any combination of 2, a minor increase in differentiation induction was detected (13 +/- 6% or less NBT-positive cells). However, when all 3 agents were combined (RA + DMSO + azaCyd), resistance was completely reversed (89 +/- 7% mature NBT-positive cells). In addition, different degrees of concentration-dependence of each agent in the combination were observed. The RA + DMSO + azadCyd combination caused a maximal accumulation of NBT-positive cells after 72 to 96 hr of incubation. These results show that the lack of competence for induction of differentiation in resistant HL-60 cells can be completely reversed by the above ternary drug combination. However, the mechanism responsible for this synergistic effect must await further elucidation of the molecular mechanisms by which such agents act.

Topics: Antineoplastic Agents; Azacitidine; Cell Differentiation; Cell Line; Decitabine; Dimethyl Sulfoxide; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Tretinoin

Interferons (IFNs) have been shown to enhance or induce differentiation in several model systems, either alone or in combination with differentiating agents. To further study these phenomena, the effects of the recombinant interferons rIFN-alpha A, rIFN-alpha D, rIFN-alpha A/D (Bgl II), rIFN-beta, and rIFN-gamma on the differentiation-producing capacities of the anticancer agents aclacinomycin A and actinomycin D, and the differentiating agent retinoic acid, were examined in the HL-60 promyelocytic leukemia cell line. The addition of IFNs to aclacinomycin A did not produce any enhancement of differentiating capacity. The differentiating capacity of actinomycin D was enhanced only by rIFN-alpha A/D (Bgl II). The differentiating capacity of retinoic acid was enhanced by all the IFNs-alpha tested, and by rIFN-beta, but not rIFN-gamma.

Topics: Aclarubicin; Cell Differentiation; Cell Division; Cell Line; Dactinomycin; Drug Synergism; Humans; Interferons; Leukemia, Myeloid, Acute; Naphthacenes; Tretinoin

Both human recombinant tumor necrosis factor alpha (TNF alpha) and all-trans-retinoic acid (RA) inhibit the in vitro clonal growth of human myeloid leukemic cells. We investigated the in vitro interaction of TNF alpha and RA with normal and a variety of leukemic myeloid cells. With the promyelocytic HL-60 cells, TNF alpha (greater than or equal to 2.5 U/mL) in combination with RA synergistically inhibited clonal growth; TNF alpha at lower concentrations (less than or equal to 1 U/mL) plus RA (10(-9) mol/L) were antagonistic in their inhibition of growth. The ability of RA (10(-8) mol/L) plus TNF alpha (2.5, 5 U/mL) to enhance differentiation of HL-60 cells paralleled their ability to inhibit clonal growth of these cells. In addition, RA (10(-9) to 10(-7) mol/L) increased the number of TNF alpha receptors on HL-60 cells 1.3- to 1.7-fold without changing the affinity for the TNF alpha receptor. With the more immature KG-1 myeloblasts, concentrations of TNF alpha greater than 10 U/mL synergistically interacted with RA to inhibit clonal growth; at lower concentrations of TNF alpha (less than 10 U/mL), RA appeared to inhibit the expected effect of TNF alpha. KG-1 cells were not induced to differentiate with either agent alone or in combination. With four of nine leukemic patients, TNF alpha in combination with RA (10(-7) mol/L) inhibited leukemic clonal growth to a greater extent than each agent alone. No marked effect of the combined treatment was seen in two other patients. The RA reversed the inhibitory action of TNF alpha on normal human granulocyte-macrophage colony forming cells (GM-CFC) and on clonal growth of leukemic cells from three patients. Our study suggests that TNF alpha and RA interact in a complex manner with normal and leukemic hematopoietic cells.

Topics: Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Hematopoiesis; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Receptors, Cell Surface; Receptors, Tumor Necrosis Factor; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We have shown previously that induced maturation of the human myeloid leukemia cell line, HL-60, is associated with a selective down-regulation of guanine ribonucleotide synthesis and depletion of intracellular guanosine triphosphate (GTP) and guanosine diphosphate (GDP) pools. We showed, furthermore, that inhibitors of the enzyme, inosine monophosphate (IMP) dehydrogenase, which catalyzes the initial rate-limiting step of guanylate synthesis from the central intermediate IMP, are potent inducers of myeloid maturation in these cells. We now show that induced maturation of HL-60 cells is prevented or impaired if intracellular concentrations of guanine ribonucleotides are maintained at high levels. HL-60 cells can utilize exogenous guanine and guanosine to maintain GTP and GDP pools through a salvage pathway that bypasses guanylate synthesis from IMP. Moreover, incubation of HL-60 cells with guanosine or guanine (10(-6) to 10(-4) mol/L) prevents both the depletion of intracellular guanine ribonucleotides and the induction of myeloid maturation caused by the IMP dehydrogenase inhibitor, tiazofurin. These findings provide strong additional support for the concept that terminal myeloid differentiation is influenced by a guanine ribonucleotide-dependent regulatory system.

Topics: Cell Differentiation; Cell Line; Dimethylformamide; Guanine; Guanine Nucleotides; Guanosine; Hematopoiesis; Humans; IMP Dehydrogenase; Leukemia, Myeloid, Acute; Ribavirin; Tretinoin

Retinoyl beta-glucuronide and retinyl beta-glucuronide, which are naturally occurring water-soluble metabolites of vitamin A, induce the granulocytic differentiation of HL-60 cells in vitro, as evidenced by an increased reduction of nitroblue tetrazolium. The relative effectiveness of various retinoids in differentiation is retinoic acid greater than retinoyl beta-glucuronide greater than retinyl beta-glucuronide. Under the selected assay conditions, retinol, hydroxyphenyl-retinamide, retinamide, and N-retinoyl-phenylalanine are essentially inactive in differentiation. At concentrations of retinoids from 10(-9) to 10(-5) M, cell viability was best with the retinoid beta-glucuronides and retinamide, less with retinoic acid and retinol, and poorest with the N-retinoyl aromatic amines. Cellular growth was depressed only slightly by retinyl beta-glucuronide and retinamide, but to a greater degree by the other derivatives. Retinoyl beta-glucuronide was hydrolyzed in part to retinoic acid, whereas retinyl beta-glucuronide was cleaved to retinol, if at all, at a very slow rate. Under the selected assay conditions, retinoic acid and the retinoid beta-glucuronides primarily induce the differentiation of HL-60 cells, whereas the N-retinoyl aromatic amines show cytotoxicity.

Topics: Cell Differentiation; Cell Division; Cell Line; Fenretinide; Humans; Leukemia, Myeloid, Acute; Nitroblue Tetrazolium; Oxidation-Reduction; Tretinoin; Vitamin A

Topics: Azaguanine; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Drug Resistance; Gene Expression Regulation; Humans; Hypoxanthine Phosphoribosyltransferase; Leukemia, Myeloid, Acute; Oncogenes; Tretinoin

We studied the effects of cis-retinoic acid (cisRA) on the clonogenic growth of samples of leukemic cells from 35 patients with acute nonlymphocytic leukemia (ANLL). We observed significant inhibition of leukemic colony growth in 17 samples by 10(-7) to 10(-6)M cisRA. However, we found that retinoid exposure resulted in striking stimulation of clonal growth in ten samples at the same drug concentrations. With the exception of cases with promyelocytic features, there was no morphologic or functional evidence that cisRA induced the leukemic blasts to differentiate. Both inhibition and stimulation were dose-dependent and observable at pharmacologically achievable levels of cisRA. Leukemic cells with monocytic features more frequently demonstrated a stimulatory response than did those without monocytic features. Depletion of T lymphocytes and monocytes did not alter the type of growth response. Assays for cellular retinoic acid-binding protein (CRABP) were performed on five samples (two with inhibitory growth responses, two with stimulatory responses, and one with no growth) and failed to reveal detectable levels of CRABP in any case. The addition of cisRA to liquid suspensions of leukemic cells produced no significant change in the number of viable cells. We conclude that the effects of cisRA on leukemic colony growth are not cytotoxic and not mediated by T lymphocytes, monocytes, or CRABP. More importantly, cisRA appears to enhance the growth of certain human leukemia cells in vitro. Taking into account the increasing use of retinoids in clinical trials for patients with leukemia, the latter findings may represent a significant cautionary note.

Topics: Bone Marrow; Carrier Proteins; Cell Differentiation; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Leukemia, Monocytic, Acute; Leukemia, Myeloid, Acute; Receptors, Retinoic Acid; Tretinoin

Retinoic acid, which induces the differentiation of HL 60 cells to granulocytes, produces a cell alkalinization from pHi = 7.03 to pHi = 7.37. The half-maximum effect of retinoic acid is observed at 10 nM. The effect of retinoic acid on the pHi develops slowly, and it precedes the differentiation of the cells. A cell alkalinization is also observed after differentiation of the cells by dimethyl sulfoxide. It is not observed using etretinate, a synthetic retinoid that does not promote the differentiation of HL 60 cells. Two pHi regulating mechanisms coexist in HL 60 cells. The Na+/H+ exchange system is the major mechanism that allows HL 60 cells to recover from an intracellular acidosis. A second mechanism is a Na-HCO3 cotransport system. During differentiation of the cells by retinoic acid, a 2-fold increase in the activity of the Na+/H+ exchange system is observed, while the activity of the NaHCO3 cotransport remains constant. The properties of interaction of the Na+/H+ exchanger with internal H+, external Na+, and Li+ as well as with amiloride and its derivatives are defined. The Na+/H+ exchanger of HL 60 cells is characterized by unusually low affinities for alkali cations and a high affinity for amiloride and its derivatives. The pHi dependence of the exchanger is not modified after differentiation by retinoic acid. It is concluded that the mechanism of activation of the Na+/H+ exchanger by retinoic acid is distinct from the short-term effect produced by mitogens and phorbol esters which change the pHi dependence of the system.

Topics: Carrier Proteins; Cell Differentiation; Cell Line; Granulocytes; Humans; Hydrogen-Ion Concentration; Kinetics; Leukemia, Myeloid, Acute; Lithium; Sodium; Sodium-Hydrogen Exchangers; Tretinoin

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.

Topics: Aclarubicin; Amanitins; Bucladesine; Calcitriol; Cell Differentiation; Cell Division; Cell Line; Cycloheximide; Dimethyl Sulfoxide; Drug Interactions; Humans; Leucine; Leukemia, Myeloid, Acute; Magnesium Deficiency; Naphthacenes; Oncogenes; Peptide Chain Elongation, Translational; Phenotype; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin

The human acute promyelocytic leukemia cell line HL-60 is induced to differentiate into morphologically and functionally mature monocytelike cells by incubation with a combination of 10 nmol/L retinoic acid (RA) and various concentrations of recombinant immune interferon (rIFN-gamma). These induced cells show marked increases in antibody-dependent cellular cytotoxicity (ADCC), antibody-coated erythrocyte (EA) rosettes, nonspecific esterase, and 5'-nucleotidase activity. rIFN-gamma alone at concentrations of 10 to 1,000 U/mL has essentially no effect on morphological maturation, nitroblue tetrazolium reduction, and immunophagocytosis. However, rIFN-gamma at these concentrations increases EA rosetting in a concentration-dependent manner that is not affected by 10 nmol/L RA. At a concentration of 1,000 U/ml, rIFN-gamma induces moderate increases in nonspecific esterase, 5'-nucleotidase, and ADCC. These parameters are markedly increased by the addition of 10 nM RA, a concentration which alone has no effect on these markers. Based on units of antiviral activity, rIFN-gamma is tenfold more active than rIFN-alpha D in inducing EA rosettes and 40-fold more active in inducing nitroblue tetrazolium reduction and immunophagocytosis. These results, indicating that combinations of rIFN-gamma or rIFN-alpha and RA synergistically induce differentiation of HL-60, suggest that this combination may have clinical utility in the treatment of patients with certain leukemias.

Topics: Cell Differentiation; Cell Line; Drug Synergism; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Recombinant Proteins; Tretinoin

Topics: Cell Differentiation; Cell Line; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Lymphotoxin-alpha; Recombinant Proteins; Rosette Formation; Tretinoin

The effect of the combination of retinoic acid (RA) and calcium ionophore A23187 on cellular differentiation was assessed in promyelocytic leukemia cell line HL-60. RA (10(-10)-2.5 X 10(-8) M) or A23187 (4 X 10(-7) M) alone produced 15-22% differentiated cells as assessed by nitroblue tetrazolium reduction. Exposure of cells for 48 hr to the combination of 4 X 10(-7) M A23187 and 10(-10)-2.5 X 10(-8) M RA resulted in 20-86% of the cells capable of reducing nitroblue tetrazolium, but with no measurable level of nonspecific esterase activity. The combination of A23187 and either dimethyl sulfoxide, 1,25-dihydroxyvitamin D3, or immune interferon failed to produce a synergistic effect on differentiation. Addition of either the calmodulin antagonists, N-(6-aminohexyl)-5-chloronaphthalenesulfonamide and trifluoperazine, or the protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, during treatment with A23187 and RA did not block differentiation. Membrane tyrosine kinase activity was measured in cells treated with A23187 and RA in a nondenaturing gel system using the exogenous substrate poly(Glu:Tyr). Membrane-bound tyrosine kinase activity was not present in untreated or RA-treated cells but was induced by A23187 treatment alone and was markedly increased in cells 48 hr after treatment with the combination of A23187 and RA. Significantly greater reduction in c-myc mRNA levels was also observed 24 hr after treatment with RA and A23187 in comparison to that observed with either agent alone. These results suggest that a Ca2+-mediated process sensitizes cells to the differentiating effect of RA and that this effect is associated with a significant reduction of c-myc expression and the induction of membrane tyrosine kinase activity in this cell line.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calmodulin; Cell Differentiation; Cell Line; Cell Membrane; Drug Synergism; Gene Expression Regulation; Hematopoiesis; Humans; Isoquinolines; Leukemia, Myeloid, Acute; Piperazines; Protein-Tyrosine Kinases; Proto-Oncogenes; RNA, Messenger; Sulfonamides; Tretinoin; Trifluoperazine

In this study, we analyzed the effect of tumor necrosis factor (TNF) on retinoic acid (RA)-induced myeloid differentiation of the promyelocytic HL-60 leukemia cell line. We show that low concentrations of the two substances, almost inactive in inducing differentiation when used separately, induce differentiation when added simultaneously to the cell cultures. Cells simultaneously expressing both monocyte/macrophage phenotype (typically induced by TNF) and granulocyte characteristics (typically induced by RA) are induced by a combination of the two factors, indicating that TNF and RA potentiate each other's activity. The results obtained using immune interferon (IFN-gamma) in combination with the two inducers suggest that the mechanism of action of TNF and IFN-gamma are possibly different. The inhibitory effect of RA on the expression of HLA class I antigens and of the high-affinity Fc receptor is potentiated by TNF but completely reversed by rIFN-gamma.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Cycle; Cell Differentiation; Cell Line; Cells, Cultured; Drug Synergism; Glycoproteins; HLA Antigens; Humans; Interferon-gamma; Leukemia, Myeloid, Acute; Receptors, Fc; Recombinant Proteins; Tretinoin; Tumor Necrosis Factor-alpha

During the induced differentiation of the human promyelocytic leukaemic cell line, HL-60, along the myelocytic lineage, DNA strand-breaks are formed. These breaks which are formed in the face of a proficient DNA repair mechanism, are only transiently maintained and subsequently become religated. The ligation of these breaks requires the activity of the nuclear adenosine diphosphoribosyl transferase (ADPRT). Inhibition of nuclear ADPRT, an enzyme totally dependent on the presence of DNA strand-breaks for its activity and required for efficient DNA repair in eukaryotic cells, blocks the religation of these breaks but not their formation. The inhibition of DNA strand ligation in the differentiating HL-60 cells results in loss of viability and cell death.

Topics: Benzamides; Cell Differentiation; Cell Line; Cell Survival; Dimethyl Sulfoxide; DNA Repair; DNA, Neoplasm; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Tretinoin

Topics: Adult; Child; Child, Preschool; Cytarabine; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin

Terminal cell differentiation of HL-60 promyelocytic leukemia cells results when they are continuously exposed to retinoic acid. This process involves an intermediate regulatory state, the precommitment memory state, which occurs before onset of differentiation or growth arrest in G0. The cellular processes occurring prior to onset of terminal differentiation can be resolved into early events anteceding development of the precommitment memory state and late events subsequent to it. While it has been suggested that retinoic acid induced early events regulate G1/0 specific growth arrest associated with terminal differentiation, the significance of induced late events is not known. Exploiting the capability of HL-60 cells to undergo either myeloid or monocytic differentiation in response to different inducers, the present studies examine the response of HL-60 cells to the sequential application of myeloid and monocytic inducers prior to onset of terminal differentiation. The results indicate that the precommitment state induced by retinoic acid is not differentiation lineage specific. Sequential application first of retinoic acid, a myeloid inducer, and then of 1,25-dihydroxyvitamin D3, a monocytic inducer, and vice versa, show that cellular choice of a specific differentiation lineage is regulated by late inducer driven events. The data support a two-step model for induction of terminal differentiation where early events anteceding precommitment regulate growth arrest and late events subsequent to precommitment regulate choice of a specific differentiation lineage. The results are of potential significance to the use of differentiation-inducing agents in chemotherapy. The potential toxicity of prolonged exposure to a single inducer might thus be mitigated by sequential brief exposures to different inducers.

