tretinoin and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Myeloid malignancies have always been at the forefront of an improved understanding of the molecular pathogenesis of cancer. In accordance, over the last years, basic research focusing on the aberrations underlying malignant transformation of myeloid cells has provided the basis for precision medicine approaches and subsequently has led to the development of powerful therapeutic strategies. In this review article, we will recapitulate what has happened since in the 1980s the use of all-trans retinoic acid (ATRA), as a first targeted cancer therapy, has changed one of the deadliest leukemia subtypes, acute promyelocytic leukemia (APL), into one that can be cured without classical chemotherapy today. Similarly, imatinib, the first molecularly designed cancer therapy, has revolutionized the management of chronic myeloid leukemia (CML). Thus, targeted treatment approaches have become the paradigm for myeloid malignancy, but many questions still remain unanswered, especially how identical mutations can be associated with different phenotypes. This might be linked to the impact of the cell of origin, gene-gene interactions, or the tumor microenvironment including the immune system. Continuous research in the field of myeloid neoplasia has started to unravel the molecular pathways that are not only crucial for initial treatment response, but also resistance of leukemia cells under therapy. Ongoing studies focusing on leukemia cell vulnerabilities do already point to novel (targetable) "Achilles heels" that can further improve myeloid cancer therapy.

Topics: Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Precision Medicine; Tretinoin; Tumor Microenvironment

Topics: Animals; Antineoplastic Agents; Benzamides; Dasatinib; Disease Models, Animal; History, 19th Century; Humans; Imatinib Mesylate; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Philadelphia Chromosome; Piperazines; Pyrimidines; Thiazoles; 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

This note mechanistically accounts for recent unexplained findings that all-trans retinoic acid (ATRA, also termed tretinoin) exerts an anti-viral effect against hepatitis C virus (HCV) in chronically infected patients, in whom ATRA also showed synergy with interferon-alpha. How HCV replication was suppressed was unclear. Both effects of ATRA can be accounted for by ATRA's upregulation of RIG protein, an 18 kDa product of retinoic induced gene-1. Increased RIG then couples ATRA to increased Type 1 interferons' production. Details of this mechanism predict that ATRA will similarly augment interferon-a activity in treating chronic myelogenous leukemia, melanoma, myeloma and renal cell carcinoma and that the addition of ribavirin and/or bexarotene will each incrementally enhance interferon-a responses in these cancers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Carcinoma, Renal Cell; Drug Screening Assays, Antitumor; Hepacivirus; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Melanoma; Models, Biological; Multiple Myeloma; Ribavirin; Tetrahydronaphthalenes; Treatment Outcome; Tretinoin

Advances in high-throughput technologies to measure genome-scale changes of genes, proteins, and other biomolecular components ('omics') in complex biological systems have dramatically revolutionized biomedical research. However, the benefits of utilizing omics information in drug development have not yet been fully realized. Fortunately, the integration of modern systems biology efforts with traditional medicine philosophies, together with integrative bioinformatics, has driven the development of a new drug discovery paradigm. Using leukemia as a disease model, therapeutic synergism between drugs and natural products has been investigated by incorporating transcriptomics and proteomics data into a network-like understanding. Here, these recent advancements will be discussed in detail, along with perspectives in the field of drug synergism.

Topics: Animals; Arsenic Trioxide; Arsenicals; Benzamides; Drug Discovery; Drug Synergism; Gene Expression Profiling; Herb-Drug Interactions; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Oxides; Piperazines; Proteome; Pyrimidines; Tretinoin

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

The rapid rise of chemical biology aimed at studying signaling networks for basic cellular activities using specific, active small molecules as probes has greatly accelerated research on pathological mechanisms and target therapy of diseases. This research is especially important for malignant tumors such as leukemia, a heterogeneous group of hematopoietic malignancies that occurs worldwide. With the use of a chemical approach combined with genetic manipulation, great progress has been achieved over the past few decades on the biological, molecular and cytogenetic aspects of leukemia, and in its diagnosis and therapy. In particular, discoveries of the clinical effectiveness of all-trans retinoic acid and arsenic trioxide in the treatment of acute promyelocytic leukemia and the kinase inhibitors Imatinib and Dasatinib in the treatment of chronic myelogenous leukemia not only make target therapy of leukemia a reality, but also push mechanisms of leukemogenesis and leukemic cell activities forward. This review will outline advances in chemical biology that help our understanding of the molecular mechanisms of cell differentiation and apoptosis induction and target therapy of leukemia.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Benzamides; Dasatinib; Growth Inhibitors; Humans; Imatinib Mesylate; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Oxides; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Thiazoles; Tretinoin

Leukemia is a multistep process involving accumulation of genetic alterations over time. These genetic mutations destroy the delicate balance between cell proliferation, differentiation, and apoptosis. Traditional approaches to treatment of leukemia involve chemotherapy, radiation, and bone marrow transplantation. In recent years, specific targeted therapies have been developed for the treatment of leukemia. The success of treatment of acute promyelocytic leukemia with All Trans Retinoic Acid (ATRA) and CML with imatinib have lead to increased efforts to identify targets that can be inhibited by small molecules for treatment of hematological malignancies. In this review, we describe the current advances in the development of targeted therapy in acute myeloid leukemia.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Cell Cycle; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Piperazines; Pyrimidines; ras Proteins; Receptor Protein-Tyrosine Kinases; Translocation, Genetic; Tretinoin

Topics: Antineoplastic Agents; Benzamides; Enzyme Inhibitors; Gene Targeting; Hematologic Neoplasms; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Tretinoin

Topics: Antineoplastic Agents; Benzamides; Clinical Trials as Topic; Cytarabine; Drug Monitoring; Fusion Proteins, bcr-abl; Gene Targeting; Genes, Wilms Tumor; Genetic Therapy; Hematopoietic Stem Cell Transplantation; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Neoplasm, Residual; Oncogene Proteins, Fusion; Pharmacogenetics; Piperazines; Pyrimidines; Recombinant Fusion Proteins; Therapeutic Equivalency; 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

Chronic myelogenous leukemia (CML) is a slowly progressive disease characterized by the overproduction of granulocytes (neutrophils, eosinophils, and basophils). A blood smear shows moderate elevations in white blood cell counts that may persist for years and be benign. Platelets are increased in number, although their function is impaired, resulting in symptoms of easy bleeding (purpura, swollen gums). Conventional medical treatment is a marrow transplant and alkylating agents, which are usually prescribed only during crisis. Several nutrients and botanicals have been studied for use in CML, including vitamin A and all-trans retinoic acid (Retin-A), vitamin D3, vitamin E, vitamin B12, indirubin (found in herbs including Indigofera tinctoria and Isatis tinctoria), and Curcuma longa. This article briefly reviews the scientific literature on the therapeutic use of these nutrients for CML.

Topics: Antineoplastic Agents; Curcumin; Humans; Indoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nutritional Support; Tretinoin; Vitamins

The beginning of this century was marked, in our specialty as in other, by two revolutions: the routine use of molecular biology tools for a better prognosis of the disease (flt3 receptor duplication in AML, mutational profile of Ig genes in CLL, gene expression profile with ARN chips in aggressive lymphomas.), and the discovery of "intelligent" molecules, targeting the tumoral cell. In this category, the most appealing is the STI571 (Gleevec , Novartis), targeting the molecular abnormality of the cells expressing bcr-abl protein: CML, ALL Ph1(+). Other molecules targeting signal transduction proteins (ras farnesylation inhibitors for example) are already in clinical trials. The increasing therapeutic use of monoclonal antibodies is also to be cited, with a special mention concerning the rituximab, used in several B lymphoid pathologies, from lymphoma to autoimmune diseases. His very good tolerance permits his use in ambulatory patients, and his combination with chemotherapy or his linkage with radioactive elements render this molecule indispensable. The other side of these molecules is their incredibly high cost, explaining the uncontrolled expenses in 2001 of hospitals hosting hematology as well as oncology activities.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Hematopoietic Stem Cell Transplantation; Humans; Imatinib Mesylate; Immunotoxins; Leukemia, Lymphoid; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Lymphoma; Myelodysplastic Syndromes; Piperazines; Pyrimidines; Tretinoin

STI571 selectively inhibits the ABL-tyrosine kinase, the activity of which is activated by the formation of chimeric BCR/ABL. A phase I study in the USA showed STI571 to be remarkably effective in cases of interferon-refractory chronic myeloid leukemia, with almost no adverse effects. STI571 may become the first choice drug prior to stem cell transplantation and interferon treatment.

Topics: Antineoplastic Agents; Clinical Trials, Phase I as Topic; Enzyme Inhibitors; Fusion Proteins, bcr-abl; Genistein; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Protein-Tyrosine Kinases; Styrenes; Tretinoin

Topics: Adult; Aged; Bone Marrow Transplantation; Child; Chromosomes, Human, Pair 22; Chromosomes, Human, Pair 9; Cytarabine; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Middle Aged; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Translocation, Genetic; 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: Animals; Cells, Cultured; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Protein Processing, Post-Translational; Proteins; Tretinoin; Tumor Cells, Cultured

While much is known about CML at both the clinical and molecular biological levels, the precise relationship between the disease at these two levels is unclear. The appearance of the fusion gene bcr-abl and disorders in the regulation of the myc gene, and perhaps other oncogenes which code for nucleoproteins, appear to play integral roles in the genesis of the chronic and blastic phases of the disease. The resistance of this disease to cytotoxic therapy appears to reflect both "classical" drug resistance and the ability of those cells which survive cytotoxic therapy to rapidly replace the killed cells thereby offsetting the effects of chemotherapy ("regrowth resistance"). The clinical evolution of the disease is compatible with two fundamentally different processes: one compatible with a deterministic chaotic model and the other involves two basically independent linear phenomena which overlap and intersect as the blastic phase appears and replaces the chronic phase.