Topics: Calcitriol; Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Monocytes; Proto-Oncogenes; Tretinoin

Control of terminal cell differentiation was studied in the HL-60 human promyeloctyic leukemia cell line. Retinoic acid is known to induce myeloid differentiation associated with GO arrest in these cells. In this case, onset of terminal differentiation occurs after an exposure period corresponding to two division cycles. This is preceded by acquisition of a precommitment memory state occurring by one division cycle. Cells in precommitment undergo accelerated onset of terminal differentiation upon reexposure to inducer. The present report shows that the precommitment state can be induced by a pulse exposure to hydroxyurea. While the hydroxyurea exposure does not itself induce terminal differentiation, the treated cells undergo accelerated onset of phenotypic differentiation and GO arrest upon exposure to retinoic acid. Thus a perturbation of S-phase specific cellular metabolism induces the early precommitment regulatory state in the course of induced HL-60 terminal myeloid differentiation. The results support a model in which initiation of a cellular program of terminal differentiation depends on an S-phase specific event associated with replication of cellular DNA and possibly involving gene amplification. Significantly, the results indicate that a conventional chemotherapeutic agent such as hydroxyurea can synergistically interact with a differentiation inducing agent such as retinoic acid. This indicates the significance of investigating the interaction between conventional S-phase specific chemotherapeutic agents and differentiation inducing agents as a potential treatment modality.

Topics: Cell Differentiation; Cell Division; Cell Line; DNA; DNA Replication; Humans; Hydroxyurea; Interphase; Leukemia, Myeloid, Acute; Tretinoin

The human promyelocytic leukemia cell line HL60 differentiates to either granulocytes or monocytes/macrophages when induced with various chemicals and lymphokines. Retinoic acid (RA) induces HL60 to differentiate to granulocyte-like cells. However, HL60/MRI cells, derived from a transplantable HL60 tumor established in athymic nude mice, differentiate to monocytoid cells when cultured with RA in vitro. HL60/MRI induced with RA are monocytes based on morphology and the expression of markers and functions specific for monocytes such as: the OKM5 monocyte-specific antigen, nonspecific esterase activity, and adhesiveness. HL60/MRI is much more sensitive to RA than is HL60. Thus, the RA concentrations that induce 50% differentiation are 0.41 nM for HL60/MRI and 37 nM for HL60, and maximum differentiation occurs at 2 days for HL60/MRI and at 4 days for HL60. While RA induces HL60/MRI to monocytoid cells, other inducers of granulocytic differentiation of HL60, such as dimethyl sulfoxide and hexamethylene bisacetamide, induce HL60/MRI to granulocytes. Furthermore, 12-O-tetradecanoylphorbol-13-acetate induces both HL60 and HL60/MRI to macrophage-like cells. The isozyme phenotypes of HL60/MRI and HL60 are identical. Cytogenetic analysis of HL60/MRI indicates that many of its normal chromosomes are triploid and that it has five abnormal chromosome markers, M1-M5, three of which, M1-M3, are seen also in HL60. This unique cell line, HL60/MRI, may be useful for studying the event(s) triggering differentiation of myelomonocytic cells and the mechanism of action of RA.

Topics: Acetamides; Animals; Antigens, Surface; Cell Differentiation; Cell Line; Chromosome Aberrations; Dimethyl Sulfoxide; Humans; Isoenzymes; Leukemia, Myeloid, Acute; Mice; Mice, Inbred BALB C; Monocytes; Neoplasm Transplantation; Tetradecanoylphorbol Acetate; Tretinoin

Cultures of the promyelocytic leukemia cell line HL-60 usually contain considerable numbers of spontaneously differentiating cells and are asynchronous in terms of cell-cycle phases. Counterflow centrifugal elutriation studies have been conducted to obtain a homogeneous cell population with regard to cell-cycle phases and stage of differentiation. Despite their small volume and probably because of their high buoyant density, differentiated cells are elutriated predominantly at higher flow rates. Accordingly, G1 cells elutriated at low flow rates are substantially free from spontaneously differentiating cells. By optimizing the technique, a population with approx. 90% G1 cells and less than 1% spontaneously differentiating cells was obtained. The yield in the fractions chosen was 5.1% of all cells recovered from elutriation. In culture, a cell population of this purity maintains a synchronous cell cycle for more than 2 days. This allows an exact determination of the time after induction when the first signs of differentiation occur. The presence of 1 microM retinoic acid (RA) causes the first significant increase of NBT-positive cells between the 24th and 27th h of culture.

Topics: Cell Cycle; Cell Differentiation; Cell Line; Cell Separation; Cell Survival; Centrifugation; Humans; Interphase; Leukemia, Myeloid, Acute; Tretinoin

We examined the differentiation activity of retinoyl beta-D-glucuronide, a biologically active physiological metabolite of vitamin A, using the human promyelocytic leukemic cell line HL-60, which can be induced to differentiate with retinoic acid. Retinoyl beta-D-glucuronide (1 microM) inhibited HL-60 cell proliferation by 55-75%, inhibited tritiated thymidine incorporation into DNA by 63-80%, and induced 38-50% of the cells to differentiate into mature granulocytes. The potency of growth inhibition and induction of differentiation by retinoyl beta-D-glucuronide was similar to that of all-trans-retinoic acid. The continuous presence of either retinoyl beta-D-glucuronide or all-trans-retinoic acid was not required to obtain maximum growth arrest and differentiation: a 1-hr exposure of HL-60 cells to the retinoids gave the same response (measured after a total incubation time of 48 hr) as a 24-hr or 48-hr continuous treatment. Retinoyl beta-D-glucuronide (0.1-0.2 mM) was 50% less cytotoxic to HL-60 cells than all-trans-retinoic acid at an equimolar concentration. Retinoyl beta-D-glucuronide was not significantly metabolized to other retinoids; retinoic acid was not formed during incubation. We conclude that retinoyl beta-D-glucuronide can arrest HL-60 cell proliferation and induce their differentiation into mature granulocytes; it may act by itself or by being hydrolyzed to retinoic acid, which could be immediately utilized and metabolized. The therapeutic use of this retinoid as an antineoplastic agent is suggested.

Topics: Cell Differentiation; Cell Division; Cell Line; DNA Replication; Humans; Kinetics; Leukemia, Myeloid, Acute; Tretinoin

We have previously demonstrated that a combination of interferon beta and a differentiation agent, dimethyl sulfoxide (DMSO), is cytotoxic for HL-60 cells, a human promyelocytic leukemic cell line. We now report that a combination of recombinant interferon alpha (Intron; Schering) and retinoic acid is synergistically cytostatic for HL-60 cells. Retinoic acid (RA) induced the differentiation of HL-60 cells into granulocytes. Interferon (IFN) alone at 1-1000 IU/ml had no effect on either differentiation or proliferation of HL-60 cells. The addition of 1000 IU/ml of IFN and 10(-7) M RA at the initiation of culture reduced the number of viable cells to 28% of that observed for cells treated with RA alone. The decreased number of cells was a result of decreased cellular proliferation, rather than of a cytotoxic effect of the combination. IFN-RA-treated cells differentiated more rapidly than cells treated with RA alone. In addition, the final percentage of mature cells was increased at day 7 in IFN-RA-treated cultures, as compared with RA-treated cells. Simultaneous treatment of the cells with IFN and RA decreased the concentration of RA needed to induce differentiation or to exert a cytostatic effect. Significant changes in the nuclear structure of RA-treated HL-60 cells after 24 h have been reported. Cells were pulsed with RA for 24 h, washed, and IFN added. At day 7, cell growth was inhibited to the same extent as that of cells continuously exposed to IFN-RA. However, while 70% of the continuously exposed cell differentiated, cells pulsed with RA and subsequently treated with IFN did not differentiate. The results of this investigation further support our findings that combinations of IFN and inducers of differentiation may be of importance in the treatment of leukemia.

Topics: Cell Differentiation; Cell Division; Cell Line; Drug Synergism; Granulocytes; Hematopoiesis; Humans; Interferon Type I; Leukemia, Myeloid, Acute; Recombinant Proteins; Time Factors; Tretinoin

Studies with human myeloid leukemia cell lines indicate that combined interferon (INF) and retinoic acid (RA) have greater effects in inhibiting cell growth and in inducing terminal differentiation than either agent alone. Consequently, we studied the effects of these agents, singly and in combination, on fresh leukemic blast cells obtained from 13 acute myelogenous leukemia (AML) patients, most of whom were subsequently treated with recombinant leukocyte-alpha A interferon (rINF-alpha A). The in-vitro response to rINF-alpha A and RA was assessed in an established myeloid leukemic blast cell clonogenic assay containing conditioned medium from phytohemagglutinin-stimulated lymphocytes. Strong inhibition of colony cell growth (greater than or equal to 50%) was observed in 4/10 cases treated with rINF-alpha A alone, but only at high concentration (greater than or equal to 2500 U/ml) and in 4/10 cases treated with RA alone (5 X 10(-8) M or 5 X 10(-7) M). Combined rINF-alpha A and RA augmented the inhibition of primary or secondary colony cell growth in 5/8 evaluable cases. Stimulation of leukemic cell differentiation was observed in 1/8 cases by rINF-alpha A alone and in 4/7 cases by RA alone. Combined rINF-alpha A and RA enhanced cell differentiation in 4/7 cases. In addition, increased inhibition of clonal cell growth and/or differentiation by RA alone was observed in 2/5 cases following in-vivo rINF-alpha A treatment. These results suggest that treatment with combined rINF-alpha A and RA may be rewarding in some cases of AML.

Topics: Cell Differentiation; Cell Division; Drug Synergism; Interferon Type I; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Recombinant Proteins; Tretinoin; Tumor Stem Cell Assay

Recently, a novel approach has been used in the treatment of leukemia: induction of the leukemic cells to undergo terminal differentiation. Based on its in vitro ability to induce differentiation in several myeloid leukemic cell lines, retinoic acid (RA) has been applied clinically in cases of myelodysplastic syndromes and acute myeloid and promyelocytic leukemia. In the present study we have determined in detail the ability of RA to induce expression of granulocytic functions in a human promyelocytic leukemia cell line (HL-60) and compared it with that of dimethylsulfoxide (DMSO). Several granulocytic characteristics (phagocytosis, surface adherence and generation of free radicals in response to phorbol-ester) were induced to the same degree by both agents. Other normal neutrophil functions, including lysozyme accumulation, spontaneous migration, chemotactic activity toward zymosan-activated serum (containing C5a), the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and spontaneous motility in semi-solid medium were induced by DMSO, but they were absent or incompletely expressed in RA-induced cells. In contrast, only RA induced migration toward leukotriene B4 (LTB4). Simultaneous treatment with RA and DMSO proved synergistic with respect to morphological maturation and several functions (e.g. NBT reduction), but complementary stimulation of other activities (e.g. chemotaxis, lysozyme content) could not be demonstrated. Furthermore, characteristics induced by DMSO (i.e., expression of C5a and FMLP receptors and accumulation of lysozyme) were inhibited by the addition of RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Adhesion; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Granulocytes; Humans; Kinetics; L-Lactate Dehydrogenase; Leukemia, Myeloid, Acute; Muramidase; Neutrophils; Phagocytosis; Tretinoin

Topics: Cytarabine; Drug Therapy, Combination; Humans; Leukemia, Myeloid, Acute; Tretinoin

Cells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulocytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70% of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkenyl-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the untreated cells, while 1,2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1,2-diacyl GPE decreased to 63%, while those of 1-alkenyl-2-acyl GPE, choline-containing glycerophospholipids, and phosphatidylinositol increased to 169, 154, and 153%, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.

Topics: Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Phospholipid Ethers; Phospholipids; Tretinoin

DNA synthesis inhibitors and vincristine greatly enhance the response of leukaemic and dysplastic cells to differentiation inducing agents such as retinoic acid (RET). Differentiation induction therapy is an attractive therapeutic approach in myelodysplasia (MDS) and in acute myeloid leukaemia (AML) in the elderly, since it should be possible to increase the production of mature cells, at the expense of precursor cells, without incurring the complications of intensive cytotoxic therapy. Single agent differentiation therapy has, however, not been highly successful. We have therefore investigated the use of synergistic combinations of agents. We treated nine patients (6 with MDS, 3 with AML) with 13-cis-retinoic acid (up to 100 mg/m2/day) in combination with either 6-thioguanine (20-40 mg/day in 14-57 day courses) or with vincristine (1-2 mg as a single injection during a four-day course of RET). Seven patients responded with an increase in the mature cells of at least one haemopoietic lineage. A concomitant decrease in marrow blasts was observed in 3/4 responding patients. The retention of dysplastic and karyotypic abnormalities and lack of a hypoplastic phase all suggested that differentiation induction was occurring in vivo. Prior failure to respond to therapy with single agents (RET in two and cytosine arabinoside in five patients) suggests that the synergy observed in vitro operates in vivo. In-vitro studies on marrow cells from seven patients demonstrated synergistic differentiation induction in 6/7 samples. The seventh patient was one of the two who did not respond clinically. The second of these clinically unresponsive patients had cells which were relatively refractory to RET in vitro, suggesting that in-vivo and in-vitro responses may be related.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Cell Differentiation; Cytarabine; Drug Synergism; Female; Humans; Karyotyping; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Thioguanine; Tretinoin; Tumor Cells, Cultured; Vincristine

Topics: Cell Differentiation; Cell Line; Culture Media; Humans; Leukemia; Leukemia, Experimental; Leukemia, Myeloid, Acute; Lymphocytes; Phytohemagglutinins; Tretinoin

Mr 145,000 nucleolar protein antigen (p145) is associated with growing cells (R. L. Ochs et al., J. Cell Biol., 101: 211a, 1985) and has been found in a broad range of human cancers (J. W. Freeman et al., Cancer Res., 46: 3593-3598, 1986). In this study the presence of nucleolar antigen p145 was examined in the human promyelocytic tumor cell line HL-60 which was induced to differentiate by retinoic acid. Differentiation was monitored by morphological changes, [3H]thymidine accumulation, the ability of cells to reduce nitroblue tetrazolium, and cell number. The monoclonal antibody to nucleolar antigen p145 produced bright immunofluorescence in all cycling interphase HL-60 cells; during mitosis only diffuse staining was detected. Nucleolar antigen p145 in HL-60 cells was undetectable after 132 h of treatment with retinoic acid. The absence of nucleolar antigen p145 was associated with an 81% decline in thymidine accumulation and apparent inactivation of ribosomal and nonribosomal DNA transcription as observed by electron microscopy. The loss in expression of the antigen also correlated with increased nitroblue tetrazolium-positive cells, appearance of morphologically distinct myeloid cells, and termination of cell proliferation. These data indicate that the expression of nucleolar antigen p145 occurred in cycling HL-60 cells but not in terminally differentiated noncycling HL-60 cells.

Topics: Antibodies, Monoclonal; Antigens; Cell Differentiation; Cell Division; Cell Line; Cell Nucleolus; Fluorescent Antibody Technique; Humans; Leukemia, Myeloid, Acute; Microscopy, Electron; Molecular Weight; Tretinoin

The differentiation of HL-60 cells induced by 1 alpha,25 dihydroxyvitamin D3 was found to be separated into two stages, i.e. commitment and promotion. Most of the HL-60 cells were committed to monocyte/macrophage lineage by pretreatment with 1 alpha,25 dihydroxyvitamin D3 (5-50 ng/ml) for 18-24 hr. The promotion in the second stage was inducer and lineage independent; treatment with 1.25% DMSO for 2 or 3 days promoted the differentiation of the committed HL-60 cells by 1 alpha,25 dihydroxyvitamin D3 into monocyte/macrophage lineage, but not granulocyte lineage.

Topics: Antibodies, Monoclonal; Antigens, Surface; Calcitriol; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Epitopes; Esterases; Granulocytes; Humans; Leukemia, Myeloid, Acute; Macrophages; Time Factors; Tretinoin

Both peripheral blood monocytes and neutrophils are known to be capable of lysing a variety of extracellular tumor and non-tumor cell targets. The HL-60 human promyelocytic leukemia cell line has served as a useful model of human granulocyte and macrophage differentiation in studies from many laboratories. We have previously reported that phorbol diesters, which induce differentiation along the macrophage pathway, stimulate HL-60 cells to become strikingly cytotoxic to a variety of red cell targets. We now report that agents known to differentiate HL-60 along the granulocyte pathway (retinoic acid, dimethyl sulfoxide, 5-azacytidine) do not, in themselves, induce HL-60 to become cytotoxic. However, previous exposure (3-5 days) to these granulocyte pathway active agents markedly enhances phorbol diester-triggered killing. This enhancement is particularly striking at decreased effector:target ratios (as low as one effector per five targets) and is also demonstrated by a shift to lower concentrations of the phorbol diester dose-response curve. Retinoic acid is the most effective of the three agents tested, although priming (previous exposure) with dimethyl sulfoxide or 5-azacytidine also markedly enhances killing. These studies demonstrate that HL-60-mediated killing may be dissected pharmacologically into at least two distinct steps and further support the utility of this model system in studies of the development of macrophage-like cytotoxic cells. This system has also proven to be useful in the characterization of cytokines which mimic the differentiation effects of retinoic acid and dimethyl sulfoxide (J. A. Leftwich and R. E. Hall, manuscript in preparation).