Topics: Antineoplastic Agents; Blast Crisis; Cell Differentiation; Chromosome Aberrations; Clone Cells; DNA, Neoplasm; Drug Resistance; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Chronic-Phase; Models, Biological; Nonlinear Dynamics; Oncogenes; Tretinoin; Tumor Cells, Cultured

The aims of this study were to evaluate the safety and efficacy of all-trans retinoic acid (ATRA) in the treatment of the accelerated and blastic phase of chronic myeloid leukemia (CML) and to evaluate in vitro correlates of biological activity. ATRA was administered in an intermittent schedule to patients with CML in the accelerated or blastic phases for a 6 week induction period, which was continued if there was evidence of clinical response or stable disease. If the patient was progressing at 6 weeks, interferon-alpha could be added to the ATRA. Laboratory correlative studies were performed prior to treatment and at intervals during treatment to evaluate effects on maturation and differentiation, and on CML progenitor cell growth by assessment of colony-forming cells (CFC). Eighteen patients were enrolled. There was 1 complete response, 1 partial response and 2 with hematological improvement. A fifth patient was stable on ATRA and interferon for several months. Laboratory data for the responders demonstrated high sensitivity of primary CFC to ATRA prior to treatment and low serial CFC counts on ATRA therapy. ATRA demonstrated clinical and hematological activity in 5 of 18 patients with the accelerated phase of CML, and there was evidence of a biological effect in laboratory studies of 3 of the 5 patients' progenitor cells. Combination therapy with other differentiating agents may be useful in this disease.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Combined Modality Therapy; Cytogenetic Analysis; Disease-Free Survival; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Interferon alpha-2; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Recombinant Proteins; Survival Analysis; Time Factors; Tretinoin

The cytogenetic responses during the first chronic phase of 11 patients with chronic myeloid leukaemia (CML) treated with all-trans retinoic acid (ATRA) + interferon (IFN) were compared with those of 9 other CML patients treated with IFN alone. Metaphase and interphase cytogenetics and a semi-quantitative polymerase chain reaction were used to evaluate the cytogenetic responses. Two of the 11 patients in the ATRA + IFN group were withdrawn, one of them because of IFN intolerance, and the other because of non-compliance. Of the 9 remaining ATRA + IFN-treated patients 6 exhibited major cytogenetic responses, 3 of which were complete. Of the 9 patients treated with IFN alone, only 2 showed major cytogenetic responses. No severe adverse effects were observed. The preliminary results suggest that the ATRA + IFN combination may be superior in achieving cytogenetic remission in the first chronic phase of CML.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytogenetic Analysis; Female; Humans; Interferon alpha-2; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Recombinant Proteins; Recurrence; Remission Induction; Tretinoin

Preclinical data have shown that all-trans retinoic acid (ATRA) with interferon-alpha (IFN-alpha) can exert significant suppressive effects on Philadelphia-chromosome (Ph)-positive cells. The aim of this study combining IFN-alpha, low-dose cytosine arabinoside (ara-C) and ATRA was to increase the proportion of patients achieving a major cytogenetic response, in comparison with a group of 140 patients previously treated with IFN-alpha plus low-dose ara-C. Forty three patients with Ph-positive CML in early chronic phase were treated with IFN-alpha 5 MU/m2 s.c. daily, low-dose ara-C 10 mg s.c. daily and ATRA 45 mg/m2 orally daily, for 7 consecutive days every other week. Overall, 76% of patients achieved a complete hematologic response (CHR). A cytogenetic response was in observed 59% (major in 38% and complete in 17%). Compared with patients treated with IFN-alpha and low-dose ara-C, those receiving additional ATRA had a lower CHR rate (p. 014), but other response rates were similar. Severe toxicities were common with the triple regimen (64%), mostly related to ATRA therapy. Two patients experienced pseudotumor cerebri; two patients had leukocytosis during the week on ATRA treatment, decreasing during the week off (one suffered a severe asthma-like reaction followed by pulmonary edema, resembling ATRA syndrome). Six patients had other unusual side-effects: aseptic necrosis of the hip (1 patient), ataxic syndrome (1 patient), paranoid syndrome (2 patients), syncopal episodes (1 patient), pure red cell aplasia (1 patient). In conclusion the results of IFN-alpha and low-dose ara-C combined with ATRA in patients with early CML-chronic phase were disappointing, due to excessive toxicity. Whether different ATRA dose schedules may result in fewer side-effects and improve hematologic and cytogenetic response remains to be determined.

Topics: Adult; Aged; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Ataxia; Combined Modality Therapy; Cytarabine; Drug Administration Schedule; Female; Femur Head Necrosis; Humans; Immunologic Factors; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytosis; Life Tables; Male; Middle Aged; Paranoid Disorders; Pilot Projects; Pseudotumor Cerebri; Pulmonary Edema; Red-Cell Aplasia, Pure; Remission Induction; Survival Analysis; Syncope; Treatment Outcome; Tretinoin

Since in vitro observations indicated that all-trans retinoic acid (ATRA), especially in combination with IFNalpha, can exert significant suppressive effects on Ph+ cells, we investigated the effects and the pharmacokinetic profile of ATRA in a selected cohort of patients with Ph+ chronic myeloid leukemia (CML) in chronic phase. Eighteen patients were treated with ATRA at a dose of 80 mg/m2/day (p.o.), divided into two equal doses after meals, for 7 consecutive days every other week for a maximum of 12 courses (1 course = 1 week on and 1 week off). Pharmacokinetic profiles of ATRA were evaluated during intermittent therapy on days 1 and 7 of course 1; on day 1 of course 2; on day 1 of course 6. Out of the 18 patients treated with ATRA, 11 (61%) went off study before the sixth course of treatment because of progressive hyperleukocytosis (seven cases), or thrombocytosis (one case), or refusal (three cases). Seven (39%) patients completed the first six courses (12 weeks) of treatment with ATRA and two of them (11%) maintained a white blood cell (WBC) <10 x 10[9]/l which was induced by the pretreatment with hydroxyurea. One patient completed the 12th course of ATRA maintaining WBC <10 x 10(9)/l, platelets <500 x 10(9)/l and spleen not palpable. The treatment with ATRA was well tolerated and only one patient discontinued the therapy because of non-hematological side-effects. The area under the concentration-time curve (AUC) decreased significantly (P< 0.001) during the first week of therapy. By adopting an intermittent dosing regimen, 1 week on/ 1 week off (1 course), at the start of courses 2 and 6, we obtained the ATRA AUCs equivalent to the ones achieved on day 1 of course 1. In conclusion, our results showed that ATRA alone appeared to be unable to control the WBC expansion in the CML patients in chronic phase. Moreover, it did not induce any remarkable cytoreductive effects on the platelet count and on the hemoglobin level. The major interest of ATRA would be in combination with other therapies. If ATRA was given in combination with IFNalpha or other agents, dose reduction of these would not be planned. On the basis of the pharmacokinetic profile, ATRA should be administered intermittently rather than continuously.

Topics: Adult; Aged; Antineoplastic Agents; Drug Administration Schedule; Female; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Chronic-Phase; Leukocyte Count; Male; Middle Aged; 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

Clofarabine (2‑chloro‑2'‑fluoro‑2'‑deoxyarabinosyladenine, CIF), a second‑generation 2'‑deoxyadenosine analog, possesses a variety of anti‑cancer activities, including the capacity to modulate DNA methylation marks. Bioactive nutrients, including resveratrol (RSV) and all‑trans retinoic acid (ATRA) have been indicated to regulate epigenetic machinery in malignant cells. The purpose of the current study was to evaluate whether the tested phytochemicals, RSV or ATRA, can improve the therapeutic epigenetic effects of CIF in chronic myeloid leukemia (CML) cells. The present study investigates, to the best of our knowledge, for the first time, the influence of CIF in combination with RSV or ATRA on the expression of relevant modifiers of DNA methylation machinery, including DNA Methyltransferase 1 (DNMT1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) in CML cells. Subsequently, the combinatorial effects on promoter methylation and transcript levels of methylation‑silenced tumor suppressor genes (TSGs), including phosphatase and tensin homologue (PTEN) and retinoic acid receptor beta (RARB), were estimated using MSRA and qPCR, respectively. The tested TSGs were chosen according to bioinformatical analysis of publicly available clinical data of human DNA methylation and gene expression arrays in leukemia patients. The K562 cell line was used as an experimental CML in vitro model. Following a period of 72 h exposure of K562 cells, the tested combinations led to significant cell growth inhibition and induction of caspase‑3‑dependent apoptosis. These observations were accompanied by DNMT1 downregulation and CDKN1A upregulation, with a concomitant enhanced decrease in DNMT1 protein level, especially after ATRA treatment with CIF. Concurrent methylation‑mediated RARB and PTEN reactivation was detected. The results of the current study demonstrated that CIF that was used in combination with the tested phytochemicals, RSV or ATRA, exhibited a greater ability to remodel DNA methylation marks and promote cell death in CML cells. These results may support the application of CIF combinations with natural bioactive agents in anti‑leukemic epigenetic therapy.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Apoptosis; Cell Line, Tumor; Clofarabine; Cyclin-Dependent Kinase Inhibitor p21; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Drug Synergism; Epigenesis, Genetic; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Resveratrol; Tretinoin

ITR‑284 is a carboxamide analog that can inhibit proliferation in human promyelocytic leukemia HL-60 cells. To understand the effects and molecular mechanisms of ITR‑284 in human erythromyeloblastoid leukemia, we treated K562 cells with different concentrations of ITR‑284 (0, 2, 4, 6, 8 and 10 nM) and all-trans retinoic acid (ATRA) (0, 0.1, 0.5, 1, 5 and 10 µM) for 24 h. The IC50 of ITR‑284 was ~10 nM in K562 cells treated for 24 h as determined by MTT assay. May-Grünwald-Giemsa staining and nitro blue tetrazolium (NBT) assays were used to determine cell morphology changes and differentiation after ITR‑284 and ATRA treatment. In addition, mRNA expression levels of hematopoietic factors, including GATA‑1, NF-E2 and GATA‑2, were elevated, while expression levels of BCR‑ABL were downregulated in K562 cells after 24 h of treatment with ITR‑284 as determined by quantitative reverse transcription polymerase chain reaction. In addition, western blot analyses showed that FOXM1, GLI 1 and c-MYC protein levels were decreased by ITR‑284. Taken together, our data show that ITR‑284 induced K562 cell differentiation, which led to decreased tumorigenesis. Our findings suggest that ITR‑284 could be a potential candidate for treating chronic myelogenous leukemia.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Survival; Dose-Response Relationship, Drug; Forkhead Box Protein M1; Fusion Proteins, bcr-abl; GATA1 Transcription Factor; GATA2 Transcription Factor; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; NF-E2 Transcription Factor, p45 Subunit; Proto-Oncogene Proteins c-myc; Thiophenes; Tretinoin; Zinc Finger Protein GLI1

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD+) levels and blocked acquired resistance by inhibiting the activity of the NAD+-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.

Topics: ADP-ribosyl Cyclase 1; Apoptosis; Benzamides; Cell Differentiation; Cell Line, Tumor; Dasatinib; DNA Damage; Drug Resistance, Neoplasm; Flow Cytometry; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Point Mutation; Protein Kinase Inhibitors; Pyrimidines; Sirtuin 1; Thiazoles; Tretinoin

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However, despite an overall significant hematological and cytogenetic response, imatinib therapy may favor the emergence of drug-resistant clones, ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression, either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid, a clinically used drug. Furthermore, we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore, combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Activation; Gene Expression Regulation, Enzymologic; HEK293 Cells; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Pyrimidines; Telomerase; Transcription, Genetic; Tretinoin

Arsenic trioxide (As(2)O(3)) has potent antileukemic properties in vitro and in vivo, but the mechanisms by which it generates its effects on target leukemic cells are not well understood. Understanding cellular mechanisms and pathways that are activated in leukemic cells to control the generation of As(2)O(3) responses should have important implications in the development of novel approaches using As(2)O(3) for the treatment of leukemias. In this study, we used immunoblotting and immune complex kinase assays to provide evidence that the kinases thousand-and-one amino acid kinase 2 (TAO2) and transforming growth factor-beta-activated kinase 1 (TAK1) are rapidly activated in response to treatment of acute leukemia cells with As(2)O(3). Such activation occurs after the generation of reactive oxygen species and regulates downstream engagement of the p38 mitogen-activated protein kinase. Our studies demonstrate that siRNA-mediated knockdown of TAO2 or TAK1 or pharmacological inhibition of TAK1 enhances the suppressive effects of As(2)O(3) on KT-1-derived leukemic progenitor colony formation and on primary leukemic progenitors from patients with acute myelogenous leukemia. These results indicate key negative-feedback regulatory roles for these kinases in the generation of the antileukemic effects of As(2)O(3). Thus, molecular or pharmacological targeting of these kinases may provide a novel approach to enhance the generation of arsenic-dependent antileukemic responses.

Topics: Acetylcysteine; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Dithiothreitol; Enzyme Activation; Genetic Variation; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Kinase Kinases; Oxides; Phosphorylation; Protein Kinases; Reactive Oxygen Species; RNA, Small Interfering; Tretinoin; U937 Cells

The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Disease Progression; Female; Gene Expression Regulation, Leukemic; Gene Silencing; Granulocytes; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Neoplastic Stem Cells; Prognosis; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-abl; Tretinoin

We investigated the possible mechanisms of All-trans retinoic acid (ATRA)-promoted apoptosis induced by alpha-tocopherol succinate (alpha-TS) in freshly isolated leukemic cells obtained from chronic myeloid leukemic patients. alpha-TS at 50 microM concentration significantly decreased superoxide dismutase (SOD) activity and reduced glutathione (GSH) by 29% and 25%, respectively, and increased lipid peroxidation level by 33%. Though 10 microM ATRA did not affect these parameters, it further significantly enhanced alpha-TS-induced changes. Bax expression in the leukemic cells was increased by treatment with ATRA, alpha-TS, and their combination to 40%, 240%, and 320%, respectively, without any change in Bcl2 and p53 expression. C-myc was down regulated by treatment with ATRA, alpha-TS and their combination to 22%, 48.5%, and 52%, respectively. In conclusion, the data reveal that enhancement of alpha-TS-induced apoptosis by ATRA in leukemic cells was through up regulation of Bax and lipid peroxidation, and down regulation of c-myc and GSH.

Topics: Adult; alpha-Tocopherol; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Drug Synergism; Female; Gene Expression Regulation, Leukemic; Glutathione; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lipid Peroxidation; Male; Middle Aged; Proto-Oncogene Proteins c-myc; Superoxide Dismutase; Tretinoin; Tumor Cells, Cultured

A 32-yr-old man with the chronic phase of chronic myeloid leukemia (CML-CP) was treated with imatinib mesylate for 6 mo. The real-time quantitative reverse transcription PCR ratio for BCR/ABL in blood mRNA (BCR/ABL RT-QPCR) decreased from an initial value of 0.0159 to a low value of 0.0012 after 3 mo, indicating complete hematologic response. During the next 3 mo, the patient progressed to a promyelocytic blast crisis, displaying leukemic cells containing both BCR/ABL and PML/RARalpha chimeric mRNAs. Complete remission was achieved by therapy with all-trans retinoic acid (ATRA) and high-dose imatinib mesylate. Using retrospective PML/RARalpha RT-QPCR with a bone marrow specimen obtained at the initial diagnosis of CML-CP, we quantified the mRNA ratio as 0.000321, suggesting that the clonal evolution of PML/RARalpha translocation occurred early in the CML-CP.

Topics: Adult; Antineoplastic Agents; Benzamides; Blast Crisis; Humans; Imatinib Mesylate; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Piperazines; Pyrimidines; Treatment Outcome; Tretinoin

Nested reverse-transcriptase polymerase chain reaction (rt-PCR) was performed on 58 leukemia patients at BIRDEM Laboratory, as a pioneering work in Bangladesh. Thirty of themwere examined for the presence of BCR-ABL being clinically and morphologically diagnosed as chronic myeloid leukemia (CML) and 28 for PML-RARalpha fusion transcripts being clinically and morphologically diagnosed as acute promyelocytic leukemia (APL/ AML M3). The cases were selected for targeted therapy with imatinib mesylate and all-Trans retinoic acid (ATRA) to treat CML and APL respectively. Samples were received either before commencement or during therapy. In the positive cases, amplified DNA products were visible after gel electrophoresis and were reported accordingly. In case of BCR-ABL, positive results were found for five out of six (83.33%) untreated cases and 11 out of 24 (45.83%) treated cases. Positive results for PML-RARalpha were found for 12 out of 14 (85.70%) untreated cases and 11 out of 16 (68.75%) treated cases. A strong positive correlation was found between duration of treatment and negativity of PCR results in both the cases. In present times, the detection of minimal residual disease in patients undergoing treatment for hematological malignancies has become an important goal, not only to monitor the effectiveness of therapy but also to detect an impending relapse. This is the first time in Bangladesh that rt-PCR method is being employed to detect or monitor the presence of abnormal fusion genes in hematological malignancies.

Topics: Adolescent; Adult; Antineoplastic Agents; Bangladesh; Benzamides; Child; Female; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Piperazines; Prospective Studies; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Treatment Outcome; Tretinoin

We investigated the in vitro efficacy of all-trans retinoic acid (ATRA) and alpha-tocopherol succinate (alpha-TS) alone and in combination on the induction of cell death in freshly isolated leukemic cells obtained from chronic myeloid leukemia (CML) patients. In vitro cytotoxicity and induction of lipid peroxidation by ATRA (10 microM) and alpha-TS (25 or 50 microM) were evaluated in primary leukemic cells by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and malondialdehyde formation respectively. Treatment of leukemic cells with alpha-TS alone or in combination with ATRA significantly (P < 0.05) decreased the cell viability in a concentration and time dependent manner as compared to peripheral blood mononuclear cells obtained from normal healthy controls. Lipid peroxidation was enhanced by 98% (P < 0.05) on combined treatment of cells with ATRA (10 microM) and alpha-TS (50 microM). ATRA alone did not enhance the externalization of phosphatidyl serine as studied by annexin-V binding using fluorescence activated cell sorter analysis, whereas in combination with alpha-TS it increased to 400% at 12 h. The treatment of leukemic cells to combination of ATRA with alpha-TS significantly decreased (P < 0.05) mitochondrial membrane potential and enhanced lysosomal destabilization. The combination of these drugs also increased mitochondrial and cytosolic reactive oxygen species (ROS) production, nitric oxide levels, and caspase-3 activity significantly and caused DNA fragmentation at 24 h in a concentration dependent manner in the leukemic cells. Our data suggest that ATRA in combination with alpha-TS efficiently induces apoptosis in leukemic cells, which may be a useful therapeutic modality in CML patients.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase 3; Cell Separation; Cell Survival; DNA Fragmentation; Drug Evaluation, Preclinical; Drug Synergism; Female; Humans; In Vitro Techniques; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lipid Peroxidation; Lysosomes; Male; Membrane Potential, Mitochondrial; Middle Aged; Tocopherols; Tretinoin; Vitamin E