Topics: Azacitidine; Cell Differentiation; Cell Line; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Leukemia, Myeloid, Acute; Macrophages; Monocytes; Phorbol 12,13-Dibutyrate; Phorbol Esters; Tetradecanoylphorbol Acetate; Tretinoin

Retinoic acid has been shown to induce large accumulations of tissue transglutaminase in cultured myeloid cells. Addition of retinoic acid to mouse resident peritoneal macrophages increased the level of tissue transglutaminase mRNA within 30-60 min. Retinoic acid also increased tissue transglutaminase mRNA levels in human promyelocytic leukemia (HL-60) cells. These studies show that retinoic acid can induce acute alterations in specific gene expression in both normal and leukemic myeloid cells.

Topics: Animals; Enzyme Induction; Gene Expression Regulation; Leukemia, Myeloid, Acute; Macrophages; Male; Mice; Molecular Weight; Protein Processing, Post-Translational; Time Factors; Transglutaminases; Tretinoin

The differentiation-inducer activity of the aromatic retinoid etretin and of its ethyl ester etretinate (Tigazon) was tested on the human myelomonocytic leukemia cell lines HL-60 and U-937 and compared to that of retinoic acid. Whereas all-trans and 13-cis isomers of retinoic acid were equally potent in inducing the differentiation of about 80% of the cells of both lines, etretin and etretinate were found to be totally ineffective. These results indicate that neither compounds should be used in vivo in the treatment of acute myelocytic leukemia patients.

Topics: Acitretin; Cell Differentiation; Cell Line; Etretinate; Humans; Leukemia, Myeloid, Acute; Stereoisomerism; Structure-Activity Relationship; Tretinoin

Topics: Azaguanine; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Drug Resistance; Humans; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Leukemia, Myeloid, Acute; Tretinoin

Differentiation of human promyelocytic leukemia cells (HL-60) in response to several classes of inducing agents is characterized by the sequential appearance of granulocytic or monocytic markers. Compounds that increase intracellular cAMP in HL-60 cells induce a program of maturation in which cells demonstrate early functional phagocytic properties. Cyclic nucleotide metabolism was studied during monocytic and granulocytic differentiation of the HL-60 cell line. In synchronous and nonsynchronous cell cultures, cyclic AMP levels were raised 300-fold or 25-fold in response to N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphate or the combination of prostaglandin E2 and theophylline, respectively. No reproducible changes in intracellular adenosine 3':5'-cyclic monophosphate levels occurred in response to retinoic acid or dimethyl sulfoxide, suggesting that changes in adenosine 3':5'-cyclic monophosphate levels alone do not mediate cell maturation induced by these compounds. Agents that increased intracellular cyclic AMP in HL-60 cells slowed the progress through the S and G2-M phase of the cell cycle, whereas other agents such as dimethyl sulfoxide stimulated cells through this same period.

Topics: Bucladesine; Cell Cycle; Cell Division; Cell Line; Cyclic AMP; Dimethyl Sulfoxide; Dinoprostone; Humans; Leukemia, Myeloid, Acute; Prostaglandins E; Protein Kinases; Theophylline; Tretinoin

Topics: Aged; Bone Marrow; Child; Cholecalciferol; Combined Modality Therapy; Cytarabine; Female; Follow-Up Studies; Humans; Interferon Type I; Leukemia, Myeloid, Acute; Male; Middle Aged; Prognosis; Tretinoin

Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; DNA Replication; Heme; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Receptors, Cell Surface; Receptors, Transferrin; Tetradecanoylphorbol Acetate; Transferrin; Tretinoin

Functional and specific histamine H2 receptors were characterized in human peripheral monocytes and in U-937 cells, before and after retinoic acid--induced differentiation into monocyte/macrophage-like cells. The relative potencies of histamine and the H1, H2 receptor agonists and antagonists studied are remarkably similar in U-937 cells and U-937 monocytes. There is no change in histamine concentration and activity of the enzymes forming and degrading histamine during monocytic-like differentiation. The results raise the possibility that histamine H2 receptors might be involved in pathophysiological regulations (proliferation/differentiation and biological function) of normal and leukemic monocytes.

Topics: Adult; Cell Differentiation; Cell Line; Cyclic AMP; Histamine; Humans; Leukemia, Myeloid, Acute; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Monocytes; Receptors, Histamine; Receptors, Histamine H2; Tretinoin

Superoxide production and chemiluminescence induced in differentiated HL-60 cells by the chemoattractant formylmethionyl-leucyl-phenylalanine: In order to study the generation of oxidative metabolites in relation to cell differentiation, dimethyl sulfoxide (DMSO) and retinoic acid (RA) differentiated HL-60 cells were stimulated with the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP). The oxidative response was measured as luminol-dependent chemiluminescence, lucigenin-dependent chemiluminescence, and cytochrome c reduction. Cells grown in the presence of DMSO or RA progressively expressed morphological changes, and when the mature cells were exposed to FMLP the cells produced oxidative metabolites. Quantitatively the HL-60 cells grown in the presence of DMSO gave rise to the most pronounced response. No correlation was obtained between superoxide production, luminol-chemiluminescence and lucigenin-dependent chemiluminescence, indicating that different aspects of the oxidative response are elucidated by the three different methods. Furthermore, the experiments show that DMSO and RA-induced differentiation of HL-60 cells leads to granulocyte-like cells with different abilities to produce oxidative metabolites, possibly due to differences in receptor function.

Topics: Cell Differentiation; Cell Line; Cytochrome c Group; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Luminescent Measurements; N-Formylmethionine Leucyl-Phenylalanine; Superoxides; Tretinoin

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10(-6) M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na]i, which reaches its maximum after 6 h, and an increase in the activity of the Na+-pump, which is reflected by an ouabain-sensitive K+ influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabain-binding sites occurs, this being accompanied by an increase in the number of K+ ions actively transported by each site. The effect of modulation of Na+-pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination, these drugs induced rapid stimulation of the Na+-pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na+]i and in the activity of the Na+-pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.

Topics: Cell Differentiation; Cell Line; Enzyme Activation; Humans; Kinetics; Leukemia, Myeloid, Acute; Neutrophils; Ouabain; Potassium; Sodium; Sodium-Potassium-Exchanging ATPase; Tretinoin

We investigated the influence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (ABA) on induction of phenotypic markers of granulocyte differentiation by retinoic acid and markers of macrophage differentiation by TPA in HL-60 cells. The differentiation of HL-60 cells towards the granulocyte lineage was assessed by hexose monophosphate shunt activity, proportion of cells capable of reducing NBT dye, and the appearance of recognizable neutrophils and bands. The effect of ABA and retinoic acid on NBT dye reduction and appearance of mature neutrophils and bands was synergistic, whereas the effects of these agents on hexose monophosphate shunt activity were additive. The differentiation inducing capacity of ABA in the presence of retinoic acid was dose-related. The influence of ABA on TPA-induced markers of macrophage differentiation was assessed by determining the proportion of adherent cells produced after treatment and by measuring acid phosphatase activity in the adherent cell fraction. In the presence of ABA, the number of cells adhering to plastic declined after day 2 of exposure to TPA, and acid phosphatase activity in adherent cells was inhibited fourfold (p = 0.01). The influence of ABA on the phenotypic markers of granulocyte and macrophage differentiation was detectable at concentrations that were not cytotoxic. The influence of ABA on HL-60 differentiation is similar to that previously reported for human bone marrow CFU-GM. Our data suggest that poly(ADP-ribose) polymerase plays a role in differentiation of HL-60 cells and that HL-60 might provide a useful model for evaluating control mechanisms involved in the differentiation of CFU-GM.

Topics: Acid Phosphatase; Benzamides; Cell Differentiation; Cell Line; Granulocytes; Hematopoietic Stem Cells; Humans; Isoniazid; Leukemia, Myeloid, Acute; Macrophages; Poly(ADP-ribose) Polymerase Inhibitors; Tetradecanoylphorbol Acetate; Tretinoin

The appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells was investigated during 1 alpha,25-dihydroxyvitamin D-3-induced monocytic differentiation. 1 alpha,25-Dihydroxyvitamin D-3-treated HL-60 cells acquired the ability to convert [1-14C]arachidonic acid to thromboxane B2 and prostaglandin E2 during monocytic differentiation. The major cyclooxygenase product synthesized by the HL-60 cells after 3-4 days exposure to 1 alpha,25- dihydroxyvitamin D-3 (48 nM) was thromboxane B2 and its production was about 19-25-times higher than that of untreated HL-60 cells. The percent conversion of thromboxane B2 from [1-14C]arachidonic acid in the 1 alpha,25-dihydroxyvitamin D-3 (48 nM, 3 day exposure)-treated HL-60 cells was about 4.4% as compared to that (about 0.3%) of the untreated cells, whereas the percent conversion of thromboxane B2 from [1-14C]prostaglandin H2 in the 1 alpha,25-dihydroxyvitamin D-3-treated cell homogenate was about 22.4% as compared to that (about 13.6%) of the untreated cell homogenate. The stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on thromboxane B2 production from [1-14C]arachidonic acid and from [1-14C]prostaglandin H2 in HL-60 cells was inhibited by the addition of cycloheximide (1 microgram/ml). However, 1 alpha,25-dihydroxyvitamin D-3 (48 nM) did not significantly stimulate the arachidonic acid release either in HL-60 cells or in 1 alpha,25-dihydroxyvitamin D-3-induced cells. These results suggest that the stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on the thromboxane production in HL-60 cells was not due to the activation of phospholipase A2 but due to the induction of fatty acid cyclooxygenase and thromboxane synthetase activities. Thromboxane A2 actively produced during the monocytic differentiation of HL-60 cells could influence the cell adhesiveness of the monocyte-macrophage-differentiated cells.

Topics: Arachidonic Acid; Arachidonic Acids; Calcitriol; Carbon Radioisotopes; Cell Differentiation; Cell Line; Cycloheximide; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Methacrylates; Monocytes; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Thromboxane B2; Time Factors; Tretinoin

The ability of retinoic acid (RA) to promote 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-initiated macrophage differentiation was examined in human promyelocytic leukemia cell line HL-60. One-hour exposure to 10 nM TPA and subsequent exposure for 48 h to 1 microM RA following removal of TPA rapidly induced the macrophage phenotype in 65% of the cells. This effect was comparable to continuous exposure for 48 h to TPA alone, but contrasted with the absence of macrophage-like cells after RA treatment alone or the induction of 10% of the cell population to a macrophage phenotype after 1-h exposure to TPA. The effect of TPA + RA was accompanied by increased cell adherence and increased nonspecific esterase activity but not by a change in the reduction of nitroblue tetrazolium. Protein kinase C (PK-C) activity was increased 35-40% in cells treated for 1 h with TPA alone or after subsequent exposure to RA. Cells treated for 48 h with RA exhibited a 2-fold increase in PK-C activity while cells exposed to TPA for 48 h lost all PK-C activity. The changes in PK-C activity in TPA + RA-treated cells were accompanied by Ca2+/phospholipid(PL)-dependent phosphorylation in vitro of pp38 which is characteristic of treatment with RA alone, as well as the Ca2+/PL-independent phosphorylation in vitro of pp82 and pp130 (vinculin) which is prevalent in cells treated continuously with TPA alone and is absent in RA-treated cells. These results indicate that the macrophage phenotype induced by TPA + RA is similar to that produced by continuous exposure to TPA alone with respect to their in vitro phosphoprotein patterns, cytochemical markers, cell adherence and morphology, but that the disappearance of PK-C is not an obligatory characteristic of these cells.

Topics: Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Macrophages; Muscle Proteins; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin; Vinculin

We have produced a murine IgM monoclonal antibody (Y201) that recognizes a cell surface antigen present on HL60 cells. Seventy percent of uninduced HL60 cells expressed Y201 antigen, while the remainder did not. There were no morphological differences between HL60 cells that expressed Y201 antigen and cells that did not express Y201 antigen. Cells with the greatest number of antigenic sites were found to have greater proliferative capacity in liquid culture and in soft agar than did HL60 cells deficient in this marker. Expression or lack of expression of the Y201 antigen is not constant over a prolonged period in that both subpopulations ultimately reproduced the original pattern of antigenic expression when grown in liquid culture. The antigen identified by Y201 was lost with terminal differentiation of HL60 cells using a variety of inducers. Loss of Y201 antigen during differentiation was associated with a decrease in proliferative capacity in soft agar. Loss of Y201 antigen by greater than 95% of differentiated HL60 cells was associated with loss of proliferative capacity. These data suggest that HL60 cells are heterogeneous in regard to proliferative capacity and that this heterogeneity is associated with expression of the cell surface antigen identified by Y201.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody Specificity; Antigens, Neoplasm; Antigens, Surface; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Flow Cytometry; Granulocytes; Humans; Leukemia, Myeloid, Acute; Monocytes; Tretinoin

A patient with acute promyelocytic leukemia (APL) and laboratory evidence of fibrinolysis who could not be treated with aggressive cytostatic regimens because of Aspergillus pneumonia was treated with cis-retinoic acid (RA), a substance that can induce differentiation and maturation of APL cells. After seven weeks of daily oral treatment, he went into complete remission, and signs of coagulopathy disappeared. Meanwhile, the Aspergillus pneumonia could be treated adequately. Based on the experience in this single patient, RA deserves further evaluation in the treatment of APL.

Topics: Adult; Aspergillosis; Bone Marrow; Humans; Leukemia, Myeloid, Acute; Male; Pneumonia; Tretinoin

The addition of retinoic acid to human promyelocytic leukemia cells in culture results in their differentiation to mature myeloid forms with acquisition of the differentiated phenotype, i.e., the ability to reduce nitroblue tetrazolium. A heavily pretreated patient with acute promyelocytic leukemia and residual malignant cells in his marrow after multiple courses of chemotherapy was given 13-cis-retinoic acid upon demonstration of both morphologic and functional differentiation of his leukemic cells by transretinoic acid in vitro. The patient achieved a complete remission and was maintained on 13-cis-retinoic acid for 1 year, when the patient relapsed with a population of cells that were resistant to retinoic acid-induced differentiation.

Topics: Adult; Cell Differentiation; Cytarabine; Humans; In Vitro Techniques; Isotretinoin; Leukemia, Myeloid, Acute; Male; Tretinoin

The capability of HL-60 cells to undergo G1/0 specific growth arrest and myeloid differentiation in response to isomers of retinoic acid (RA) having an altered alkyl chain was determined. At a concentration where beta-all trans RA induces myeloid differentiation and G1/0 specific growth arrest, 11,13-dicis and 9,13-dicis isomers failed to induce significant phenotypic differentiation, assayed by the inducible superoxide production characteristic of mature myeloid cells, but could induce moderate G1/0 specific growth arrest. The 9-cis and 11-cis isomers induced both phenotypic differentiation and G1/0 specific growth arrest. The occurrence of G1/0 specific growth arrest without phenotypic differentiation indicates that the induced cellular programs leading to phenotypic differentiation and G1/0 specific growth arrest are not tightly coupled. Within the 48-h period usually required by beta-all trans RA to induce onset of phenotypic differentiation and G1/0 specific growth arrest, all isomers could complete early events in the cellular programs leading to G1/0 specific growth arrest and phenotypic differentiation, but the dicis isomers could not complete late events in the phenotypic differentiation program. The capability of dicis isomers to drive late events in the G1/0 specific growth arrest program was also compromised. Characteristic early and late changes in Ca2+ binding cytosolic proteins induced by RA and its isomers were consistent with their early and late capabilities. Failure of the dicis isomers to induce differentiation was thus due to a failure in late events associated with aberrations in cytosolic, Ca2+ binding proteins. The results suggest a model in which RA presents two signals to induce HL-60 terminal differentiation.