Recent progress in molecular biology has led to an increase of prognostic markers and development of molecular-targeted therapy in pediatric malignancies. Previous treatment including stem cell transplantation showed a remarkable cure rate, however, some patients are resistant to such therapy. Recently, all-trans retinoic acid (ATRA) for acute promyeloblastic leukemia, imatinib for chronic myeloid leukemia, and rituximab for B-cell malignant lymphoma serve to improve the clinical outcome of these patients. Furthermore, molecular-targeted therapies including tyrosine kinase inhibitor, farnesyl transferase inhibitor, methylation inhibitor, and histone deacetyl enzyme inhibitor, were applied for clinical study. For pediatric malignancies, in addition to molecular-targeted therapy against leukemia, molecular-targeted therapies, mainly tyrosin kinase inhibitors, were applied to neuroblastoma and various types of sarcomas. Recent progress in prognostic molecular marker and molecular-targeted therapy against pediatric malignancies was here reviewed.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzamides; Biomarkers, Tumor; Child; Enzyme Inhibitors; fms-Like Tyrosine Kinase 3; Hematologic Neoplasms; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Loss of Heterozygosity; Piperazines; Prognosis; Protein-Tyrosine Kinases; Pyrimidines; Quinolones; Rituximab; Tretinoin

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

We show that common heterochromatin antigenic protein markers [HP1alpha, -beta, -gamma and mono-, di-, and trimethylated histone H3 lysine 9 (H3K9)], although present in human blood progenitor CD34+ cells, differentiated lymphocytes, and monocytes, are absent in neutrophil granulocytes and to large extent, in eosinophils. Monomethylated and in particular, dimethylated H3K9 are present to variable degrees in the granulocytes of chronic myeloid leukemia (CML) patients, without being accompanied by HP1 proteins. In patients with an acute phase of CML and in acute myeloid leukemia patients, strong methylation of H3K9 and all isoforms of HP1 are detected. In chronic forms of CML, no strong correlations among the level of histone methylation, disease progression, and modality of treatment were observed. Histone methylation was found even in "cured" patients without Philadelphia chromosome (Ph) resulting from +(9;22)(q34;q11) BCR/ABL translocation, suggesting an incomplete process of developmentally regulated chromatin remodeling in the granulocytes of these patients. Similarly, reprogramming of leukemia HL-60 cells to terminal differentiation by retinoic acid does not eliminate H3K9 methylation and the presence of HP1 isoforms from differentiated granulocytes. Thus, our study shows for the first time that histone H3 methylation may be changed dramatically during normal cell differentiation. The residual histone H3 methylation in myeloid leukemia cells suggests an incomplete chromatin condensation that may be linked to the leukemia cell proliferation and may be important for the prognosis of disease treatment and relapse.

Topics: Acute Disease; Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Cell Differentiation; Cell Proliferation; Chromatin; Chromobox Protein Homolog 5; Disease Progression; Granulocytes; Histones; HL-60 Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Methylation; Middle Aged; Tretinoin

Transglutaminase 2 (TG2) is a GTP-binding protein with transglutaminase activity. Despite advances in the characterization of TG2 functions and their impact on cellular processes, the role of TG2 in Human chronic myelogenous leukemia K562 cell line is still poorly understood. To understand the biological significance of TG2 during the differentiation of K562 cells, we established and characterized K562 cells that specifically express TG2. Non-transfected K562 cells showed the increase of membrane-bound-TG2 level after 3 days in the response to Hemin and all trans-retinoic acid (tRA), indicating that membrane recruitment of TG2 is occurred during the erythroid differentiation. However, membrane recruitment of TG2 in TG2-transfected cells revealed within earlier time period, compared with that in vector-transfected cells. The ability of membrane-bound-TG2 to be photoaffinity-labeled with [alpha-32P]GTP was also increased in TG2-transfected cells. TG2-transfected cells activated Akt phosphorylation and inactivated ERK1/2 phosphorylation, compared with vector-transfected cells. Furthermore, phosphorylation of CREB, one of the Akt substrates, was increased in TG2-transfected cells and this phenomenon was confirmed by RT-PCR analysis of several marker genes related with erythroid lineage in the absence of PI3K specific inhibitor, Wortmannin, indicating that PI3K/Akt signaling pathway also involved in the differentiation of the cell. Finally, as results of benzidine positive staining as well as hemoglobinization analysis, overexpression of TG2 revealed acceleration of the erythroid differentiation of K562 cells. Taken together, there was no increased TG2 expression level in the response of Hemin/tRA and delayed differentiation in vector transfected cells than in TG2-transfected cells, suggesting that suppression of TG2 expression may retard the erythroid differentiation of K562 cells. Therefore, our study may give a new insight for another aspect of the development of this disease.

Topics: Androstadienes; Cell Differentiation; CREB-Binding Protein; Enzyme Activation; Enzyme Inhibitors; Erythroid Precursor Cells; Genetic Vectors; GTP-Binding Proteins; Hemin; Humans; K562 Cells; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Protein Glutamine gamma Glutamyltransferase 2; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Trans-Activators; Transfection; Transglutaminases; Tretinoin; Wortmannin

To explore whether the oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation.. Intracellular mean fluorescence intensity was measured by flow cytometry.. After treatment with FITC-labeled G3139 at the concentration of 0.60 mumol.L-1 for 4 h, the G3139 uptake into peripheral blood mononuclear cell and bone marrow mononuclear cell in hematological tumor patients was significantly higher than that in normal control. There was different uptake of G3139 among the malignant hematological tumor cell strains, and the uptake in cells derived from monocyte, B lymphocyte and myeloid cell was much higher than that in cells derived from T lymphocyte. After treatment with all-trans retinoic acid (ATRA), HL60 cell proliferation was markedly inhibited and the uptake of G3139 decreased significantly.. Hematological tumor cells were capable of taking up oligonucleotide, and the oligonucleotide uptake in hematological tumor cells is related to its cellular species and its activation.

Topics: Biological Transport; Cell Division; Genes, bcl-2; HL-60 Cells; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; Lymphoma, Non-Hodgkin; Oligonucleotides, Antisense; Thionucleotides; Tretinoin; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Humans; Interferons; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Remission Induction; Tretinoin

Combination of STI571, a tyrosine kinase inhibitor, with other drugs may be beneficial in the treatment of chronic myeloid leukaemia (CML). We measured the effects of STI571, AG490, farnesyltransferase inhibitor (FTI), interferon alpha (IFN-alpha), cytosine arabinoside (Ara-C) and all-trans retinoic acid (ATRA), singly and in combination, on clonogenic leukaemic cell proliferation. STI571, IFN-alpha and ATRA each reduced proliferation by 50-60%; AG490, FTI and Ara-C had less effect. Comparing the observed and expected (i.e. additive) effects of drug combinations showed STI571 + FTI, STI571 + AG490 and IFN-alpha + ATRA were additive; STI571 + IFN-alpha, IFN-alpha + Ara-C and STI571 + AG490 + FTI were less than additive. Thus, STI571 + FTI, STI571 + AG490 and IFN-alpha + ATRA may be better combination therapies for CML than STI571 + IFN-alpha, IFN-alpha + Ara-C or STI571 + AG490 + FTI.

Topics: Alkyl and Aryl Transferases; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Benzamides; Cell Division; Cytarabine; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Imatinib Mesylate; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Stem Cells; Tretinoin; Tyrphostins

A 51-year-old male who had intractable leg ulcers during treatment for chronic myelogenous leukaemia with hydroxyurea (HU) is reported. His leg ulcers were treated by application of tretinoin tocoferil (TT) ointment after surgical debridement; good results were obtained. Although stopping HU administration is vitally important, surgical debridement and TT application are effective for treating leg ulcers due to HU.

Topics: Antineoplastic Agents; Debridement; Drug Combinations; Humans; Hydroxyurea; Leg Ulcer; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Ointments; Tretinoin; Vitamin E

Topics: Antineoplastic Agents; Antioxidants; Benzamides; Breast Neoplasms; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Genes, BRCA1; Genes, BRCA2; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lung Neoplasms; Male; Mastectomy; Ovarian Neoplasms; Ovariectomy; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Receptors, Retinoic Acid; Smoking; Tobacco Smoke Pollution; Tretinoin; Vitamins

The treatment of CML with IFN alpha is limited due to resistance against this substance. Recent studies with different cells than chronic myelogenous leukemic cells revealed a synergistic effect of a combined use of Retinoids (RA) and IFN alpha. The purpose of the study was to detect possible interactions of IFN alpha and RA in CML considering also the effect of the BCR-ABL gene-product. Therefore, we investigated three CML cell lines in their proliferation after incubation with IFN alpha and Retinoids alone and in combination. We measured low susceptibility to IFN alpha but a marked influence of the Retinoids. In combination, the growth inhibition was enhanced potentially in response to an increased efficacy of IFN alpha. Even solely, ineffective concentrations of both substances lead to decreased proliferation.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Drug Synergism; Humans; In Vitro Techniques; Interferon-alpha; Isotretinoin; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Thymidine; Tretinoin; Tumor Cells, Cultured