Topics: Calcium-Binding Proteins; Cell Cycle; Cell Differentiation; Cell Line; Cell Nucleus; Cytosol; Humans; Isomerism; Kinetics; Leukemia, Myeloid, Acute; Molecular Weight; Neoplasm Proteins; Structure-Activity Relationship; Time Factors; Tretinoin

The effect of the combination of two different agents on myeloid differentiation was studied. Human promyelocytic cells (HL-60) and monoblast-like cells (U-937) were treated with 1-B-D arabinofuranosyl cytosine (Ara-C) alone, or in combination with retinoic acid (trans-RA). The known dual potentiality of HL-60 promyelocytes was confirmed by their ability to mature into granulocytes following induction by retinoic acid and into monocyte-like cells following treatment with 1-B-D arabinofuranosyl cytosine. The U-937 cell line differentiated to monocyte-like cells with either of the two drugs. The differentiation induced by Ara-C involved an irreversible step after 24-h incubation with the drug, was concentration dependent, and far more superior on U-937 cells than on HL-60 cells. The outcome of these two cell lines after treatment with both Ara-C and trans-RA was also different: this combination maintained the monocyte differentiation in the HL-60 cell line, and resulted in a higher sensitivity of U-937 cells to Ara-C, as indicated by a cell response to low Ara-C concentrations and a more rapid expression of monocyte specific properties.

Topics: Antibodies, Monoclonal; Cell Differentiation; Cell Line; Cells, Cultured; Cytarabine; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Synergism; Humans; In Vitro Techniques; Isomerism; Leukemia, Myeloid, Acute; Nitroblue Tetrazolium; Tretinoin

The dynamics in levels of HL-60 (a human promyelocytic leukemia cell line) c-myc RNA due to retinoic acid-induced myeloid differentiation were measured. An increase in levels of c-myc RNA occurred early in this process when the cells are known to be in a precommitment early regulatory state. The increased levels of c-myc RNA occurred before any G1-0-specific growth arrest or phenotypic differentiation. Cells in G1 and S had similar levels of c-myc RNA during this process. Onset of growth arrest and phenotypic differentiation preceded an apparent decline of c-myc RNA levels. Levels of c-myc RNA decreased only in advanced cultures with growth arrest and differentiation essentially completed. The kinetics and cell cycle dependence of the early increase in c-myc levels paralleled previously reported nuclear structural changes characteristic of the precommitment state. Since c-myc encodes a putative nuclear matrix protein, the results suggest a regulatory role for increased c-myc expression in mediating the nuclear structural change characteristic of precommitment early in the retinoic acid-induced process of HL-60 terminal myeloid differentiation. The results argue against a change in c-myc RNA levels as a requirement for G1 to S transit or for G1-0-specific growth arrest during terminal differentiation. In contrast, the results argue for a putative regulatory role for c-myc in induction of the precommitment early regulatory state. C-myc may thus act in a homeotic regulatory capacity during HL-60 terminal myeloid differentiation.

Topics: Cell Cycle; Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Proto-Oncogenes; RNA, Neoplasm; Tretinoin

It has been found that leukemia cells can be induced by various agents [e.g., by retinoic acid (RA)] to mature to a nonproliferative end stage. It has also been found that normal mature granulocytes produce a chalone-like "hemoregulatory peptide (HP)" which seems to be involved in the inhibitory proliferation control of myelopoietic cells. In view of the intended use of maturation induction treatment as an alternative to current antileukemic therapy it appeared to be of interest to know if granulocytes, obtained by RA treatment of the promyelocytic leukemia cell line HL-60, would produce normal HP or if their transformed phenotype would cause production of deviant regulatory peptide(s). It was found that conditioned media from RA-treated HL-60 cells inhibited myeloid proliferation but strongly stimulated the growth of erythroid and lymphoid cells. A low molecular weight thiol-containing peptide was isolated which inhibited colony formation by normal granulocyte-macrophage committed stem cells but unlike HP had no effect on (untreated) HL-60 cells themselves. It was also shown that the HL-60 RA peptide is chemically different from HP in terms of molecular size, electrophoretic mobility, composition, and NH2-terminal sequence, which was determined as glutamine-aspartic acid-proline. It is concluded that differentiated HL-60 cells produce hemoregulatory factor(s) with properties different from those of normal HP. The implication of a possible abnormal regulatory behavior of induced leukemic populations is discussed with respect to leukemia therapy by differentiation induction.

Topics: Cell Differentiation; Cell Line; Erythropoiesis; Granulocytes; Growth Inhibitors; Hematopoiesis; Humans; Leukemia, Myeloid, Acute; Lymphocytes; Molecular Weight; Oligopeptides; Peptides; Tretinoin

During treatment of HL-60 myeloid leukemia cells in culture with polar solvents or retinoic acid at a concentration inducing terminal maturation in 90-95% of the cells, there is a rapid decline (within 2 h) in the Vmax for influx of the folate analogue, [3H]methotrexate. Following 24 h of exposure to these agents, there is no effect on growth, but influx Vmax is reduced by 70%. After 7 days of exposure, influx Vmax is reduced 90-95%. A similar time course was seen for the reduction in intracellular levels of dihydrofolate reductase, a marker of cellular proliferation. Both the extent of terminal maturation (as determined by the extent of nitro blue tetrazolium reduction) and decrease in influx showed the same dependence on the concentration of inducer. In contrast to the effect seen on influx Vmax, both influx Km and mediated efflux of [3H]methotrexate remained unchanged in HL-60 cells exposed to inducers of maturation. Finally, evidence is presented for the coupling of this alteration on [3H]methotrexate influx with commitment of HL-60 cells to terminal maturation. This evidence shows that the effect on folate analogue influx precedes commitment and documents the irreversible nature of the reduction in influx once the majority of the cells exposed to inducer were committed to the process of maturation. The possible relevance of these results to the process of neoplastic transformation is discussed.

Topics: Acetamides; Biological Transport, Active; Cell Differentiation; Cell Line; Cells, Cultured; Folic Acid; Humans; Kinetics; Leukemia, Myeloid, Acute; Mathematics; Methotrexate; Tretinoin

A new human myeloid leukemia cell line, PL-21, consisting of promyelocytes, was microscopically and immunohistochemically studied for their maturation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid (RA). More than 80% of PL-21 cells cultured for 4 days with 10 ng/ml of TPA became macrophage-like cells with functional and histochemical properties consistent with monocytes/macrophages. The cells adhered to the plastic flask, developed phagocytic activity and macrophage-specific intracytoplasmic alpha subunit of S-100 protein, and had reduced myeloid-specific cytochemical markers. In contrast, RA-treated PL-21 cells displayed mature neutrophil-like morphology after 7 days of exposure. Most of the cells had reduced nitro blue tetrazolium and acquired phagocytic activity with persistence of myeloid-specific cytochemical markers such as peroxidase, naphthol-AS-D chloroacetate esterase, and Sudan black B. These results indicate that the PL-21 cell line ca be induced to mature into two directions of macrophages and neutrophils by chemical inducers, and will provide a useful tool for studying the differentiation of leukemic cells and searching for other differentiation inducers.

Topics: Cell Differentiation; Cell Line; Histocytochemistry; Humans; Leukemia, Myeloid, Acute; Microscopy, Electron; Nitroblue Tetrazolium; Phagocytosis; S100 Proteins; Tetradecanoylphorbol Acetate; Tretinoin

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.

Topics: Dimethyl Sulfoxide; Doxorubicin; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Myeloid, Acute; Membrane Proteins; Phorbols; Phosphoproteins; Phosphorylation; Protein Kinases; Tetradecanoylphorbol Acetate; Tretinoin

Two variants of HL-60 promyelocytic leukemia cells (HSC, OCI) that were indistinguishable by morphology, cell surface markers, DNA histograms, and by their inability to reduce nitroblue tetrazolium, were induced to differentiate by retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and by phytohemagglutinin-leucocyte conditioned medium (PHA-LCM). Only OCI cells were induced to differentiate to mature granulocytes by TPA. Both cell lines expressed, however, the monocytic associated cell surface antigen detected by MO1 monoclonal antibody in response to TPA. MO1 expression was detected as early as 32 hours after initiation of differentiation by TPA, whereas partial morphologic changes were apparent only after 72 hours. Induction of differentiation by retinoic acid led to a significant inhibition of colony formation in HSC variant (from 1522 +/- 60 to 523 +/- 20/10(4) cells plated) and in the OCI variant (from 628 +/- 20 to 185 +/- 33 colonies/10(4) cells plated). The addition of PHA-LCM further inhibited colony growth of both RA-induced cell lines (155 +/- 7/10(4) cells plated in HSC, and 59 +/- 4 in OCI). PHA-LCM by itself reduced HL-60 colony numbers in a dose-related manner, and also increased the expression of MO1 on noninduced HSC and OCI cells. These observations suggest that differentiation of HL-60 cells is not necessarily accompanied by concomitant change in morphology, cell surface characteristics, and proliferation potentials, and may be dependent on different degrees of cellular commitment. They also suggest a role for growth factors in the induction to maturation of leukemic cells.

Topics: Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Antigens, Surface; Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphokines; Male; Tetradecanoylphorbol Acetate; Tretinoin

The relationship between calcium ions and the differentiation of human promyelocytic leukemia HL-60 cells was investigated. Proliferation of HL-60 cells incubated in calcium-free medium was inhibited without cell differentiation. On the other hand, incubation with 100 microM verapamil markedly inhibited cell proliferation and caused slight cell differentiation into monocytes. Both calcium-free medium and 100 microM verapamil enhanced HL-60 cell differentiation after treatment with 1 nM 1 alpha,25-dihydroxyvitamin D3, 1 nM beta-all-trans-retinoic acid, or 0.75% dimethyl sulfoxide. However, no enhancement was obtained by treatment with 1 nM 12-O-tetradecanoylphorbol-13-acetate. The free cytosolic calcium concentration was measured by the intracellularly trappable fluorescent calcium indicator, quin 2. The increase of intracellular calcium induced by 250 nM ionomycin was completely blocked by 100 microM verapamil in calcium-free medium, suggesting that the high concentration of verapamil (100 microM) blocks the intracellular calcium mobilization in HL-60 cells. Therefore, the enhancing effect of calcium deprivation or verapamil of HL-60 cell differentiation seemed to be closely related to the inhibition of intracellular calcium mobilization. This speculation is supported by the finding that 50 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, an intracellular calcium antagonist, also enhanced HL-60 cell differentiation induced by 1 alpha,25-dihydroxyvitamin D3, beta-all-trans-retinoic acid, or dimethyl sulfoxide.

Topics: Calcitriol; Calcium; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Ethers; Gallic Acid; Humans; Ionomycin; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin; Verapamil

Acute leukemia is the result of a defect in the process of normal cellular differentiation. Human leukemia cell lines (HL60, RDFD-2) have been established which can be induced to differentiate into phenotypically mature cells by a variety of agents. Recent evidence suggests that cyclic adenosine 3'-5'-monophosphate (cAMP) and the cAMP dependent protein kinase (cAMP-dPK) may be intimately involved in myeloid differentiation. The addition of low levels of a wide variety of inducers of a diverse chemical nature, dimethylformamide (DMF), retinoic acid (RA), actinomycin D (ACT-D) or hypoxanthine (HPX) prior to the addition of 8-bromo-cyclic adenosine 3'-5' monophosphate (8-Br-cAMP), cholera toxin (CT) or the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) results in marked potentiation of differentiation of both HL60 and RDFD cells as manifested by the acquisition of the antigen OKM-1, the ability to reduce nitroblue tetrazolium or expression of the chemotactic receptor. Potentiation of differentiation is also observed when 8-Br-cAMP, CT or IBMX is added prior to the addition of either RA, DMF, ACT-D or HPX. These results suggest a role for cAMP in myeloid differentiation.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Cell Differentiation; Cell Line; Cholera Toxin; Cyclic AMP; Dactinomycin; Dimethylformamide; Drug Synergism; Humans; Hypoxanthine; Hypoxanthines; Leukemia, Myeloid, Acute; Time Factors; Tretinoin

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.

Topics: Acyltransferases; Bucladesine; Cell Line; Dimethyl Sulfoxide; Dimethylformamide; Dose-Response Relationship, Drug; Enzyme Induction; Humans; Hypoxanthine; Hypoxanthines; Leukemia, Myeloid, Acute; Retinol-Binding Proteins; Time Factors; Transglutaminases; Tretinoin

HL60 cells induced to differentiate into myeloid cells by retinoic acid exhibited a 300-fold increase in transglutaminase (TGase) activity which peaked on day 5. HL60 cells induced to differentiate into monocytes by a phorbol ester tetradecanoylphorbol-12-myristate-13-acetate (TPA) had a greater than 840-fold increase in TGase activity on day 7. In contrast, cells induced to differentiate along the myeloid pathway by dimethyl sulfoxide (DMSO) exhibited no increase in TGase activity. Elevation of TGase activity appears to be characteristic of monocyte differentiation and retinoic acid-induced myeloid differentiation but not of myeloid differentiation in response to DMSO.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Macrophages; Monocytes; Phorbols; Tetradecanoylphorbol Acetate; Time Factors; Transglutaminases; Tretinoin

A leukemic cell line (HL60) and acute myeloblastic leukemia (AML) cells from six patients were co-cultured with normal marrow cells to assess their effects on growth of normal CFU-GEMM. The effects of the following inducers: 12-0-tetradecanoyl-phorbol-13-acetate (TPA), retinoic acid (RA), dimethylsulphoxide (DMSO), 1-25 (OH) D3 (Vitamin D3) and PHA-LCM on both the HL60 and AML cells, were studied. Inhibition of growth of normal CFU-GEMM was observed in the co-cultures in the presence of 1 X 10(4) HL60 or AML leukemic cells/ml. This inhibition was reversed by pretreating the HL60 line with vitamin D3, TPA and RA. No effect on growth of CFU-GEMM was noted when DMSO and PHA-LCM were used. AML cells were morphologically induced to differentiate by TPA or RA in all six cases. In three cases, reversal of inhibition of growth of normal pluripotent hemopoietic progenitors occurred and in three the inhibition of growth persisted. Regulation of inhibition by different inducers did not seem to correlate in all cases with morphological differentiation.

Topics: Cell Differentiation; Cell Line; Cholecalciferol; Culture Techniques; Dimethyl Sulfoxide; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Leukocyte Count; Neoplastic Stem Cells; Phytohemagglutinins; Stem Cells; Tetradecanoylphorbol Acetate; Tretinoin

Human promyelocytic leukemia cells (HL-60) mature into functional granulocytes in response to in vitro treatment with several classes of chemical agents. Compounds that increase intracellular adenosine 3':5' -cyclic monophosphate induce a modified program of maturation in which the cells demonstrate functional properties characteristic of mature phagocytic cells while remaining morphologically immature. We compared the developmental programs initiated by two well-studied inducers, retinoic acid and dimethyl sulfoxide, with the programs initiated by two inducers known to raise intracellular adenosine 3':5' -cyclic monophosphate; N6, O2-dibutyryl adenosine 3':5' -cyclic monophosphate and the combination of prostaglandin E2 and theophylline. In response to the increase in intracellular adenosine 3':5' -cyclic monophosphate, the cells ceased proliferation, expressed chemotactic receptors and demonstrated stimulated enzyme release within 24 h. Chemotaxis, adherence, NBT reduction and superoxide production appeared by 72 h, although the cells remained unchanged morphologically. A similar developmental program was induced by dimethylsulfoxide, but appearance of the markers was delayed by 48 h. Expression of these markers was delayed and incomplete in response to retinoic acid.

Topics: Bucladesine; Cell Differentiation; Cell Division; Cell Line; Chemotaxis; Dinoprostone; Granulocytes; Humans; Leukemia, Myeloid, Acute; Oxidation-Reduction; Prostaglandins E; Receptors, Formyl Peptide; Receptors, Immunologic; Superoxides; Tretinoin

Induction of hematopoietic differentiation was investigated in human promyelocytic leukemia cells [HL-60] using two lipophilic vitamins, retinoic acid and 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Both vitamins suppressed proliferation and induced differentiation of HL-60 cells, but 1 alpha,25(OH)2D3 was 70- to 100-fold more potent than was retinoic acid on a molar basis. Simultaneous treatment with suboptimal concentrations of 1 alpha,25(OH)2D3 (0.12 to 1.2 nM) and retinoic acid (10 to 100 nM) showed additive effects in reducing nitroblue tetrazolium, a common marker for monocyte-macrophage and granulocyte differentiation. For the study of alternative differentiation of the cells by the two vitamins, we used monoclonal antibodies specific for either human monocyte-macrophages or granulocytes and other markers specific for macrophage differentiation such as alpha-naphthyl acetate esterase activity and adherence to the dish surface. HL-60 cells were induced to differentiate alternatively into macrophages by 1 alpha,25(OH)2D3 or into granulocytes by retinoic acid. When HL-60 cells were treated with various concentrations of 1 alpha,25(OH)2D3 (1.2 to 120 nM) in the presence of 1000 nM retinoic acid which is a concentration sufficient to induce maximal granulocyte differentiation, the appearance of the markers for monocyte-macrophage differentiation by 1 alpha,25(OH)2D3 was not at all affected by the retinoic acid. These results indicate that 1 alpha,25(OH)2D3 and retinoic acid have additive effects in inducing differentiation of HL-60 cells, but monocyte-macrophage differentiation by 1 alpha,25(OH)2D3 occurs much more readily than does granulocyte differentiation by retinoic acid.