Cytogenetic responses of 11 chronic myeloid leukemic (CML) patients during the first chronic phase, treated with the combination of all-trans retinoic acid (ATRA) + interferon (IFN) were compared to 9 other CML patients of phase one, treated with interferon monotherapy. Metaphase and interphase cytogenetics and a semiquantitative polymerase chain reaction (PCR) were used to evaluate the cytogenetic responses. Two of the 11 patients in the ATRA + interferon treated group were withdrawn, one of them because of interferon intolerance, and the other because of compliance failure. Among the 9 ATRA + interferon treated patients 6 major cytogenetic responses could be detected and 3 of them were complete. Of the 9 patients treated with IFN monotherapy only 2 major cytogenetic responses could be registered. No severe adverse effects were observed. The first results suggest that the ATRA + interferon combination may be superior in achieving cytogenetic remission in the first chronic phase of CML.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Female; Humans; Interferons; Interphase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Polymerase Chain Reaction; Remission Induction; Treatment Outcome; Tretinoin

Although interferon (IFN)-alpha has no specific inhibitory effect on the plating efficiency of granulocyte-macrophage colony-forming cells (CFU-GM) from patients with chronic myeloid leukaemia (CML), it does selectively inhibit the replating ability (secondary colony formation) of CML CFU-GM. Thus, amplification of CFU-GM may be a target for IFN-alpha and other agents used in the treatment of CML. Here we examined whether cytarabine (Ara-C) or all-trans retinoic acid (ATRA) exert similar effects and whether they might in combination with IFN-alpha enhance its efficacy. We found that Ara-C preferentially inhibits the formation of CML CFU-GM compared to normal CFU-GM, but this inhibition was not increased by addition of IFN-alpha. When Ara-C was added to cultures containing IFN-alpha, the inhibition of replating by CML progenitors was abrogated. ATRA increased significantly the plating efficiency of normal CFU-GM. The addition of IFN-alpha to ATRA had no effect on CML or normal colony numbers. However, addition of ATRA to cultures containing IFN-alpha reversed the selective inhibition of CML CFU-GM replating seen in cultures containing IFN-alpha alone. In four IFN-alpha/Ara-C experiments, secondary CML patient-derived colonies were examined by fluorescence in situ hybridisation (FISH). All of them were Ph chromosome positive. No significant effects on CFU-GM production were observed when CML primitive haemopoietic progenitor cells were investigated in a delta (delta) assay. Thus we conclude that combining IFN-alpha with Ara-C or ATRA neutralises the effect of IFN-alpha on CML CFU-GM. This observation provides a rationale for treating patients with alternating courses of IFN-alpha and Ara-C or ATRA, rather than giving either of these two agents in combination with IFN-alpha.

Topics: Cell Division; Cytarabine; Hematopoietic Stem Cells; Humans; In Situ Hybridization, Fluorescence; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Tretinoin

Chronic myeloid leukemia (CML) is a hematological malignancy resulting from clonal expansion and massive accumulation of leukemic myeloid cells that retain differentiation and maturation capacity. Since CML cell accumulation has been related to apoptosis inhibition by the product of the BCR-ABL gene, attempts to eradicate leukemic cells would require therapeutic drugs able to overcome this inherent resistance. Here, we investigated in vitro the apoptotic effect of all-trans retinoic acid (ATRA) and cytosine arabinoside (ARA-C), employed alone, in combination or in sequence, on freshly isolated cells from 10 patients with chronic-phase CML. Our cell cultures showed that both ATRA and ARA-C were able to induce apoptosis in CML cells, even if ARA-C resulted more effective than ATRA. The combined use of ATRA and ARA-C seemed to have only an additive effect while the sequential use did not show any advantage. These in vitro observations indicate that ATRA and ARA-C may be effective in reducing CML cells through apoptosis induction, suggesting that it could be worthwhile to examine ATRA and ARA-C combinations in the therapy of CML.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Cell Differentiation; Cell Separation; Cytarabine; DNA Fragmentation; Electrophoresis, Agar Gel; Female; Granulocytes; Humans; Immunophenotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Chronic-Phase; Macrophage-1 Antigen; Male; Middle Aged; Sialic Acid Binding Ig-like Lectin 3; Tretinoin; Tumor Cells, Cultured

Unlike solid organ transplantation, Kaposi's sarcoma (KS) occurs rarely following hematopoietic stem cell transplantation (HSCT). In fact, only 5 cases of KS have been reported after allogeneic or autologous HSCT. The usual treatment combines a substantial decrease in, or elimination of, immunosuppressive therapy along with local measures such as surgical excision, cryotherapy or radiation therapy. A 46-year-old woman with chronic myelogenous leukemia who had received an allogeneic HSCT previously from an HLA-identical sibling, presented on day +814 with human herpes virus-8-associated KS involving her left lower extremity. She had been on continuous immunosuppressive therapy since her transplant because of chronic graft-versus-host disease. The intensity of immunosuppressive therapy was decreased once a diagnosis of KS had been established. However, the nodular lesions continued to progress in size and number. Therefore, a course of irradiation was administered to sites of bulk disease on her legs. Furthermore, thalidomide was initiated along with a topical retinoid, alitretinoin 0.1% gel applied twice daily to the nonirradiated lesions. This approach yielded a partial response in both irradiated and nonirradiated lesions over the course of the following 7 months. Both thalidomide and alitretinoin 0.1% gel appear to be beneficial in HSCT-associated KS and exhibit tolerable side effects.

Topics: Administration, Oral; Administration, Topical; Adult; Alitretinoin; Antineoplastic Agents; Child; Female; Hematopoietic Stem Cell Transplantation; Herpesvirus 8, Human; Humans; Immunosuppressive Agents; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Polymerase Chain Reaction; Sarcoma, Kaposi; Thalidomide; Tretinoin

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Female; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Pilot Projects; Tretinoin

Topics: Animals; Cell Division; Cell Line; Drug Therapy, Combination; Fusion Proteins, bcr-abl; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Oncogene Proteins; Philadelphia Chromosome; Transfection; Tretinoin

Reduced or absent neutrophil alkaline phosphatase (NAP) activity is a common feature of neutrophilic granulocytes from patients with chronic myeloid leukemia (CML). In this study we examined whether NAP activity could be restored in vitro by stimulating CML cells with different promoters such as all-trans-retinoic acid (ATRA), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). The results obtained indicated that ATRA and G-CSF, either alone or in combination, were effective in inducing NAP activity in CML cells, whereas GM-CSF was not. Further, NAP restoration in ATRA- and G-CSF-treated cultures was accompanied by increased morphologic differentiation of the CML clone. It might be concluded that the CML clone could be driven in vitro by ATRA and G-CSF both to achieve granulocytic maturation and to correct functional NAP-related defects.

Topics: Alkaline Phosphatase; Cell Separation; Enzyme Induction; Female; Growth Substances; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Neutrophils; Tretinoin; Tumor Cells, Cultured

We established two novel cell lines, designated as IMS-BC1 and IMS-BC2, from two patients with chronic myelogenous leukemia in blast crisis. The two cell lines were positive for CD13 and CD33 and negative for CD34 and HLA-DR by surface marker analysis. IMS-BC1 had four Philadelphia (Ph1) chromosomes and a breakpoint within the 3'-portion of M-bcr, and IMS-BC2 had five Ph1 chromosomes and two breakpoints within the 3'- and 5'-portions of M-bcr. Both cell lines' growth activities were moderately suppressed by IFN-alpha. The proliferation of IMS-BC2 was inhibited by IFN-gamma and apoptosis was induced within 72 h, while IMS-BC1 was resistant to IFN-gamma. Fibronectin inhibited the proliferation of the two cell lines at higher than 10 micrograms/ml, but only IMS-BC2 showed apoptosis. Transforming growth factor-beta inhibited the proliferation of IMS-BC2 resulting in apoptosis, while it inhibited that of IMS-BC1 moderately but failed to induce apoptosis. All-trans retinoic acid (ATRA) inhibited the proliferation of IMS-BC2 at very low concentration (10(-17) mol/l) and induced apoptosis at doses higher than 10(-9) mol/l within 72 h without terminal differentiation, while IMS-BC1 was completely resistant to ATRA. The two cell lines showed different responses to growth inhibitory cytokines and factors. These cell lines should prove useful in the analysis of mechanisms of apoptosis induced by growth inhibitory cytokines and factors.

Topics: Adolescent; Adult; Antigens, CD; Antineoplastic Agents; Apoptosis; Cell Division; Humans; Interferon-gamma; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The retinoblastoma tumor-suppressor gene, RB, has been implicated in tumor suppression, in regulation of the cell cycle, and in mediating cell differentiation. RB is necessary for hematopoiesis in mice, and aberrant RB-expression is associated with the progress and prognosis of leukemia. We have used antisense oligonucleotides, established clones stably expressing an antisense RB construct, and also established clones over expressing the retinoblastoma protein (pRb) to study the role of RB expression in monocytic differentiation induced by all-trans retinoic acid (ATRA) or 1-alpha-25-dihyroxycholecalciferol (Vit D3) in the monoblastic cell line U-937 and erythroid differentiation induced by transforming growth factor beta1 (TGFbeta1) and hemin in the erythroleukemic cell line K562. A reduction in pRb production in antisense RB-transfected U-937 clones was shown. Antisense oligonucleotides as well as expression of the antisense RB construct suppressed differentiation responses to ATRA or Vit D3, as judged by the capability to reduce nitro blue tetrazolium, by the appearance of monocyte-related cell surface antigens and by morphologic criteria. K562 cells showed decreased differentiation response to TGFbeta1, but not to hemin, when incubated with antisense oligonucleotides. U-937 antisense RB-transfected cells were also suppressed in their ability to upregulate levels of hypophosphorylated pRb when induced to differentiate. Although U-937 cells incubated with antisense oligonucleotides and clones expressing the antisense RB construct were hampered in their ability to differentiate on incubation with ATRA or Vit D3, the induced G0/G1-accumulation was similar to differentiating control cells treated with ATRA or Vit D3. Intriguingly, U-937 clones overexpressing RB were also inhibited in their differentiation response to ATRA or Vit D3 but not inhibited in their ability to respond with G0/G1 accumulation when induced with these substances. The results indicate that pRb plays a role in induced differentiation of U-937 cells as well as K562 cells involving mechanisms that, at least partially, are distinct from those inducing G1 accumulation.

Topics: Animals; Antigens, Differentiation; Antigens, Neoplasm; Calcitriol; Cell Differentiation; G1 Phase; Gene Expression Regulation, Leukemic; Genes, Retinoblastoma; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Large B-Cell, Diffuse; Mice; Monocytes; Neoplasm Proteins; Oligonucleotides, Antisense; Retinoblastoma Protein; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.

Topics: Apoptosis; Bone Marrow; Diterpenes; Drug Synergism; Farnesol; Flow Cytometry; Gefarnate; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Molecular Structure; Myelodysplastic Syndromes; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured; Vitamin K; Vitamin K 1; Vitamin K 2

All-trans retinoic acid (ATRA) has recently been shown to synergize with the inhibitory effect of interferon alpha (IFN alpha) on the growth of malignant cells isolated from solid tumors. We investigated whether ATRA could potentiate the inhibitory effects of IFN alpha on the proliferation of leukemic progenitors in chronic myeloid leukemia (CML). CD34+ cells from chronic phase, newly diagnosed patients, were incubated in short-term liquid culture with ATRA, IFN alpha or a combination of both molecules and then plated on semi-solid cultures for colony-forming cell assay. IFN alpha was found to inhibit preferentially the generation of late progenitors. ATRA at a concentration of 10(-8) M was found strongly to inhibit CFU-M colonies. Addition of ATRA to IFN alpha dramatically potentiated the inhibitory effects of INF alpha on CFU-GM growth. In the presence of both molecules the inhibition of day 14 CFU-GM from CD34+ cells was lowered to 27 +/- 4% of control. CFU-M colonies were completely inhibited. RT-PCR analysis of the colonies resulting from the action of the combination IFN alpha plus ATRA showed the presence of an increased number of BCR-ABL-negative colonies relatively to what was observed with IFN alpha alone. FISH analysis showed a higher percentage of Ph-negative cells in the ATRA plus IFN alpha-treated samples, confirming PCR experiments. These results indicate that, in vitro, the combination of IFN alpha and ATRA effectively inhibits CFU-GM colony formation in CML and suggest that it has a potential interest for the treatment of CML.

Topics: Antigens, CD; Antigens, CD34; Cell Division; Colony-Forming Units Assay; Drug Synergism; Fusion Proteins, bcr-abl; Hematopoietic Stem Cells; Humans; Interferon Type I; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Trans retinoic acid (RA) has proven to be a potent therapeutic agent in the treatment of acute promyelocytic leukemia. Unfortunately, other subtypes of acute myelogenous leukemia are resistant to the antiproliferative and differentiating effects of RA. In this report, we describe a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that not only totally inhibits the proliferation of RA-resistant leukemic cell lines HL-60R and K562 but also induces apoptosis in these cells. Exposure of HL-60R to CD437 results in the rapid (within 30 minutes) increase of the cyclin-dependent kinase inhibitor p21(waf1/cip1) as well as GADD45 mRNA. Manifestations of CD437-mediated programmed cell death are noted within 2 hours, as indicated by both the cleavage and activation of the CPP32 protease and cleavage of poly (ADP-ribose) polymerase. This is followed by cleavage of bcl-2 and internucleosomal DNA degradation. HL-60R cells do not express the retinoid nuclear receptor RAR beta and RAR gamma and express a truncated RAR alpha. Thus, CD437 induction of p21(waf1/cip1) and GADD45 mRNAs and apoptosis occurs through a unique mechanism not involving the retinoid nuclear receptors. CD437 represents a unique retinoid with therapeutic potential in the treatment of myeloid leukemia.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Differentiation; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; DNA Fragmentation; Drug Resistance, Neoplasm; GADD45 Proteins; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Naphthalenes; Neoplasm Proteins; Neoplastic Stem Cells; Poly(ADP-ribose) Polymerases; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoids; Tretinoin; Tumor Cells, Cultured

Retinoids have significant antiproliferative effect against chronic myelogenous leukemia (CML) cells in vitro. We conducted a pilot study to investigate the clinical effect of all-trans retinoic acid (ATRA) in patients with CML. Thirteen patients with Philadelphia chromosome (Ph)-positive CML in late chronic phase (n=7), accelerated phase (n=5), or blastic phase (n=1) were treated. All had been previously treated and 12 (92%) had disease refractory to interferon-alpha therapy. They received ATRA 175 mg/m2 orally in two divided doses daily until disease progression. The median duration of therapy was 56 days (range 11 to 190). Only one patient in late chronic phase had a transient decrease in WBC counts; all other patients in late chronic phase showed no response to therapy. Four of the five patients in accelerated phase showed evidence of antileukemia effect manifested by a decrease in bone marrow and/or peripheral blood blasts, promyelocyte and/or basophil percentages. In all cases the response was transient. The patient in blastic phase had no evidence of antileukemic effect. The treatment was well tolerated with the major side-effects being headache, nausea, dry skin, and dry mucosal membranes. One patient required dose reductions due to toxicity. We conclude that in this population of patients with extensively treated, advanced stage, Ph-positive CML, ATRA alone is ineffective for long-term therapy. The antileukemia effect seen in some patients warrants further investigation of retinoids in other schedules and in combinations in patients with CML.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Female; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Pilot Projects; Tretinoin

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

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

We investigated the effect of all-trans retinoic acid (ATRA) alone and in combination with interferon-alpha (IFN-alpha) on the granulocyte-macrophage (GM) colony formation of peripheral blood progenitors isolated from patients with chronic myeloid leukemia (CML) (n = 12) or other myeloproliferative disorders (n = 10) as well as from healthy controls (n = 7). The ATRA or IFN-alpha alone inhibited slightly, but not significantly, the GM colony growth in CML. Granulocyte-macrophage colony formation decreased significantly (P<0.05) when ATRA and IFN-alpha were combined (114 +/- 96 versus 74 +/- 53 colonies/10(4) mononuclear cells). The combination did not have any inhibitory effect on the other MPDs. In healthy controls, ATRA or IFN-alpha alone or their combination stimulated GM colony growth, the increase being from 22 +/- 9 to 39 +/- 16 colonies for ATRA (P<0.05), up to 47 +/- 12 colonies for IFN-alpha (P<0.05) and up to 50 +/- 19 colonies for the combination (P<0.05). In conclusion, ATRA combined with IFN-alpha inhibits GM colony growth in CML. This combination may be worth testing clinically as a treatment of CML.

Topics: Adult; Aged; Aged, 80 and over; Cell Division; Female; Hematopoietic Stem Cells; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Tretinoin

The cDNA of the human wild-type p53 tumor suppressor gene was constitutively overexpressed in the leukemic cell line K562 (which lacks detectable amounts of p53 protein) in order to investigate the consequences for growth and differentiation. Several stable clones were established by transfection of the expression vector pc53SN3. Expression of p53 protein was characterized by biosynthetic labeling and immunoprecipitation with the monoclonal antibodies pAb 1801 (reacting with wild-type and mutant human p53), pAb 240 (reacting with mutant human p53) and pAb 1620 (reacting with wild-type human p53). All clones which were 1801+, 240-, 1620- or 1801+, 240-, 1620+ were defined as "wild-type-like p53-expressing" clones. Our results show that expression of p53 protein is compatible with continuous proliferation of K562 cells. The growth characteristics of wild-type-like p53-expressing clones did not differ from that of control clones. However, the former were more sensitive than p53-negative control clones to growth inhibition by tumor necrosis factor (TNF), a cytokine with a potential role in growth and differentiation of myeloid leukemic cells. In addition, a 2- to 4-fold increase of the amount of hemoglobin, a marker of erythroid differentiation, was observed when wild-type-like p53 protein-expressing clones were incubated with TNF. This suggests that differentiation is the mechanism responsible for the increased TNF sensitivity of these clones. Our results support a role for p53 in mediating growth inhibitory and differentiation inducing signals by TNF.

Topics: Antibodies, Monoclonal; Apoptosis; Base Sequence; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Gene Expression; Genes, p53; Hemoglobins; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Sequence Data; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Juvenile chronic myelogenous leukemia (JCML) is a myeloproliferative disease in which morbidity and mortality are primarily caused by nonhematopoietic organ failure from myelomonocytic infiltration or by failure of the normal bone marrow. Morphologic evidence of maturation arrest, karyotypic abnormalities, and progression to blast crisis are infrequent events. Viral infections and other reactive processes can initially mimic the clinical course of JCML, creating diagnostic problems. Because of the rarity of JCML and technical limitations, formal clonality studies have not been reported previously. Nine female JCML patients were identified by clinical criteria, characteristic 'spontaneous' in vitro cell growth, and negative cultures and titers for various viral agents. Peripheral blood and bone marrow samples were obtained at the time of diagnosis for cell separation and RNA and DNA isolation. To assess clonality, X-chromosome inactivation patterns were evaluated using three different, recently developed polymerase chain reaction-based clonality assays. All nine female JCML patients showed evidence for monoclonal origin of mononuclear cells at the time of diagnosis. Cell separation studies further traced the monoclonal origin back to at least the most primitive myeloid progenitor cell. Reversion to a polyclonal state was demonstrated after bone marrow transplant and also in one patient following treatment with 13-cis retinoic acid. This demonstration of clonality in JCML delineates it from the reactive processes and provides a basis for molecular genetic strategies to identify causally associated mutations.