Topics: Calcitriol; Cell Differentiation; Cell Line; Drug Synergism; Granulocytes; Humans; Leukemia, Myeloid, Acute; Macrophages; Monocytes; Receptors, Calcitriol; Receptors, Steroid; Tretinoin

The effects of differentiating agents on the activity and phosphorylation pattern produced by phospholipid- and Ca2+-dependent protein kinase (PL-Ca-PK) were examined in human promyelocytic leukemia cell line HL-60. Dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increased the appearance of mature myelocytic (DMSO and RA) or monocytic [1,25(OH)2D3] cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the appearance of adherent macrophage-like cells. Coincident with the appearance of differentiated cells induced by DMSO, RA, and 1,25(OH)2D3 was an increase in PL-Ca-PK activity. In contrast, TPA treatment resulted in the rapid disappearance of PL-Ca-PK and the induction of phospholipid- and Ca2+- (PL-Ca-) independent protein kinase activity. The phosphorylation pattern resulting from endogenous PL-Ca-PK in extracts from cells treated with DMSO, RA, or 1,25(OH)2D3 showed a prominent phosphorylated protein of molecular weight 37,000 (pp37) and 38,000 (pp38) which was related to the appearance of the myelocyte/monocyte phenotype. pp37 and pp38 were also present in TPA-treated cells, but their phosphorylation was no longer dependent on the presence of phospholipid and calcium. Cells treated with DMSO and RA also exhibited a PL-Ca-dependent pp21 which was barely evident in 1,25(OH)2D3-treated cells and thus represented a myeloid cell marker. Also present was a prominent PL-Ca-dependent pp19 which remained unchanged following treatment with DMSO, RA, and 1,25(OH)2D3, but which diminished markedly in TPA-treated cells. On the other hand, TPA-treated cells exhibited a characteristic pp130 which was antigenically related to the actin binding protein, vinculin. These results indicate that there are characteristic PL-Ca-dependent phosphorylated proteins indicative of mature myelocytic and monocytic cells, as well as PL-Ca-independent phosphorylated proteins characteristic of the macrophage-like phenotype.

Topics: Calcitriol; Cell Differentiation; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Myeloid, Acute; Macrophages; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Kinases; Tetradecanoylphorbol Acetate; Tretinoin

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Light; Nitroblue Tetrazolium; Oxidation-Reduction; Scattering, Radiation; Tetrazolium Salts; Tretinoin

The myeloid lineage to which HL-60 promyelocytic leukemia cells will differentiate in response to a chemical differentiation inducer can be switched by altering the pH of the growth medium. Cells passaged previously at pH 7.2 become neutrophiles, and those passaged previously at pH 7.6 become eosinophiles after 5 to 7 days of culture in the presence of 0.5 mM butyric acid. Butyric acid and its analogues are unique in that all other chemical maturation inducers tested, such as dimethyl sulfoxide and retinoic acid, promote neutrophilic differentiation regardless of the prior culture history of the cells. This suggests that lineage commitment and maturational commitment are mechanistically separate processes in this multipotential cell line and can be independently manipulated experimentally.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Cells, Cultured; Dimethyl Sulfoxide; Eosinophils; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid, Acute; Neutrophils; Structure-Activity Relationship; Tretinoin

Glycosaminoglycans (GAGs) play an important role in cell-cell and cell-substratum interactions, and undergo specific changes during neutrophil development. Previous studies (Luikart, S.D., Maniglia, C. A., and Sartorelli, A. C. Cancer Res., 44: 2907-2912, 1984) have shown that both dimethyl sulfoxide and 4-beta-phorbol-12-beta-myristate-13-alpha-acetate decreased GAG production by a hypoxanthine-guanine phosphoribosyl transferase-deficient clone of HL-60 promyelocytic leukemia cells prior to the appearance of a mature myeloid or monocytoid phenotype. To expand these investigations further, GAGs were analyzed by cetylpyridinium chloride precipitation and DEAE-Sephacel ion-exchange chromatography after labeling of parental HL-60 cultures with [35S]sulfate and D-[3H]glucosamine for 6 h, following treatment with 1 microM all-trans retinoic acid (RA). Chondroitin sulfate represented the major GAG species produced, although endo-beta-galactosidase-sensitive undersulfated macromolecules which possibly might be keratan sulfate, were also identified. GAG production decreased over a time period of 144 h in culture. RA treatment reduced the amount of radiolabeled cell-associated GAGs by 50% after 48, 96, and 144 h of exposure. In contrast, commitment to myelocytic maturation of the majority (i.e., approximately 60%) of the cells occurred between 72 and 96 h of RA treatment. Concurrently with the appearance of mature granulocytic cells, two-thirds of the radiolabeled GAGs were recovered from the medium, compared to one-third in untreated cultures, a phenomenon that resulted in an overall alteration in the distribution of GAGs. When RA was removed by washing after either 48 h (i.e., precommitment to differentiation) or 96 h (i.e., postcommitment to differentiation), a 1.5- to 3.5-fold increase in GAG production was noted 48 h later; this increase was unrelated to the medium change or to alterations in cell cycle distribution. The amounts of endo-beta-galactosidase-sensitive macromolecules were unaltered. Thus, although 1 microM RA inhibited the synthesis of chondroitin sulfate by HL-60 leukemia cells, this inhibition was reversible by removal of the drug and appeared to be unrelated to the commitment to myelocytic maturation.

Topics: Cell Differentiation; Cells, Cultured; Glycosaminoglycans; Humans; Leukemia, Myeloid, Acute; Tretinoin

Analysis of commitment of human promyelocytic leukemia HL-60 cells to terminal granulocytic maturation induced by dimethyl sulfoxide (DMSO) or retinoic acid (RA) was accomplished using biochemical measurements and a plasma clot clonal assay system that permits a high plating efficiency of 40 to 60%. Commitment to granulocytic maturation occurs very rapidly. When cells are exposed to these inducers for only 8 to 18 h, an interval much shorter than a single generation time, and are then subcultured in inducer-free plasma clots, they demonstrate a decrease in proliferative capacity and form colonies composed of mature nitroblue tetrazolium (NBT)-positive cells along with occasional colonies containing both NBT-positive and NBT-negative cells; in both types of colony, the NBT-positive cells are widely dispersed. Undifferentiated HL-60 cells give rise to compact NBT-negative colonies of large size without cell migration. HL-60 cell differentiation induced by either DMSO or RA is associated with a progressive decline in both DNA and RNA synthesis; this includes transcriptional inactivation of ribosomal DNA sequences. In contrast to DMSO, which induces development primarily of metamyelocytes, RA treatment leads to the accumulation of more mature band and segmented neutrophils; sequential exposure of cells pretreated with DMSO to RA alone fails to cause rapid appearance of segmented neutrophils. From these studies, we conclude that HL-60 cells become very rapidly committed to terminal maturation and that DMSO and RA appear to induce granulocytic maturation via two different mechanisms.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; DNA, Neoplasm; Granulocytes; Humans; Kinetics; Leukemia, Myeloid, Acute; RNA, Neoplasm; Tretinoin

The time-dependent dynamics of nuclear structure, cell cycle transit and arrest, and cellular differentiation were studied using the human promyelocytic leukemia cell line HL-60. Myeloid differentiation of HL-60 cells was induced by 10(-6) M beta, all trans, retinoic acid (RA). During exponential growth the cells had G1, S, G2 and M durations of 9, 11, 0.5 and 0.5 h respectively. Significant growth arrest in the G1/0 phase of the cell cycle was apparent after 48 h of RA exposure or after two division cycles. Thereafter, cells arrested in G1/0 with wide dispersion in times of arrest which extended over several cell cycle generation times. The kinetics of phenotypic differentiation, detected by phorbol myristate acetate inducible superoxide production, paralleled those of G1/0 growth arrest with similar lag and dispersion. These kinetics are consistent with a model hypothesizing the existence of an S-phase differentiation control (DC) point regulating both terminal proliferation and differentiation. Before any cell differentiation or termination of cell proliferation occurred, the nuclei of RA-treated cells underwent a structural change detected by narrow-angle light scatter measured with flow cytometry. Narrow-angle light scatter was transiently reduced, reaching a nadir at 24-48 h and returning to control values at 96 h. This change was independent of cell cycle phase or total nuclear protein content. It was associated with a morphological change of the nuclear membrane from a smooth to dimpled or pitted structure. These findings focus attention on the potential significance of nuclear structural reorganization as an early event during cell differentiation.

Topics: Cell Cycle; Cell Differentiation; Cell Division; Cell Line; Cell Nucleus; DNA, Neoplasm; Granulocytes; Humans; Leukemia, Myeloid, Acute; Tretinoin

Recombinant human leukocyte interferon (IFN-alpha A) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-alpha A is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-alpha A and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represents a novel means of reducing resistant leukemic cell populations.

Topics: Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Drug Synergism; Humans; Interferon Type I; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin

Sub-lines of the cultured human promyelocytic leukemia cell line HL-60 were individually selected for their ability to sustain exponential growth in the presence of 3 structurally-unrelated inducers of granulocytic differentiation - retinoic acid (RA), dimethylsulfoxide (DMSO), and 6-thioguanine (6TG). Selections were made by step-wise augmentation to final drug concentrations of 10(-3)mM RA, 169mM (1.2%) DMSO and 0.12mM (20 micrograms ml-1) 6TG. In addition to growth resistance, cells in each sub-line displayed variable cytodifferentiation resistance to each of the 3 selective agents, which was quantitated as the ratio of the concentration of drug required to induce differentiation in 50% of the cells in each resistant sub-line versus comparably-passaged wild-type HL-60 cells. The levels of resistance/cross-resistance were as follows: RA-resistant (res) sub-line greater than 2700-fold to RA, 1.3-fold to DMSO and greater than 1.5-fold to hypoxanthine (HXN; the noncytotoxic purine base inducer analogue of 6TG); DMSO-res sub-line 2.5-fold to DMSO, 137-fold to RA and greater than 1.5-fold to HXN; and 6TG-res sub-line greater than 1.5-fold to HXN, 9-fold to RA and 1.6-fold to DMSO. These sub-lines were not cross-resistant to sodium butyrate (NaBut), a monocyte inducer, or to 12-0-tetradecanoylphorbol 13-acetate (TPA), a macrophage inducer. HL-60 sub-lines selected by exposure to a single high concentration of 5-bromo-2'-deoxyuridine (BUdR; 3.3 X 10(-2)mM) or oubain (Ou; 5 X 10(-3)mM) were not or were slightly cross-resistant to either granulocyte or monocyte inducers. Although some variations in line/sub-line phenotype were observed, this was minor compared to the quantitative variations in response to individual inducing agents. The RA-res and 6TG-res sub-lines contained numerous double minute chromosomes (indicators of amplified genes) which were either absent or present in much smaller numbers in the parental wild-type cells or in the other drug-resistant sub-lines. There was little change or a decrease in the amplification level of the known amplified oncogene c-myc in the various drug-resistant sub-lines compared to wild-type HL-60 cells. These results (a) confirm that the neutrophilic granulocytic and monocytic/macrophagic differentiation programs in HL-60 cells are mechanistically different and separable; (b) suggest that both agent-specific and common quantitative alterations contribute to the mechanism(s) for resistance to granulocyte differentiation

Topics: Bromodeoxyuridine; Butyrates; Butyric Acid; Cell Count; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Drug Resistance; Gene Amplification; Humans; Hypoxanthine; Hypoxanthines; Leukemia, Myeloid, Acute; Oncogenes; Ouabain; Phenotype; Tetradecanoylphorbol Acetate; Thioguanine; Tretinoin

Both human beta-interferon (IFN-beta) and retinoic acid (RA) are able to induce the differentiation of the human histiocytic lymphoma cell line U937, but neither one alone can effectively eliminate the leukemic cells. When U937 cells are incubated with a combination of IFN-beta (200 units/ml) and RA (0.1-1.0 microM), extensive cell death can be observed as early as day 3 posttreatment, and IFN-beta alone at a concentration as high as 10(4) units/ml is ineffective. These data suggest that there is a strong synergistic cell killing effect between IFN-beta and RA against the U937 cells. This effect is so highly selective that similar enhancement has not been detected using a closely related cell line, HL-60, and RA enhances neither IFN-alpha nor IFN-gamma in the killing of U937 cells. The mechanism of such synergism is unknown in the present study, but it appears that cytostasis or promotion of differentiation alone cannot account for this phenomenon since neither activity is enhanced to any appreciable extent.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cell Line; Cell Survival; Drug Synergism; Humans; Interferon Type I; Leukemia, Myeloid, Acute; Lymphoma; Tretinoin

Using a system of sequential daughter cell transfers in semisolid medium, we have analyzed self-renewal and differentiation of human promyelocytic leukemia cells (HL60) in presence of all-trans retinoic acid and human placental conditioned medium (HPCM). We find that retinoic acid induces coordinated losses of self-renewal potential which are followed by phenotypic differentiation. The latter occurs as an all-or-none event and is reversible in the presence of HPCM. Thus, HL60 cells that apparently had terminally differentiated (as estimated by the ability to reduce nitroblue tetrazolium [NBT]) can lose their differentiation marker and reenter the proliferative pool.

Topics: Cell Differentiation; Cell Division; Cell Line; Culture Media; Humans; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Placenta; Tretinoin

A method for clonal analysis has been developed which allows the characterization of the number and type of progeny cells produced by each single cell arising during clonal evolution. The method is based on a symmetry of self-renewal exhibited by sister cells of the human promyelocytic leukemia cell line -HL60-. This permits the use of one of the sister cells to measure the potential for self renewal of the other. Using a system of sequential daughter cell transfers in semisolid medium, we have analysed self-renewal and differentiation in individual clones exposed to all-trans retinoic acid or dimethylsulfoxide (DMSO). We find that in clones exposed to chemical inducers of differentiation commitment occurs as an all-or-none event which is preceded by coordinated but reversible losses of self-renewal potential. It is concluded that the differentiation pathway of HL60 cells has two distinct portions. These are, first, a predeterministic portion, reflected by coordinated but reversible losses of self-renewal potential, and second, a deterministic portion, reflected by irreversible phenotypic differentiation.

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Colony-Forming Units Assay; Dimethyl Sulfoxide; Granulocytes; Humans; Leukemia, Myeloid, Acute; Probability; Tretinoin; Tumor Stem Cell Assay

An alternative to a cell-kill strategy for eradication of acute myelogenous leukemia, is to restore normal differentiation. Vitamin A derivatives demonstrate differentiation-inducing activity both in vitro and in vivo on promyelocytic leukemic cells. We tested the ability of 13-cis retinoic acid to reduce proliferation and induce differentiation in 10 samples from patients with acute non-promyelocytic leukemia. DNA synthesis and leukemia colony formation were affected to varying degrees by a prolonged exposure to the vitamin A compound. Morphologically and cytochemically no differentiation was determined either after 48 h in suspension cultures or 7 additional days in semi-solid cultures. Alkaline leukocyte phosphatase, a biochemical marker of differentiation, was significantly increased in five samples. DNA synthesis in these samples was significantly reduced as compared to samples failing to express alkaline leukocyte phosphatase following 13-cis retinoic acid treatment. DNA synthesis of these same 5 samples was also strongly inhibited by Ara-C. Expression of alkaline leukocyte phosphatase following 13-cis retinoic acid exposure may be a useful indicator for cells amenable to 13-cis retinoic acid or Ara-C treatment.

Topics: Adult; Aged; Alkaline Phosphatase; Cell Differentiation; Cell Division; Cytarabine; Female; Humans; Isotretinoin; Leukemia, Myeloid, Acute; Leukocytes; Male; Middle Aged; Thymidine; Tretinoin; Tritium

In-vitro studies of leukemic cells with retinoic acid and a therapeutic clinical trial with its derivative, etretinate, in a 58 yr-old male patient with 15;17 translocation-positive acute promyelocytic leukemia (APL) in relapse are reported. Actinomycin D was used in combination. Bone marrow promyelocytes from the patient prior to etretinate and actinomycin D matured morphologically in the liquid culture with retinoic acid; 98% were matured myeloid cells after 6 days at a concentration of 10(-6) M of retinoic acid as compared with 2% in the control culture. Positive NBT reactions were seen in none of the cells in the latter but in 95% of the cells in the former. Actinomycin D, when added alone, only induced NBT positivity, but, when used in combination with retinoic acid, increased both NBT positivity and morphologically matured cells. The patient was treated daily with 2 micrograms/kg of actinomycin D (or 20 mg/m2 or 33 mg/m2 cytosine arabinoside after the 6th day) in 24-h infusions and per oral 90 mg/body of etretinate. No effectiveness was observed both morphologically and clinically. The patient expired 15 days after the initiation of etretinate. Thus, a discrepancy existed in the response of leukemic cells from this relapsed patient with APL to the in-vitro and in-vivo attempts to include differentiation by retinoids and actinomycin D.