Topics: Adult; Bone Marrow; Bone Marrow Transplantation; Cell Separation; Child; Child, Preschool; Clone Cells; DNA; Dosage Compensation, Genetic; Female; Glucosephosphate Dehydrogenase; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; Phosphoglycerate Kinase; Polymerase Chain Reaction; Polymorphism, Genetic; Receptors, Androgen; Tretinoin; X Chromosome

The membrane expression of CD45RA and CD45RO on fresh leukaemic cells taken from 529 cases of acute haemopoietic malignancies, including 117 B-origin acute lymphoblastic leukaemia (B-origin ALL), 37 T-origin acute lymphoblastic leukaemia (T-origin ALL0, 297 de novo acute myeloid leukaemia (AML), 42 refractory anaemia with excess of blasts in transformation (RAEB-T) and 36 myeloid blastic phase of chronic myelogenous leukaemia (CML-BP-my), was analysed. B-origin ALLs were characterized by the lack of the RO isoform along with the consistent presence of RA. Conversely, a differential expression of the two isoforms was detected in different subsets of T-origin ALL, in that T-stem cell leukaemias (T-SCL: CD7+, CD4-, CD8-, CD1-) preferentially expressed CD45RA whereas conventional T-acute lymphoblastic leukaemias (T-ALL: CD7+, CD4+ and/or CD8+ and/or CD1+) were consistently marked by CD45RO. Within myeloid malignancies, most of AMLs displayed CD45RA, while a substantial group of CML-BP-my preferentially exhibited CD45RO. As a general rule, a reciprocal exclusion of the two isoforms was observed in AML as well as in ALL. Nevertheless, a frequent coexpression of CD45RA and CD45RO was observed in CD14+ AML. In vitro treatment with all-trans retinoic acid (ATRA) was able to promote a switch from CD45RA to CD45RO expression in 27 de novo AML, independently from morphological subtyping. To our knowledge, this is the first report on CD45 isoform expression in a large series of patients with acute leukaemia. The knowledge of the differential expression of CD45RA and CD45RO can ameliorate our classificative approach to haematological malignancies, as well as disclose new multiple overlap points between normal and leukaemic cell differentiation.

Topics: Acute Disease; Anemia, Refractory, with Excess of Blasts; Cell Differentiation; Hematopoiesis; Humans; Immunophenotyping; Isomerism; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukocyte Common Antigens; Leukocytes; Myelodysplastic Syndromes; Tretinoin

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

Topics: Animals; Base Sequence; Butyrates; Butyric Acid; Calcitriol; Cell Differentiation; Cell Line; Cell Nucleus; Colonic Neoplasms; Cytoplasm; Dimethyl Sulfoxide; Growth Inhibitors; HL-60 Cells; Humans; Interleukin-6; Kidney; Leukemia Inhibitory Factor; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphokines; Mice; Repetitive Sequences, Nucleic Acid; Stem Cells; Telomerase; Teratocarcinoma; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Clonal proliferation in agar, cell maturation and cell cycle characteristics were studied on peripheral blood cells from a patient with chronic myeloid leukaemia (CML) in blast crisis. Studies were performed in normal conditions and after incubation with all-trans retinoic acid 10(-6) M. At the time of the study the patients showed 300 x 10(9)/leukocytes/L (40% blast and promyelocytes). Cytogenetic studies showed 90% metaphases with t (9; 22) and t (18; 21). DNA index was 1.36. In agar cultures 450 CFU/2 x 10(5)/L cells, plus abnormal clusters were obtained, in the presence of conditioned media, and 115 normal CFU-GM after addition of all-trans retinoic acid 10(-6)M. Addition of retinoic acid to cellular suspension in liquid cultures decreased the number of immature cells from 20 to 2% in 5 days and decreased the number of cells in "S" phase from 33 to 11% after 8 days. These results show cytodinamic abnormalities in patients with CML in blast crisis and potential reversibility of these alterations by all-trans retinoic acid.

Topics: Blast Crisis; Cell Differentiation; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 21; DNA, Neoplasm; Humans; Immunologic Factors; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Neoplastic Stem Cells; Philadelphia Chromosome; S Phase; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

CD38 is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B-lymphocytes. In myeloid cells, CD38 is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of CD38. Recently we reported that all-trans-retinoic acid (ATRA) is a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells. Here we report that ATRA-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid-alpha receptor (RAR alpha). ATRA failed to induce CD38 expression in a mutant subclone of the HL-60 myeloid leukemia cell line (designated HL-60R) that is relatively resistant to ATRA-induced granulocytic differentiation. Retroviral vector-mediated transduction of RA receptor (RAR alpha) into this HL-60R subclone completely restored the sensitivity of these cells to ATRA in terms of their ability to express CD38. In contrast, CD38 expression was not inducible by ATRA in HL-60R cells, transfected with a functional RAR beta, RAR gamma, or RXR alpha receptor. Induction of CD38 in acute promyelocytic and acute myeloblastic leukemia cells was independent of ATRA-induced cytodifferentiation. Following culture with ATRA, increased CD38 protein levels were also observed in normal CD34+ bone marrow cells, but not on normal circulating granulocytes. From these results, we conclude that CD38 is ATRA inducible in myeloid leukemia cells and normal CD34+ bone marrow cells. This effect is independent of differentiation and is mediated by RAR alpha in HL-60 cells, suggesting a similar role for RAR alpha in CD38 expression in other hematopoietic cells.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Antigens, CD; Antigens, Differentiation; Bone Marrow; Cell Differentiation; Cell Line; Clone Cells; Dose-Response Relationship, Drug; Drug Resistance; Flow Cytometry; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Hematopoietic Stem Cells; Humans; Interferon-gamma; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Membrane Glycoproteins; Receptors, Retinoic Acid; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured

Multidrug Resistant (MDR) plays a major role in chemoresistance. Alpha Interferon (IFN) and all trans retinoic acid (ATRA) have antiproliferative effect and IFN regulates several genes, some of them implicated in the regulation of MDR gene expression. We have studied the modulations of adriamycin cytotoxicity by IFN and/or ATRA on K 562 (MDR-) and resistant to adriamycin K 562 adri (MDR+) cell lines. We observed an important increase of adriamycin cytotoxicity on both K 562 and K 562 adri by low dose of IFN and ATRA. Studies of MDR gene expression shows an increase in K 562 adri after exposure to IFN or ATRA. So the observed effect is not due to a down regulation of MDR gene expression but probably to the own antiproliferative effect of IFN and ATRA in combination with the cytotoxicity of adriamycin.

Topics: Antineoplastic Combined Chemotherapy Protocols; Base Sequence; Cell Death; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Sequence Data; Tretinoin; Tumor Cells, Cultured

Granulocyte-macrophage colony forming units (CFU-GM) from patients with advanced stage chronic myelogenous leukemia (CML), i.e. in blastic crisis (BC) or accelerated phase (AP), were inhibited by all-trans-retinoic acid (tRA) approximately 1000-fold more potently than those from chronic phase (CP) CML patients (median IC50 = 10(-9) M tRA for six CML-AP/BC cases vs > 10(-6) M tRA for seven CML-CP cases). A similar activity pattern was observed for the stereoisomer 13-cis-RA (cRA). There was no apparent correlation of CFU-GM retinoid sensitivity with cloning efficiency or other colony characteristics. Interferon alpha-2a (INF alpha) alone strongly inhibited CFU-GM growth in all four CML-AP/BC cases (IC50 < or = 250 IU/ml) and three out of seven CML-CP cases (IC50 < or = 500 IU/ml), but there was little or no interactive effect between various concentrations of tRA and INF alpha (50 IU/ml) on CFU-GM from either CML-AP/BC or CML-CP cases. These results suggest that CML-AP/BC CFU-GM have some intrinsic molecular alteration(s) which markedly enhances their responsiveness to tRA and cRA, which may be clinically exploitable.

Topics: Adult; Aged; Base Sequence; Blast Crisis; Female; Granulocytes; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophages; Male; Middle Aged; Molecular Sequence Data; Tretinoin; Tumor Stem Cell Assay

This paper describes the treatment of promyelocytic blast crisis of chronic myelogenous leukemia with all-trans-retinoic acid (ATRA). The patient, a 22-year-old male, was diagnosed to have APL and had been treated with busulfan and then with three and half years interferon (IFN) alpha in the chronic phase. A cytogenetic study of blast cells showed the t(1;17) (p11;q11) translocation as the second chromosomal abnormality without morphological abnormality of chromosome 15. Molecular analysis showed cells to have a chimera gene consisted of PML and retinoic acid receptor alpha genes. Though maturation and differentiation of leukemic cells were seen after ATRA therapy, hematological complete remission did not occur. The ineffectiveness of ATRA may be dut to different pathological conditions from de novo APL, or progressive reduction in plasma ATRA concentration as reported by Muindi et al. When our case was compared with a similar case reported by Wiernick et al., both cases were treated with IFN alpha in the chronic phases, had no t(15;17) translocation involving No.1 chromosome abnormality and did not develop complete remission after ATRA therapy.

Topics: Adult; Blast Crisis; Gene Rearrangement; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Male; Translocation, Genetic; Tretinoin

This presentation discusses the role that proliferative advantage plays in making both the chronic and blastic phases of CML resistant to therapy. A case is made for the addition of "regrowth" inhibitors between courses of chemotherapy as a means of increasing the efficacy of therapy by suppressing or reducing the proliferative advantage that the target cells enjoy over those cells which one would like to repopulate the hematopoietic system.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blast Crisis; Carrier Proteins; Cell Division; Drug Resistance; Drug Synergism; Gene Expression Regulation, Leukemic; Genes, myc; Humans; Immunologic Factors; Interferons; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Chronic-Phase; Membrane Glycoproteins; Neoplasm Proteins; Neoplastic Stem Cells; Selection, Genetic; Treatment Failure; 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

The effect of forbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the growth of cultured human chronic promyelocytic leukemia (K562) cells has been studied using cells growing in a medium consisting of RPMI 1640 supplemented with 10% fetal serum and with or without retinoic acid. All the used concentrations of TPA (100.0, 10.0, 1.0 and 0.1 ng/ml) cause the expected inhibition of proliferation of these cells. Moreover, a block of proliferation of K562 cells became stronger after cotreatment with TPA and retinoic acid, although this acid itself did not have any effect on proliferation and differentiation of K562 cells.