Topics: Cell Differentiation; Dactinomycin; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Male; Middle Aged; Tretinoin

The differentiation of human leukemic HL-60 cells from their predominantly promyelocyte form to a neutrophil-like state can be induced by the addition of dimethylsulfoxide (DMSO) or retinoic acid (RA) to the growth medium. The binding of human recombinant interferon IFN-alpha A to the undifferentiated and differentiated HL-60 cells was investigated. Within 2 days after the addition of DMSO or retinoic acid to growing HL-60 cells, the binding of IFN-alpha A to treated cells increases significantly relative to its binding to untreated cells. The difference in binding of IFN-alpha A between the treated and untreated cells continues to increase for at least 3 days. Analysis of binding curves of IFN-alpha A to neutrophil-like and promyelocytic HL-60 cells leads to the conclusion that the increased binding of [125I]IFN-alpha A to neutrophil-like cells is primarily the result of an increase in the number of binding sites on these cells.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Interferon Type I; Kinetics; Leukemia, Myeloid, Acute; Neutrophils; Tretinoin

Topics: Cell Line; Cell Transformation, Neoplastic; Cytoskeleton; Humans; Leukemia, Myeloid, Acute; Pseudopodia; Tretinoin

Topics: Calcitriol; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin

In a case of acute promyelocytic leukemia (APL), the expression of terminal deoxynucleotidyl transferase (TdT), an early lymphoid marker, was detected. Double-fluorescent staining for the myeloid-specific antigens VIM-2 and VIM-D5 in combination with specific antiserum for TdT suggested a mixed leukemic cell population consisting of a morphologically, cytochemically, and immunologically promyelocytic component (80%) and a lymphoid, TdT+ component (20%) that was myelomonocytic in morphology but otherwise without any evidence of nonlymphoid nature. Fluorescent-activated cell analysis revealed that a greater number of cells reacted with monoclonal anti-T antibodies (OKT3, OKT6, and OKT11) than could be identified as lymphoid by TdT expression. As confirmed by double-staining fluorescence microscopy, a large fraction of the promyelocytic leukemia cells were biphenotypic, expressing both myeloid and lymphoid markers (50% positive for VIM-D5 and OKT6, 30% positive for VIM-D5 and OKT3). Subsequently, in vitro differentiation experiments were performed. While treatment of the cells with GCT-conditioned medium favored proliferation, with only a weak and delayed promotion of the cells towards maturation as reflected by enhanced expression of the mature T-marker T3 but persistent expression of the thymocyte antigen, exposure to all-trans and 13-cis retinoic acid resulted in marked differentiation of both the myeloid and the lymphoid cell characteristics. Retinoid treatment resulted in the loss of TdT, a partial disappearance of the T6-antigen, and the expression of the late T cell antigen T3 by almost 70% of the cells. In addition, myeloid maturation was obvious from the morphologic appearance of the cells, as well as from the expression of the OKM1-associated antigen by a majority of the cells. This report concerns a unique case of APL in which, for the first time, a coexistence of promyelocytic and lymphoid elements was detected, with exposure of the cultured leukemic cells to retinoic acid inducing maturation along both the myeloid and the lymphoid lineage.

Topics: Antigens, Surface; Cell Transformation, Neoplastic; Culture Media; DNA Nucleotidylexotransferase; DNA Nucleotidyltransferases; Female; Fibroblasts; Granulocytes; Hematopoietic Stem Cells; Humans; Hybrid Cells; Leukemia, Myeloid, Acute; Middle Aged; Phenotype; T-Lymphocytes; Tretinoin

Topics: Cell Differentiation; Cell Line; Culture Media; Drug Synergism; Glycoproteins; Granulocytes; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphokines; Monocytes; T-Lymphocytes; Tretinoin

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (alpha). Addition of a sublethal concentration of Aphidicolin (0.4 microM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. The level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. The inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G1 and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. Of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.

Topics: Antibiotics, Antineoplastic; Aphidicolin; Cell Differentiation; Cell Division; Cell Line; Diterpenes; DNA Replication; Drug Interactions; Flow Cytometry; Humans; Kinetics; Leukemia, Myeloid, Acute; Tretinoin

Numerous agents induce differentiation and maturation of neoplastic and dysplastic myeloid cells in vitro and some of these agents have been used with limited success in the treatment of patients with myelodysplastic syndromes (MDS) and myeloid leukaemias. We recently proposed that physiological and pharmacological agents which enhance differentiation and maturation in vitro act by two fundamentally different routes: (1) by hastening the progression through various differentiation/maturation steps; (2) by slowing proliferation (usually by inhibition of DNA synthesis). In order to test this thesis we looked for synergistic effects on differentiation using pairs of agents from the two groups in cultures of cells from myelodysplastic and acute myeloid leukaemia (AML) patients and from normal marrow donors. The results with three MDS, two AML and three normal samples show that combinations of differentiation inducing agents (retinoic acid, N-methylformamide) with DNA synthesis inhibitors (6-mercaptopurine, cytosine arabinoside and aphidicolin) produce a differentiation inducing effect equivalent to that of 10-100, or even 1000 fold higher concentrations of single agents. Myelotoxic effects in vitro were not synergistic. The use of these synergistic combinations should greatly enhance the usefulness of differentiation inducers in the therapy of MDS and myeloid leukaemia.

Topics: Antineoplastic Combined Chemotherapy Protocols; Aphidicolin; Bone Marrow; Bone Marrow Diseases; Cell Differentiation; Cells, Cultured; Cytarabine; Diterpenes; DNA; Drug Synergism; Formamides; Humans; Leukemia, Myeloid, Acute; Mercaptopurine; Preleukemia; Syndrome; Tretinoin

Induction of differentiation of the HL-60 human promyelocytic leukemia cell line by retinoic acid or 1 alpha,25(OH)2D3 was analyzed under the condition in which cellular DNA synthesis was inhibited by several antimetabolites or blocked by thymidine. The results demonstrate that differentiation occurs in the absence of DNA synthesis and that some inhibitors of DNA synthesis may enhance the differentiation of HL-60 cells by the above inducers. Among the antimetabolites used, the enhancement of induction of differentiation by hydroxyurea was shown to be more effective than that of Ara-C or aphidicolin. The effect of thymidine blockage was similar to that of hydroxyurea. These different effects may be due to the different points at which the cell cycle is blocked by these agents. These results seem to be common in both the differentiation of the granulocytic line induced by retinoic acid and of the macrophage line induced by 1 alpha,25(OH)2D3. The present study also suggests that combination treatment with the inhibitor of DNA synthesis and the inducer of differentiation could be beneficial in the clinical therapy of leukemia. The mode of action of clinical low-dose Ara-C treatment is also discussed.

Topics: Antibiotics, Antineoplastic; Aphidicolin; Calcitriol; Cell Differentiation; Cell Division; Cell Line; Cytarabine; Diterpenes; DNA Replication; Humans; Hydroxyurea; Kinetics; Leukemia, Myeloid, Acute; Tretinoin

We have investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on iron uptake into human hematopoietic cell lines K562, U937, and HL-60. TPA inhibited both cell growth and iron uptake by these cell lines. This effect was rapid, which is typical of phorbol esters which are biologically active, and it occurred at very low concentrations of TPA. This effect of TPA was dependent upon an inhibition of the transferrin-binding capacity as estimated on intact cells. However, experiments with transferrin binding on cell samples dissolved in 1% Triton X-100 showed that TPA-treated cells exhibited a transferrin-binding capacity similar to that of control cells. On the basis of this result, it is suggested that TPA modified a part of transferrin receptors present in the cells; as a result of this modification, these receptors became unavailable for binding transferrin, but they remained physically present in the cell. Other compounds capable of inducing the differentiation of leukemic cells, such as dimethyl sulfoxide, butyrate, retinoic acid, and 1 alpha,25-dihydroxy-vitamin D3, did not acutely inhibit iron uptake. We also investigated the effect of TPA on transferrin receptors in a cellular system in which phorbol esters stimulate cell proliferation. At 16 X 10(-9) M, TPA markedly stimulated the proliferation of T-lymphocytes. However, in spite of this marked stimulation of cell proliferation, TPA-stimulated lymphocytes exhibited a transferrin-binding capacity much inferior to cells stimulated by other mitogens, such as phytohemagglutinin.

Topics: Biological Transport; Calcifediol; Calcitriol; Cell Line; Dimethyl Sulfoxide; Heme; Humans; Iron; Iron Radioisotopes; Kinetics; Leukemia, Myeloid, Acute; Phorbol Esters; Phorbols; Receptors, Cell Surface; Receptors, Transferrin; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Transferrin; Tretinoin

The permanent promyelocytic cell line HL-60 was subjected to stimulation with dimethyl sulfoxide (DMSO) and retinoic acid (RA), as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and lymphokine conditioned media for the induction of granulocytic or monocytic differentiation, respectively. Cells were investigated cytochemically using alpha-naphthylacetate esterase (acid esterase; AcE), naphthol AS-D chloroacetate esterase, and peroxidase reactions. In addition, the granulocyte or monocyte specific isoenzyme patterns of AcE as an intracytoplasmic property and the immunoreactivity to monoclonal antibodies recognizing granulocytes and monocytes (Ki-M2, Ki-M5) or monocytes alone (Ki-M1) were considered. The results indicated that HL-60 cell line bear the potency to evolve into granulocytes as well as monocytes. Additional studies performed on normal human bone marrow stained for AcE led to the conclusion that the myeloid cell line remains bipolar until the maturation stage of promyelocytes. Myelocytes being AcE positive only in 11.5 +/- 5.0 are heterogeneous and display the first indications of separated monocytic or granulocytic differentiation.

Topics: Antibodies, Monoclonal; Cell Differentiation; Cell Line; Concanavalin A; Dimethyl Sulfoxide; Esterases; Granulocytes; Humans; Immunoenzyme Techniques; Leukemia, Myeloid, Acute; Lymphocytes; Lymphokines; Monocytes; Peroxidase; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin

We have studied the role of ADP-ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL-60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP-ribose)n synthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP-ribose)n synthetase was decreased in HL-60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP-ribose)n synthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N'-methylnicotinamide (N'-Met-NAm), agents which decrease ADP-ribosylation. Treatment of HL-60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N'-Met-NAm was more potent than NAm in inducing maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N'-Met-NAm also potentiated the maturation of HL-60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose-response curve for RA; maturation was observed at 10(-11) M RA in the presence of either 2 mM NAm or 0.2 mM N'-Met-NAm while 10(-9) M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N'-Met-NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N'-Met-NAm decreased by about 75% the amount of endogenous (ADP-ribose)n in a selected fraction of chromosomal proteins which included histone H1 and the nonhistone high mobility group proteins. The results of this study support the concept that ADP-ribosylation of chromosomal proteins influences the regulation of human myeloid cell maturation.

Topics: Acyltransferases; Adenosine Diphosphate Ribose; Cell Division; Cell Line; Cell Nucleus; Humans; Kinetics; Leukemia, Myeloid, Acute; NAD+ Nucleosidase; Niacinamide; Nucleoproteins; Nucleoside Diphosphate Sugars; Phagocytosis; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Transglutaminases; Tretinoin

The kinetic composition of the polyadenylated RNAs of leukemic myeloblasts and HL60 promyelocytes was examined by the cDNA-poly (A) +RNA reassociation technique before and after the induction of differentiation with retinoic acid. The data obtained in the homologous hybridizations show the following main features: the sequence complexity of the total poly(A) +RNA molecules is 72,000 different sequences in leukemic myeloblasts, 64,000 in HL60 promyelocytes before induction and 26,000 after treatment of HL60 with retinoic acid; the number of sequences reacting as abundant is clearly higher in HL60 promyelocytes (5,000) than in leukemic myeloblasts (1,000), and the number decreases sharply after induction (300); the repetition frequency of the abundant and rare components is almost unchanged between leukemic myeloblasts and HL60 promyelocytes, while it is markedly increased after induction with retinoic acid. The heterologous reactions show that the observed differences in complexity are mainly related to the missing of rare sequences. Moreover, an important portion of sequences already present in leukemic myeloblasts has a definitely higher repetition frequency in HL60 promyelocytes. Finally, the most abundant sequences in HL60 cells after induction are already present before treatment. The data presented here suggest that, during human myeloid differentiation, important, possibly transcriptional, regulatory mechanisms of gene expression are active long after the first commitment event of the hemopoietic undifferentiated stem cell.

Topics: Adult; Base Sequence; Cell Differentiation; DNA; Female; Granulocytes; Humans; Leukemia, Myeloid, Acute; Nucleic Acid Hybridization; Poly A; RNA; RNA, Messenger; Transcription, Genetic; Tretinoin

When human myeloid leukemia HL-60 cells were induced to differentiate into mature cells by dimethyl sulfoxide or retinoic acid, the amount of myeloperoxidase activity per cell decreased to 20 to 30% of that of uninduced cells, and the rate of myeloperoxidase biosynthesis decreased to an undetectable level in 19 h after induction of differentiation. After 19-h exposure to an inducer, the cells could not resume myeloperoxidase synthesis on further incubation in inducer-free medium. When polysomes and mRNAs prepared from untreated and treated cells were translated in rabbit reticulocyte lysates, the former showed myeloperoxidase polypeptide synthesis, and the latter did not. These results indicate that the inability of induced cells to synthesize myeloperoxidase is due to the absence of myeloperoxidase mRNA.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Genes; Humans; Kinetics; Leukemia, Myeloid, Acute; Molecular Weight; Peroxidase; Peroxidases; Tretinoin

The question of whether the initial regulatory event, which directs an uncommitted precursor cell toward terminal differentiation, is cell cycle phase specific was examined using the human promyelocytic leukemia cell line, HL-60. While the HL-60 system does not reflect all of the features of normal hematopoiesis, it does provide a relatively well-defined in vitro experimental system which can be useful for examining aspects of the differentiation process. HL-60 cells were induced to undergo myeloid differentiation by retinoic acid. The subsequent differentiation kinetics of HL-60 populations initially enriched in different cell cycle phases was measured. This was compared to the cellular uptake of retinoic acid as a function of cell cycle position. If the initial differentiation-regulating event were cell cycle phase independent, then the kinetics of differentiation would be independent of the cell cycle status of the initial population. Flow cytometric cell sorting, based on cellular narrow angle and orthogonal light scatter intensity spectra, was used to select G1-enriched and S + G2 + M-enriched cell populations without pharmacological perturbation. These two populations were each induced to undergo myeloid differentiation with 10(-6) M beta-all-trans-retinoic acid. The kinetics of G1/0 arrest associated with terminal cell differentiation, as well as phenotypic differentiation, assayed by development of oxidative metabolism, was measured for both populations. The kinetics of differentiation differed for the two populations, indicating that the initial differentiation-regulating event was cell cycle phase specific. For both of the initial cell populations, significant phenotypic differentiation followed approximately 24 hr after enrichment in the relative number of S-phase cells. When exponentially proliferating HL-60 cells were exposed to a 1-hr pulse of 10(-5) M [3H]retinoic acid and then flow cytometrically sorted by DNA content, cells in late S + G2 + M had an approximately 10-fold higher uptake than cells in G1 or early S. The results indicate that cellular regulation of myeloid differentiation first becomes responsive to the inducer, retinoic acid, in S phase when uptake is enhanced.

Topics: Biological Transport; Cell Cycle; Cell Differentiation; Cell Line; DNA, Neoplasm; Flow Cytometry; Humans; Kinetics; Leukemia, Myeloid, Acute; Superoxides; Tetradecanoylphorbol Acetate; Tretinoin

In order to determine if cyclic adenosine 3':5'-monophosphate- (cyclic AMP)-dependent protein kinase has a role in the expression of chemically induced differentiation of HL-60 cells, levels and subcellular distribution of this enzyme were studied during this process. Cyclic AMP binding protein and stimulated kinase activities increased moderately in cytosol and more markedly in nucleosol and nonhistone chromatin-associated protein fractions of cells induced to differentiate with dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate. Retinoic acid induced similar cytosolic changes but less marked intranuclear increases. Nuclear increases did not occur in the differentiation-resistant subline, HL-60 Blast II, treated with dimethyl sulfoxide. DEAE-cellulose chromatography, as well as photoaffinity labeling and gel electrophoresis, disclosed higher ratios of type I to type II kinase in cytosol than in intranuclear fractions. Differences of the qualitative binding protein patterns between cytosol and nucleosol were enhanced following chemically induced differentiation. Dibutyryl cyclic AMP increased cytoplasmic and nuclear binding protein levels when given alone or in combination with retinoic acid or dimethyl sulfoxide, and it enhanced differentiation. These results suggest that intranuclear cyclic AMP-dependent protein kinase is associated with the expression of the differentiative program in HL-60 cells.