Topics: Cell Division; Depression, Chemical; Dose-Response Relationship, Drug; Drug Synergism; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured

Topics: Adult; Blast Crisis; Carrier Proteins; Chimera; Chromosome Banding; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Cloning, Molecular; Gene Rearrangement; Humans; Interferon-alpha; Karyotyping; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Male; Neoplasm Proteins; Nuclear Proteins; Promyelocytic Leukemia Protein; Receptors, Retinoic Acid; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

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

Acute myeloid leukemia blasts express dual affinity (high and low) granulocyte-macrophage colony-stimulating factor (GM-CSF) binding, and the high affinity GM-CSF binding is counteracted by excess interleukin-3 (IL-3). Neutrophils express a single class of GM-CSF-R with intermediate affinity that lack IL-3 cross-reactivity. Here we demonstrate the differentiation associated changes of GM-CSF binding characteristics in three models representative of different stages of myeloid maturation. We find that high affinity GM-CSF binding is converted into intermediate affinity binding, which still cross-reacts with IL-3, beyond the stage of promyelocytes. During terminal maturation towards neutrophils, IL-3 cross-reactivity is gradually lost. We sought to determine the mechanism underlying the affinity conversion of the GM-CSF-R. Northern and reverse transcriptase-polymerase chain reaction analysis of GM-CSF-R alpha and -beta c (KH97) transcripts did not provide indications for the involvement of GM-CSF-R splice variants in the formation of the intermediate affinity GM-CSFR complex. In COS-cell transfectants with increasing amounts of beta c in the presence of a fixed number of GM-CSF-R alpha chains, the high affinity GM-CSF binding converted into intermediate affinity GM-CSF binding. These results are discussed in view of the concept that increasing expression of beta c subunits may cause alternative oligomerization of the GM-CSF-R alpha and -beta c subunits resulting in the formation of intermediate rather than high affinity GM-CSFR alpha.beta c complexes.

Topics: Acute Disease; Animals; Base Sequence; Cell Line; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Macromolecular Substances; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Recombinant Proteins; RNA, Neoplasm; Sequence Deletion; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Recently three isoforms of the mouse retinoic acid receptor (mRAR beta 1, mRAR beta 2, mRAR beta 3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5'-untranslated (5'-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRAR beta 1/beta 3 is encoded in the first two exons (E1 and E2), while exon E3 includes N-terminal sequences of the mRAR beta 2 isoform. We have determined the structure of the human RAR beta 2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both 'zinc-fingers' of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.

Topics: Amino Acid Sequence; Base Sequence; Carrier Proteins; Exons; Genome, Human; Genomic Library; Humans; Introns; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Sequence Data; Open Reading Frames; Receptors, Retinoic Acid; Restriction Mapping; 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

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

We examined steady-state levels of mRNA for alkaline phosphatase in neutrophils (NAP) treated with granulocyte colony-stimulating factor (G-CSF). The amount of mRNA for NAP was shown to increase after 6 hours of culture with G-CSF when no increase in NAP activity was yet observed, and the transcript was the greatest after 20-24 h of culture with G-CSF. Treatment of neutrophils with both G-CSF and retinoic acid augmented the amount of mRNA for NAP over the amount obtained by G-CSF alone, which was most marked at 24 h. These results show that both G-CSF-mediated NAP induction and its potentiation by retinoic acid are due to the increased levels of mRNA for NAP.

Topics: Alkaline Phosphatase; Drug Synergism; Gene Expression Regulation; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neutrophils; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The in vitro differentiation of cultured primary bone marrow cells from patients with acute promyelocytic leukemia (M3) and chronic myelocytic leukemia (CML) induced by retinoic acid (RA) and TPA was studied. The results indicated that both the M3 and CML bone marrow cells bipotently differentiated into either myeloid or macrophage-monocytic lineage in response to the inducers. On M3 cells the effect of TPA was more potent than RA, and TPA could inhibit the phenotype of myeloid terminal differentiation induced by RA but not vice versa. However, RA could overcome the TPA-induced inhibition of myeloid terminal differentiation of CML cells. These experiments provide a useful model for studying the molecular mechanism of hematopoietic cell differentiation.

Topics: Bone Marrow; Cell Differentiation; Drug Synergism; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Two recent reports have described major clinical benefits from all-trans-retinoic acid (tRA) therapy of patients with promyelocytic leukemia (APL). This paper describes the first patient with a blast crisis of chronic myelogenous leukemia (CML-BC) who responded to oral tRA therapy. In vitro marrow studies, including clonogenic assays, immunopheno-typing, cytogenetics and premature chromosome condensation together with chromosome painting provided evidence for the in vivo differentiation and maturation of the malignant cells. The patient achieved a partial remission with reversal of all clinical features of disease, including normalization of peripheral blood counts, complete resolution of fever, fatigue and splenomegaly, and marked maturation of the bone marrow. This response to tRA in CML-BC is unique, and broadens the spectrum of diseases which may respond to retinoids.

Topics: Antineoplastic Agents; Blast Crisis; Bone Marrow; Cell Differentiation; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Remission Induction; Tretinoin; Tumor Cells, Cultured

Human K562 cells, a multipotential cell line of hematopoietic origin, were found to differentiate towards a macrophage-like cell when incubated in the presence of phorbol myristate acetate (PMA). Differentiation was accompanied by the expression and secretion of a plasminogen activator inhibitor (PAI). Incubation of the cells in the presence of other agents (dimethyl sulfoxide, retinoic acid, vitamin D3) capable of inducing differentiation towards other phenotypes did not lead to the induction of PAI expression. The inhibitor induced by PMA was partially purified and it exhibited biochemical characteristics similar to PAI-2. The molecule is a glycoprotein with a pI of 5.8. Northern blot analysis of mRNA isolated from control and PMA-treated cells revealed the presence of an approximately 2-kilobase mRNA from treated cells that hybridized to a PAI-2-specific synthetic oligonucleotide. This mRNA did not hybridize with a PAI-I-specific oligonucleotide and was absent from control cells. These observations are consistent with the literature regarding PAI-2 expression by cells of the monocyte-macrophage lineage. Therefore, commitment of this multipotential cell line towards the macrophage lineage results in specific activation of the PAI-2 gene. Further analysis of the regulatory elements of the PAI-2 gene may provide additional insight into the relationship between this gene and the macrophage phenotype.

Topics: Base Sequence; Cell Differentiation; Cholecalciferol; Dimethyl Sulfoxide; DNA Probes; Gene Expression Regulation, Neoplastic; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrophages; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Phenotype; Plasminogen Activators; Plasminogen Inactivators; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Poly(A) polymerase activity was markedly elevated in CML in the blastic phase, moderately high in the accelerated phase and low in the chronic phase. The activity was significantly higher in the myeloid crisis than in the lymphoid crisis and elevated with increasing ratio of blasts in leukemia cases. In TPA or retinoic acid-treated leukemia cells poly(A) polymerase activity was decreased. These results suggest that poly(A) polymerase activity changes, depending on the maturation of leukemic cells and the assay of this enzyme activity may be useful for the early detection of the exacerbation of CML cases.

Topics: Blast Crisis; Bone Marrow; Cell Line; Humans; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nucleotidyltransferases; Polynucleotide Adenylyltransferase; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The activity of liposome-associated retinoic acid was analyzed on in vitro cultured tumor cell lines and compared to the antiproliferative effects of free retinoic acid. It was found that liposome-associated retinoic acid is about 300 times more active than free retinoic acid in inhibiting in vitro cell growth of leukemic and melanoma cell lines. An increased activity of retinoic acid (10-20 times) was also obtained after premixing of this compound with empty liposomes, demonstrating that the retinoic acid efficiently interacts with liposomes which may facilitate solubility and cell uptake of retinoids.

Topics: Animals; Cell Division; Drug Carriers; Freeze Fracturing; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Liposomes; Melanoma; Mice; Microscopy, Electron; Phosphatidylcholines; 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

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

HL-60 promyelocytic leukemic cells were induced to differentiate by the combination of two alternative inducers: phorbol-12-myristate 13-acetate (PMA) and either dimethyl sulfoxide (DMSO) or retinoic acid (RA). Simultaneous exposure to optimal concentrations of PMA and either DMSO or RA potentiated PMA-induced differentiation into monocyte-macrophages. Granulocytic inducers combined with lower concentration of PMA competed with the latter for the differentiation pathway, producing monocyte-macrophages, granulocytes, paramyeloid and giant multinucleated cells. Lineage specificity of cells treated sequentially with two discrete exposures to alternative inducers depended on the order of exposure. The first exposure initiated differentiation into the pathway specific for the inducer used. The second exposure determined lineage specificity and stimulated terminal differentiation. Thus, treatment with RA for 24-72 h followed by PMA resulted in monocyte-macrophage differentiation; reversed order of exposure resulted in granulocytic differentiation. The switch in the differentiation pathway occurred at the relatively advanced stages of differentiation.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Drug Synergism; Granulocytes; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes, Mononuclear; Tetradecanoylphorbol Acetate; 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