Topics: Bucladesine; Carrier Proteins; Cell Differentiation; Cell Division; Cell Line; Cyclic AMP Receptor Protein; Dimethyl Sulfoxide; Humans; Kinetics; Leukemia, Myeloid, Acute; Protein Kinases; Subcellular Fractions; Tetradecanoylphorbol Acetate; Tretinoin

We have studied the effects of retinoic acid (RA) on bone marrow leukemic cells from children with acute nonlymphocytic leukemia (ANLL) at the time of diagnosis, and on cells from four ALL/lymphoma cell lines (common-ALL, pre-B-ALL, T-ALL, and Burkitt's lymphoma) derived from children with these diseases. Cells were cultured in methylcellulose medium with clinically attainable concentrations (0.25-2.0 microM) of RA for two weeks prior to colony and cluster quantitation. Myeloid progenitor cells (CFU-GM) obtained from children with hematologically normal bone marrows were also cultured with RA. Of 19 patients with ANLL whose cells formed colonies, 16 (84%) were inhibited by RA; three patients showed either increased or unchanged colony numbers with RA. RA had a similar effect on both ANLL cluster and colony growth. RA (1-2 microM) also inhibited colony growth of the pre-B-ALL, common-ALL, and Burkitt's lymphoma lines; the T-ALL line and normal bone marrow CFU-GM were not inhibited. The inhibitory effects of RA on pediatric ANLL bone marrow cells and on some ALL/lymphoma cell lines compared with CFU-GM indicate that RA may be of value in the treatment of these malignancies in children.

Topics: B-Lymphocytes; Bone Marrow; Burkitt Lymphoma; Cell Division; Cell Line; Child; Colony-Forming Units Assay; Hematopoietic Stem Cells; Humans; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; T-Lymphocytes; Tretinoin

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Macrophage-1 Antigen; Nitroblue Tetrazolium; Receptors, Complement; Tretinoin

A decrease in the expression of the myc proto-oncogene of HL-60 cells has been reported as an accompaniment of myeloid differentiation induced by either dimethylsulfoxide or retinoic acid. We report herein that several inhibitors of poly(ADP-ribose)-polymerase induced myeloid differentiation in HL-60 cultures. Studies on the expression of the c-myc gene in total cell RNA populations indicate that expression of this gene is inversely correlated with the state of differentiation, either myeloid or monocytic, of the cultured cells independent of the inducer and the rate of cell proliferation.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Kinetics; Leukemia, Myeloid, Acute; Nucleic Acid Hybridization; Oncogenes; Poly(ADP-ribose) Polymerase Inhibitors; Proto-Oncogene Mas; RNA, Neoplasm; Tretinoin

The anticonvulsant drug 1-methyl-1-cyclohexanecarboxylic acid ( MCCA ) has been shown to cause maturation of murine neuroblastoma cells in vitro at concentrations that are pharmacologically achievable. HL-60 human promyelocytic leukemia cells cultured with this drug underwent a dose-dependent decrease in growth. Similarly, neutrophilic differentiation, based on morphologic criteria and the acquisition of the ability to reduce nitroblue tetrazolium and phagocytose yeast, was observed. Valproic acid, a clinically available anticonvulsant that is chemically related to MCCA , likewise inhibited growth and promoted maturation of HL-60 cells, although only at concentrations above the recommended therapeutic blood levels. MCCA was additive in its ability to induce differentiation of HL-60 with retinoic acid, another compound that induces differentiation at pharmacologic concentrations. MCCA , or similar branched-chain fatty acids, may be useful in the treatment of human leukemia, particularly in combination with other differentiation-inducing drugs.

Topics: Anticonvulsants; Antineoplastic Agents; Cell Differentiation; Cell Division; Cells, Cultured; Cyclohexanecarboxylic Acids; Humans; Leukemia, Myeloid, Acute; Neutrophils; Tretinoin; Valproic Acid

2',5'-Oligoadenylate (2-5A) synthetase, which polymerizes adenosine triphosphate into 2-5A, is induced upon treatment of cells with interferon (IFN) and is thought to be involved in its antiviral and anticellular action. We report here that retinoic acid (RA) enhanced the level of this enzyme in two human transformed cell lines, WISH and Namalva. Like IFN, RA induced 2-5A synthetase activity in a time- and dose-dependent manner. Addition of anti IFN-alpha, -IFN-beta, or -IFN-gamma antibodies to the medium concomitantly with RA did not prevent such induction; therefore, the effect of RA is clearly not mediated through the induction and externalization of IFN. Pretreatment of cells with actinomycin D inhibited 2-5A synthetase induction by RA, suggesting that RA increased the transcription of the 2-5A synthetase gene. In WISH cells, the growth of encephalomyocarditis virus was inhibited by RA treatment, which is consistent with the hypothesis that 2-5A synthetase plays an important role in the antiviral action of IFN, at least in encephalomyocarditis virus replication. When the anticellular effects of IFN and RA were compared to their ability to induce 2-5A synthetase activity in four human cell lines, there was no strict correlation between the amplitude of the enzyme activity induced and the extent of the antiproliferative effect. It is concluded that the 2-5A system is probably not the only pathway responsible for the antiproliferative effect of both substances. We further suggest that the induction of 2-5A synthetase by IFN and RA might be connected with at least some of the similarities observed between other biological effects of both compounds.

Topics: 2',5'-Oligoadenylate Synthetase; Burkitt Lymphoma; Cell Division; Cell Line; Cell Transformation, Neoplastic; Enzyme Induction; Female; Fibroblasts; Humans; Interferon Type I; Interferon-gamma; Leukemia, Myeloid, Acute; Placenta; Pregnancy; Tretinoin

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca2+ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.

Topics: Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Leukemia, Myeloid, Acute; Tretinoin

A 60-kilodalton protein was identified in chromatin digested by micrococcal nuclease during retinoic acid-induced differentiation of human leukemia (HL-60) cells to mature-like granulocytes. The protein was not detected in a retinoic acid-resistant variant of the HL-60 cell line treated with retinoic acid, in HL-60 cells induced with dimethyl sulfoxide, or in normal human granulocytes. This protein may have an important role in the regulation of retinoic acid-induced leukemic cell differentiation.

Topics: Cell Line; Cell Transformation, Neoplastic; Centrifugation, Density Gradient; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Granulocytes; Humans; Leukemia, Myeloid, Acute; Neoplasm Proteins; Nucleosomes; Tretinoin

The effect of various classes of differentiation-inducing agents on macromolecular synthesis was studied in a human myeloblastic leukemia cell line (ML-1). Antineoplastic drugs such as 1-beta-D-arabinofuranosylcytosine, daunorubicin, and actinomycin D caused early inhibition of DNA synthesis, which generally preceded the accrual of differentiation markers. In contrast, retinoic acid and conditioned medium from mitogen-stimulated leukocytes caused a delayed decline in DNA synthesis, which accompanied the appearance of maturing morphology. With 12-O-tetradecanoylphorbol-13-acetate, the decline in DNA synthesis was temporally linked to the onset of maturation, and this agent evidenced some properties of both the antineoplastic agents and the more physiological inducers, retinoic acid and conditioned medium. Antineoplastic agents and conditioned medium, when applied simultaneously, induced differentiation in an additive or synergistic manner, simulating the effects of 12-O-tetradecanoylphorbol-13-acetate. RNA and protein synthesis continued during maturation induced with all these agents, although a partial reduction in RNA synthesis was observed at later time points (greater than or equal to 24 hr). Agents incapable of inducing differentiation, such as cordycepin and cycloheximide, were characterized by a lack of sustained inhibition of DNA synthesis and/or by early (3 hr) inhibition of RNA or protein synthesis. The decline in DNA synthesis caused by the inducing agents was accompanied by decreased cell cycle progression, cells accumulating largely in G1 phase. With daunorubicin and actinomycin D, block of the G1-S transition was evident at 24 hr, whereas with conditioned medium and retinoic acid, accumulation in G1 occurred in a progressive fashion, greater than 77% of cells residing in this phase on Day 6. Maximal inducing doses of 12-O-tetradecanoylphorbol-13-acetate (greater than 80% differentiation) caused an accumulation of cells in G1, as well as an accumulation of cells with a G2-M-phase DNA content (approximately 40%). These observations indicate that early inhibition of DNA synthesis, with sparing of RNA and protein synthesis, is characteristic of the differentiation-inducing antineoplastic drugs examined. These agents may induce differentiation by inhibition of the proliferation path, whereas conditioned medium and retinoic acid may act by the stimulation of differentiation paths. Differentiation can be enhanced by the simultaneous application of

Topics: Cell Cycle; Cell Differentiation; Cell Line; Cycloheximide; Cytarabine; Dactinomycin; Daunorubicin; DNA Replication; Humans; Kinetics; Leucine; Leukemia, Myeloid, Acute; Protein Biosynthesis; Thymidine; Transcription, Genetic; Tretinoin; Tritium; Uridine

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type l cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type l cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.

Topics: Carrier Proteins; Caseins; Cell Differentiation; Cell Line; Fluorides; Histones; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Phosphorylation; Protamines; Protein Kinases; Proteins; Time Factors; Tretinoin

A 30-year-old woman was diagnosed as having an acute promyelocytic leukaemia in September 1981. Chemotherapeutic courses of daunomycin, ara-C, thioguanine and prednisolone were administered, resulting in a complete remission. A relapse occurred in January 1982, and chemotherapy did not lead to a second complete remission; neutropenia persisted with a marked left shift of the marrow granulopoiesis. Courses of chemotherapy were given throughout the study. In September 1982, marrow promyelocytes markedly increased, making up 51% of the nucleated cells. One month later the situation was unchanged, and 13-cis-retinoic acid (1 mg/kg) was administered by mouth. Gradually the marrow proportion of promyelocytes decreased to normal levels. The peripheral blood and marrow were still normal after 20 weeks of treatment with retinoic acid. Thus, retinoic acid seemed to have been inducing differentiation in an abnormally increased, maturation-deficient population of promyelocytes in a patient with acute promyelocytic leukaemia.

Topics: Adult; Bone Marrow; Cell Count; Cell Differentiation; Female; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Tretinoin

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL-60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation-induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.

Topics: Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; G(M1) Ganglioside; G(M2) Ganglioside; Globosides; Glycosphingolipids; Humans; Leukemia, Myeloid, Acute; Macrophages; Tretinoin

Topics: Antibodies, Monoclonal; Antigens, Surface; Cell Differentiation; Cell Line; Cells, Cultured; Granulocytes; Humans; Leukemia, Myeloid, Acute; Monocytes; Phorbols; Tetradecanoylphorbol Acetate; Tretinoin

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Kinetics; Leukemia, Myeloid, Acute; Membrane Potentials; Neutrophils; Tetradecanoylphorbol Acetate; Tretinoin

Various human and mouse myeloid leukemia cell lines can differentiate to mature myeloid or monocytoid cells in response to different agents. The myeloblastic leukemia of the RFM/Un mouse (the RF.AML line) was studied here to determine its ability to differentiate after in vitro and in vivo treatment. The RF.AML cells were passed in vivo by i.v. or i.p. injection of freshly harvested leukemic spleen cells or in vitro-passaged leukemia cells. The cells proliferated in the spleen and peritoneal cavity. The RF.AML cells had the appearance of myeloblasts or myelomonoblasts on Wright's stain, had slight positivity for peroxidase, and lacked staining for nonspecific esterase. The cells grew in suspension in vitro with a doubling time of 48 hr. Various phorbol diester tumor promotors inhibited proliferation and incorporation of thymidine into the RF.AML cells. Phorbol myristate acetate (10 to 100 nM) caused the cells to adhere to plastic, and enhanced the phagocytic ability of the cells for Candida albicans. The RF.AML cells had specific receptors for phorbol dibutyrate, binding 0.37 +/- 0.03 (S.E.) pmol of [3H]phorbol dibutyrate/10(6) cells after a 2-hr incubation at 4 degrees with 50 nM [3H]phorbol dibutyrate. Thirty-three to 300 nM dexamethasone caused 19 to 37% of the cells to become nonspecific esterase positive and enhanced their phagocytosis of C. albicans. Likewise, 0.5 or 1.0 microM 13-cis-retinoic acid, or 0.6 or 1.2% dimethyl sulfoxide enhanced the phagocytic ability of the RF.AML cells but had no effect on the adherence, proliferation, or nonspecific esterase activity. None of the treatments induced lysozyme activity in the cells or rendered the RF.AML cells able to produce H2O2 in response to phorbol myristate acetate treatment in vitro. In vivo treatment of mice with RF.AML present with phorbol myristate acetate or dexamethasone did not induce differentiation of the RF.AML cells or alter the survival of the animals. Thus, although the RF.AML cells differentiate in vitro in response to various agents, in vivo differentiation was not seen in this model.

Topics: Animals; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Differentiation; Cell Line; Dexamethasone; Dimethyl Sulfoxide; DNA Replication; Female; Leukemia, Experimental; Leukemia, Myeloid, Acute; Mice; Mice, Inbred Strains; Muramidase; Phorbol Esters; Protein Kinase C; Receptors, Drug; Receptors, Immunologic; Tetradecanoylphorbol Acetate; Tretinoin

Mitogen-stimulated mononuclear blood cells produce differentiation inducing factors (DIFs) for the promyelocytic cell line HL-60. We report that DIF is produced constitutively by a malignant T lymphocyte line HUT-102. DIF was purified 7,000-fold from HUT-102 conditioned media by utilizing ion-exchange chromatography with DEAE-Sepharose, gel chromatography, Blue-Sepharose chromatography, and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation is susceptible to protease treatment, has a molecular weight of 46,000, as determined by SDS-PAGE and approximately 55,000 by gel filtration, has an isoelectric point of approximately 5.2, does not adhere to lectin-Sepharose and is resistant to periodate oxidation, and is free of colony-stimulating factor. DIF induced maturation of HL-60 into phagocytizing nitro blue tetrazolium reducing cells with the morphological characteristics of myelomonocytic or monocyte-like cells. An activity, co-chromatographing with DIF, acts synergistically with retinoic acid to induce maturation not only of HL-60, but also of the monoblast-like cell line U-937 (measured as percentage of cells reducing NBT).

Topics: Cell Line; Cell Transformation, Neoplastic; Chromatography, Agarose; Drug Stability; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Lymphokines; Nitroblue Tetrazolium; T-Lymphocytes; Tretinoin

Insulin binding activity and its changes in relation to terminal differentiation were studied in the HL60 human promyelocytic cell line, and in myeloid cells from both normal bone marrow and chronic myeloid leukemia (CML) patients. After treatment with dimethylsulfoxide, the HL60 line began to differentiate into more mature myeloid cells. Treated and untreated HL60 cells were found to possess specific insulin receptors with characteristics similar to those of monocytes and granulocytes. Dimethylsulfoxide induced a progressive decrease in insulin binding, parallel to the increase in the proportion of differentiated cells. Myeloid cells from CML patients were used to study insulin binding characteristics during spontaneous differentiation. They were separated on Ficoll Hypaque into a light fraction, containing mostly undifferentiated cells, and a dense fraction, containing mostly granulocytes, with similar specific insulin receptor characteristics. Insulin binding capacity, however, was twice as high in the light fraction. To compare binding activity during normal and leukemic myeloid differentiation, cells from normal bone marrow and CML peripheral blood were fractioned by BSA density gradient into enriched fractions of one predominant cell type. Insulin binding decreased in the course of both differentiations. These findings indicate that leukemic immature myeloid cells possess a high number of specific insulin receptors, and that insulin binding decreases during both spontaneous and chemically induced terminal differentiation.

Topics: Bone Marrow; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Insulin; Kinetics; Leukemia, Myeloid, Acute; Receptor, Insulin; Tretinoin

The activities of five glycosyltransferases were measured following the induction of HL60 cells to differentiate to mature myeloid forms or to macrophages by the addition of retinoic acid or a phorbol ester, respectively. Gal-T-II, Fuc-T-I and (NeuAc-T-I) are all increased and Fuc-T-II decreased in activity upon treatment with RA. Gal-T-I and Fuc-T-II are decreased and Gal-T-II increased in activity upon with TPA treatment. The increases in enzyme activities with RA are measurable as early as 1 day but while Fuc-T-I and NeuAc-T-I are fully elevated at 2 days, Gal-T-II shows a biphasic rise with initial elevation by day 2 and a further rise at days 3 to 5. The rises in Gal-T-II are due to increases in the enzyme form present in uninduced cells.

Topics: Cell Differentiation; Cell Line; Fucosyltransferases; Galactosyltransferases; Hexosyltransferases; Humans; Kinetics; Leukemia, Myeloid, Acute; Neuraminidase; Phorbols; Tetradecanoylphorbol Acetate; Tretinoin

We have assayed glycosyltransferase activities during the granulocytic and macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells. Functional granulocytic differentiation was assayed by the decarboxylation of 2-deoxyglucose in addition to nitroblue tetrazolium reduction. Dimethylsulfoxide (DMSO) treated HL-60 cells, induced to granulocytic differentiation, had higher 2-deoxy-glucose decarboxylation activity, and contained less sialyltransferase (ST), more fucosyltransferase (FT), and more N-acetylglucosaminyltransferase (NGT) activities than untreated cells. HL-60 cells treated with another granulocytic differentiator, retinoic acid, also had higher 2-deoxyglucose decarboxylation activity, and contained less ST, more FT, and more NGT activities than untreated cells. In contrast, cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reported to differentiate HL-60 to macrophage-like cells, but did not show an increased level of 2-deoxyglucose decarboxylation activity, but contained more galactosyltransferase (GT) and FT activities as compared to untreated cells. These findings suggest that the alterations of glycosyltransferase levels during the differentiation of precursor cells may not depend upon different inducers, but are characteristic of the phenotypic expression of the mature cell type.

Topics: Cell Line; Cell Transformation, Neoplastic; Decarboxylation; Deoxyglucose; Dimethyl Sulfoxide; Fucosyltransferases; Galactosyltransferases; Hexosyltransferases; Humans; Leukemia, Myeloid, Acute; Sialyltransferases; Substrate Specificity; Tetradecanoylphorbol Acetate; Tretinoin

The surface changes occurring in three acute myeloid leukemia cell lines (HL60, ML3, and KG1) induced to differentiate by a variety of agents (dimethylsulfoxide, retinoic acid, 12-O-tetradecanoylphorbol-13-acetate, and factors present in lymphocyte conditioned medium) were probed using monoclonal antibodies that are differentiation stage- and lineage-specific. In all cases, the differentiated phenotype was defective and varied with the inducing agent and the cell line used. HL60 proved to be the most sensitive to the effect of the inducers. Retinoic acid was better than DMSO, and TPA was better than the medium factors in the ability to induce granulocytic and monocytic differentiation, respectively, in HL60 cells. These findings indicate that the differentiation block in acute myeloid leukemias is heterogeneous and that each cell line has different phenotypic characteristics that are responsible for the extent of differentiation obtained with a given inducer. These results also suggest that the extent of the differentiation response in vitro may be improved by the use of more suitable inducers for each specific leukemic line.

Topics: Antibodies, Monoclonal; Antigens, Surface; Cell Differentiation; Cell Line; Culture Media; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Lymphocytes; Tetradecanoylphorbol Acetate; Tretinoin

Leukemia cells from patients with acute non-lymphocytic leukemia were treated with various inducers of differentiation of the human promyelocytic leukemia cell line HL-60. All cells in 14 specimens tested underwent morphological, functional and histochemical changes after treatment with some inducers of differentiation of HL-60 cells, but the most effective inducer varied for different specimens. These results suggest that treatment with some inducers should be effective for inducing most acute myeloid leukemia cells to differentiate into morphologically and functionally mature granulocytes and macrophages.

Topics: Adult; Aged; Cell Differentiation; Cell Line; Cells, Cultured; Child; Child, Preschool; Female; Granulocytes; Humans; Leukemia, Monocytic, Acute; Leukemia, Myeloid, Acute; Macrophages; Male; Middle Aged; Monocytes; Tetradecanoylphorbol Acetate; Tretinoin

HL-60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). After the addition of an inducer, the HL-60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation. Within 3 hours after the addition of TPA to HL-60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.

Topics: Butyrates; Butyric Acid; Cell Count; Cell Cycle; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Macrophages; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Kinetics; Leukemia, Myeloid, Acute; Protein Kinases; Tretinoin

Two HL60 clones (C12 and C13) totally insensitive to differentiation induction by dimethylsulfoxide (Me2SO) are described. They have been growing continuously in the presence of the inducer for more than 6 months. The morphological and cytochemical features of the two populations are quite similar to those of the original HL60 cell line, whereas a different karyotype with marked hyperploidy (modal chromosome number of 86 for C12 and 82 for C13) was detected. An antigenic pattern analogous to that of the native HL60 cell line was found in C12 and C13 populations using three monoclonal antibodies differently reactive to myeloid cells. Both clones can be induced to differentiate by retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). The pattern of differentiation was assessed by morphological, cytochemical, phenotypical and functional markers. Differentiation of C12 cells by RA and TPA was similar to that observed with native HL60 cells, whereas C13 cells showed lower degrees of sensitivity to RA and TPA. The data presented suggest the existence of different mechanisms for induction of differentiation by Me2SO, RA and TPA. In addition, they are in accordance with previous observations of different degrees of inducibility to differentiation among leukemic cell populations in culture.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Drug Resistance; Humans; Kinetics; Leukemia, Myeloid, Acute; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin

We describe in vitro studies and a therapeutic trial of retinoic acid (RA) in a patient with acute promyelocytic leukemia (APL) refractory to chemotherapy. Bone marrow promyelocytes from the patient, prior to RA, matured morphologically in liquid culture with RA (97% maturing myeloid cells compared with 26% in control cultures at 7 days). RA-cultured cells displayed leukocyte alkaline phosphatase activity and cytoplasmic maturation (by electron microscopy). Retinoic-acid-treated cells, compared to controls, demonstrated increased functional maturation, with phagocytosis of opsonized zymosan (90% versus 10%) and production of superoxide (measured by nitroblue tetrazolium reduction) in response to phorbol ester, opsonized zymosan, or the chemotaxin F-met-leu-phe. There was no evidence of active proliferation in the cultures. RA-treated cells continued to show 15;17 chromosomal translocation after 7 days in culture. The patient was treated with oral 13-cis-retinoic acid (100 mg/sq m/day) for 13 days. During that time, the peripheral white blood count rose from 300 cu mm to 6,700 cu mm, and the maturing myeloid cell count rose from 54 cu mm to 3,800 cu mm. Bone marrow maturing cells increased from 1.8% to 8.0%. Despite the increasing number of maturing myeloid cells, the patient died on day 13 from disseminated candidiasis. These data confirm that RA induces maturation of leukemic promyelocytes in vitro and suggest that similar maturation is achievable in vivo. We suggest that oral retinoic acid may be a useful adjunct in the treatment of APL.

Topics: Adult; Bone Marrow; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Leukemia, Myeloid, Acute; Leukocyte Count; Male; Tretinoin

The differentiating action of 1 alpha,25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3] in hematopoietic cells was examined in 3 tumor cell lines. 1 alpha,25-(OH)2D3 induced common differentiation-associated properties in macrophages and granulocytes similarly in mouse myeloblastic leukemia cells (M1), human promyelocytic leukemia cells (HL-60) and human histiocytic monoblast-like lymphoma cells (U937). 1 alpha,25(OH)2D3 markedly induced alpha-naphthyl acetate esterase activity, a typical marker of monocyte-macrophages, in M1 and HL-60 cells. In HL-60 and U937 cells, the vitamin also induced binding of the monoclonal antibody MAS 072, specific for monocyte-macrophages, but not of MAS 067, specific for granulocytes. These results clearly indicate that 1 alpha, 25(OH)2D3 induces differentiation of all cell lines examined preferentially along the monocyte-macrophage pathway.

Topics: Calcitriol; Cell Differentiation; Cell Line; Dexamethasone; Granulocytes; Humans; Kinetics; Leukemia, Myeloid, Acute; Macrophages; Monocytes; Tetradecanoylphorbol Acetate; Tretinoin

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.

Topics: Animals; Carrier Proteins; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cytosol; Diterpenes; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Mice; Receptors, Retinoic Acid; Retinyl Esters; Tretinoin; Vitamin A

Topics: Butyrates; Cell Differentiation; Cell Line; Cell Membrane; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid, Acute; Lymphocytes; Receptors, Cell Surface; Receptors, Transferrin; Tretinoin

Topics: Cell Line; Enzyme Induction; Humans; Kinetics; Leukemia, Myeloid, Acute; NAD+ Nucleosidase; Tretinoin

The ornithine decarboxylase inhibitor DL-alpha-difluoromethyl ornithine inhibited a proliferation-associated increase in ornithine decarboxylase activity in cultured human promyelocytic leukemia cells, resulting in a marked suppression of cell proliferation and subsequent cell loss. It also inhibited increases in ornithine decarboxylase activity associated with the phorbol ester-induced conversion of promyelocytic HL-60 cells to monocyte-like cells and the retinoic acid-induced conversion to granulocyte-like cells. However, the inhibition of ornithine decarboxylase activity did not prevent cellular differentiation. These results suggest that polyamine biosynthesis has a specific role in cell proliferation rather than in inducing differentiation that is not accompanied by proliferation. The data also demonstrate that cessation of proliferation in HL-60 cells is not necessarily associated with differentiation.

Topics: Carboxy-Lyases; Cell Adhesion; Cell Differentiation; Cell Division; Cells, Cultured; Female; Granulocytes; Humans; Leukemia, Myeloid, Acute; Macrophages; Ornithine Decarboxylase; Polyamines; Tetradecanoylphorbol Acetate; Tretinoin

Retinoic acid and retinol induced functional and morphological differentiation of human promyelocytic leukemia cells (HL-60) into mature granulocytes, but did not induce functional or morphological differentiation of mouse myeloid leukemia cells (M1). Cellular retinoic acid-binding protein, but not retinol-binding protein, was detected on HL-60 cells. Neither binding protein could be detected on M1 cells. These results suggest that retinoic acid-binding protein may be necessary for induction by retinoids of functional and morphological differentiation of myeloid leukemia cells.

Topics: Animals; Carrier Proteins; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Induction; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Muramidase; Neoplasm Proteins; Rats; Receptors, Retinoic Acid; Retinol-Binding Proteins; Tretinoin

Myeloid differentiation is induced in HL-60 promyelocytic leukemia cells by dimethyl sulfoxide, retinoic acid, hypoxanthine, and a number of other chemical agents. We questioned whether the induction process was associated with changes in lipid synthesis. With [14C]acetate, a precursor for all cell lipids, a decrease in sterol and phospholipid synthesis (but not triglyceride synthesis) was observed within the first 5 hr after exposure to inducer, a time prior to inhibition of DNA synthesis or cessation of cell growth. Similarly, the membrane fraction of HL-60 cells exhibited decreased incorporation of newly synthesized lipid. Synthesis of phosphatidylcholine from choline as well as from the transmethylation of membrane phosphatidylethanolamine was also inhibited by myeloid inducers. In contrast, neither sterol nor phospholipid degradation was stimulated under these conditions. Both cholesterol and lanosterol were synthesized by growing HL-60 cells, but cholesterol esters were not. Synthesis of sterols was subject to feedback inhibition by cholesterol in the medium, but such feedback inhibition did not affect differentiation in the presence of myeloid inducers and did not alter the effect of myeloid inducers on phospholipid synthesis. Removal of dimethyl sulfoxide at 16 hr permitted a return to normal lipid synthesis and prevented differentiation, whereas removal of dimethyl sulfoxide at 40 hr was followed by continued inhibition of lipid synthesis and progressive differentiation. These studies demonstrate that the induction of myeloid differentiation is associated with an early inhibition of the synthesis of those lipids which are normally a part of cell membranes.

Topics: Acetates; Cell Differentiation; Cell Division; Cells, Cultured; Choline; Dimethyl Sulfoxide; DNA, Neoplasm; Humans; Leukemia, Myeloid, Acute; Membrane Fluidity; Methionine; Phospholipids; Sterols; Tretinoin

Mouse myeloid leukemia cells (M1) could be induced to differentiate into mature macrophages and granulocytes with dexamethasone or proteinaceous inducer. Retinoic acid inhibited functional and morphological differentiation of M1 cells, but the pyridyl analog of retinoic acid had no effect. M1 cells could be induced to produce a factor(s) inhibiting their own differentiation to macrophage- and granulocyte-like cells by retinoic acid but not by its pyridyl analog. This factor(s) inhibited induction by inducers of phagocytic activity, locomotive activity, lysozyme activity, and morphological changes in M1 cells. The production of the inhibitory factor(s) by M1 cells incubated with retinoic acid was inhibited by a low concentrations (5--10 ng/ml) of actinomycin D. The inhibitory factor seemed to be a protein(s), since it was susceptible to heat treatment and proteases. The effect of retinoic acid in inducing production of the inhibitory factor(s) by M1 cells seemed to be reversible, since it was low on washing the cells with fresh medium. Therefore, induction of this inhibitory factor may be involved in the mechanism of inhibition of functional and morphological differentiation of M1 cells by retinoic acid.

Topics: Animals; Cell Differentiation; Cells, Cultured; Dexamethasone; Leukemia, Experimental; Leukemia, Myeloid, Acute; Mice; Phagocytosis; Tretinoin

The recent finding that retinoic acid induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line, HL-60, prompted an investigation of the sensitivity to this inducer of human myelocytic leukemia cells in primary suspension culture. Of the 21 leukemic specimens, only cells from the two patients with acute promyelocytic leukemia differentiated in response to retinoic acid. After an incubation period of 5--7 days in 1 microM retinoic acid, the cells from these two patients showed extensive morphological and functional maturation. Thus, because it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes, this compound may have therapeutic utility in the treatment of acute promyelocytic leukemia.

Topics: Adolescent; Adult; Aged; Cell Differentiation; Cell Survival; Cells, Cultured; Granulocytes; Humans; Leukemia, Myeloid, Acute; Middle Aged; Nitroblue Tetrazolium; Prostaglandins E; Time Factors; Tretinoin

Topics: Acid Phosphatase; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Fibrinolysis; Humans; Leukemia, Myeloid, Acute; Muramidase; Tretinoin

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Blood Cells; Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Rabbits; Tetradecanoylphorbol Acetate; Tretinoin

The human leukemic cell lines HL60 and K562, were induced to differentiate terminally by chemical agents. The isoenzyme patterns of lactate dehydrogenase (LD) in the cells before and after differentiation were determined electrophoretically on agarose gels. In general, treatment of the leukemic cells with inducers of differentiation resulted in a quantitative shift of the isoenzyme pattern towards anodic or cathodic forms. This was correlated with the conversion of the chemically treated cells to morphologically more normal cells, as verified by light microscopy and/or synthesis of hemoglobin. The LD isoenzyme patterns of the chemically differentiated cells were: (a) characteristic for the particular cell type obtained rather than for the nature of the inducer used; and (b) not similar to those of normally differentiated cells of the corresponding lineage, indicating that incomplete differentiation had occurred.

Topics: Acetamides; Butyrates; Cell Differentiation; Cell Line; Cell Survival; Diamines; Dimethyl Sulfoxide; Erythropoiesis; Granulocytes; Hematopoiesis; Humans; Isoenzymes; L-Lactate Dehydrogenase; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin

The Hl-60 cell line, derived from a patient with acute promyelocytic leukemia, proliferates continuously in suspension culture and consists predominantly of promyelocytes. These cells can be induced to differentiate to morphologically and functionally mature granulocytes by incubation with a wide variety of compounds, including butyrate and hypoxanthine and polar planar compounds such as dimethylsulfoxide and hexamethylene bisacetamide. However, these compounds are either nonphysiological or induce HL-60 at concentrations that are markedly greater than physiological. We have now found that retinoic acid is the most potent inducer of granulocytic differentiation of HL-60. This compound induces differentiation at concentrations 1/10(3) to 1/10(6) the concentration of other inducers and at concentrations that are physiological. These findings prompted an investigation of the sensitivity to retinoic acid of fresh human myelocytic leukemia cells in primary suspension culture. Of 21 leukemic specimens, only cells from the 2 patients with acute promyelocytic leukemia differentiated in response to retinoic acid. Thus, as with HL-60, it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes and may have therapeutic utility in the treatment of acute promyelocytic leukemia. In addition, our results suggest that retinoic acid may also be involved in the differentiation of certain hematopoietic cells.

Topics: Cell Differentiation; Cell Line; Humans; Leukemia, Myeloid, Acute; Tretinoin

The fibrinolytic activity of human acute leukemia HL 60 and K 562 cells was studied by the (I25)I-fibrin plate method. The treatment of this cell lines with chemical inducers of differentiation such as Dimethyl sulfoxide (DMSO), 12-0-tetradodecanoilphorbol 13-acetate (TPA), and retinoic acid was found in some instance to affect such fibrinolytic activity. Increased activity was found during the myeloid differentiation induced by retinoic acid of HL 60, while the DMSO, another inducer of myeloid differentiation, and TPA, an inducer of macrophagic differentiation, both produced a decreased of the lytic activity. In the treatment with retinoic acid of th K 562 cells, which do not differentiate by such compound, we failed to show an increase of the fibrinolytic activity. Our data suggest that changes of the fibrinolytic activity of HL 60 are related to the process of differentiation.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Fibrinolysis; Granulocytes; Humans; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin

The specific insulin binding activity of human promyelocitic HL 60 cell line during the myeloid and macrophagic differentiation induced by chemical compounds was investigated. Dimethyl sulfoxide (DMSO) and retinoic acid myeloid induced differentiation in HHL 60 cells was accompanied by a marked decrease of insulin receptors. In K 562 cell line, where DMS O has no effect on differentiation, the number of insulin receptors was only slightly affected. 12-0-tetradodecanoil phorbol 13-acetate (TPA) induced macrophagic differentiation of HL 60 cell line was also accompanied by a decrease of insulin binding activity. Our results support the hypothesis that during the process of terminal differentiation a decrease of insulin receptors occurs.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Granulocytes; Humans; Insulin; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Macrophages; Receptor, Insulin; Tetradecanoylphorbol Acetate; Tretinoin