tretinoin and Leukemia--Myeloid

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

The success of all-trans retinoic acid (ATRA) therapy for acute promyelocytic leukemia (APL) provides a rationale for using retinoic acid (RA)-based therapy for other subtypes of acute myeloid leukemia (AML). Recently, several studies showed that ATRA may drive leukemic cells efficiently into differentiation and/or apoptosis in a subset of AML patients with an NPM1 mutation, a FLT3-ITD, an IDH1 mutation, and patients overexpressing EVI-1. Because not all patients within these molecular subgroups respond to ATRA and clinical trials that tested ATRA response in non-APL AML patients have had disappointing results, the identification of additional biomarkers may help to identify patients who strongly respond to ATRA-based therapy. Searching for response biomarkers might also reveal novel RA-based combination therapies with an efficient differentiation/apoptosis-inducing effect in non-APL AML patients. Preliminary studies suggest that the epigenetic or transcriptional state of leukemia cells determines their susceptibility to ATRA. We hypothesize that reprogramming by inhibitors of epigenetic-modifying enzymes or by modulation of microRNA expression might sensitize non-APL AML cells for RA-based therapy. AML relapse is caused by a subpopulation of leukemia cells, named leukemic stem cells (LSCs), which are in a different epigenetic state than the total bulk of the AML. The survival of LSCs after therapy is the main cause of the poor prognosis of AML patients, and novel differentiation therapies should drive these LSCs into maturity. In this review, we summarize the current knowledge on the epigenetic aspects of susceptibility to RA-induced differentiation in APL and non-APL AML.

Topics: Acetylation; Acute Disease; Antineoplastic Agents; Apoptosis; Cell Differentiation; Gene Expression Regulation, Leukemic; Histones; Humans; Leukemia, Myeloid; MicroRNAs; Nucleophosmin; Tretinoin

All-trans retinoic acid therapy of acute promyelocytic leukemia represents the most successful example of differentiation-induction therapy in clinical oncology. However, acute promyelocytic leukemia represents only a small minority (10-15%) of the myeloid leukemias. Recent studies provide significant insight into why some myeloid leukemias respond dramatically to all-trans retinoic acid mediated differentiation therapy, whereas others do not.. Utilizing in-vitro experimental models of all-trans retinoic acid triggered myeloid leukemia differentiation, specific genes that are important regulators of granulocytic differentiation have been identified including transcription factors, apoptosis regulators, protein synthesis inhibitors and protein degradation factors. Moreover, recent studies have identified repressive chromatin marks generated by the aberrant, acute promyelocytic leukemia specific promyelocytic locus gene-retinoic acid receptor alpha (PML-RARalpha) fusion protein as well as the specific enzymes that mediate these chromatin changes.. The molecular basis for PML-RARalpha- mediated leukemogenesis is complex involving both the repression of numerous potential target genes and critical 'off promoter' functional activity of this fusion protein. The acute promyelocytic leukemia specific repressive chromatin marks related to PML-RARalpha activity may be present in other myeloid leukemias as well. This suggests alternative approaches for treating myeloid leukemia involving therapeutic agents that inhibit heterochromatin formation and enhance transcriptional activity. All-trans retinoic acid or related compounds may also play a significant role in enhancing hematopoietic stem cell self-renewal as well as the production and differentiation of regulatory T cells.

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

For decades, retinoic acid has been known to alter the proliferation and differentiation of myeloid cells. Currently, retinoic acid is a front-line agent in the treatment of certain forms of acute myelogenous leukemia. In this review, we focus on recent advances in our understanding of the mechanisms by which retinoids affect growth and proliferation of myeloid cells and contribute to the pathogenesis of leukemia. We have not attempted to summarize the related clinical literature.. The past 2 years have yielded important understanding of the mechanisms by which retinoids and their nuclear receptors interact with other signal transduction pathways and transcription factors to modify chromatin, alter gene expression, and participate in normal myeloid differentiation and leukemogenesis. Important advances regarding cell biology, molecular biology, biochemistry, and animal studies of retinoids and myeloid differentiation are reviewed.. Greater understanding of the role of retinoids and their receptors in myeloid cell growth and differentiation provides important insight into normal myelopoiesis. These findings have resulted in successful rational approaches to the treatment of acute leukemia and provide the promise of improved treatments in the near future.

Topics: Animals; Cell Differentiation; Cell Transformation, Neoplastic; Chromatin; Gene Expression Regulation; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Mice; Myeloid Cells; Myelopoiesis; Myeloproliferative Disorders; Oncogene Proteins, Fusion; Protein Processing, Post-Translational; Receptors, Retinoic Acid; Retinoids; Signal Transduction; Transcription Factors; Tretinoin

Valproic acid (VPA) has been used as an anticonvulsant for decades. Recently, it was demonstrated that VPA also acts as a histone deacetylase inhibitor and induces differentiation and apoptosis in a variety of malignant cells in vitro. The effect of VPA on tumor cells differs according to cell type, degree of differentiation, and underlying genetic alterations. Clinical trials with VPA have focused on acute myeloid leukemia and the myelodysplastic syndromes. When it was used as monotherapy or in combination with all-trans retinoic acid, which synergizes in vitro, VPA achieved hematologic improvement in a subset of patients. Similar to other inhibitors of histone deacetylases, complete or partial remissions rarely were observed. In this report, the authors reviewed the in vitro and in vivo data obtained with VPA, and they considered possible combination regimens aimed at improving therapeutic efficacy.

Topics: Acute Disease; Apoptosis; Cell Line, Tumor; Drug Synergism; Drug Therapy, Combination; Histone Deacetylase Inhibitors; Humans; Leukemia, Myeloid; Tretinoin; Valproic Acid

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

Acute myeloid leukemia (AML) is characterized by the arrest of differentiation leading to the accumulation of immature cells. This maturation arrest can be reversed by certain agents. Although differentiation therapy for patients with acute promyelocytic leukemia (APL) using all-trans retinoic acid (ATRA) has been established, the clinical response of AML patients other than those with APL to ATRA is limited. We must consider novel therapeutic drugs against other forms of AML for the development of a differentiation therapy for leukemia. Regulators that play an important role in the differentiation and development of plants or invertebrates may also affect the differentiation of human leukemia cells through a common signal transduction system, and might be clinically useful for treating AML. Cotylenin A, a plant growth regulator, is a potent and novel inducer of the monocytic differentiation of human myeloid leukemia cell lines and leukemia cells freshly isolated from AML patients.

Topics: Animals; Antigens, Differentiation; Antineoplastic Agents, Phytogenic; Cell Differentiation; Cholecalciferol; Diterpenes; Drug Screening Assays, Antitumor; Drug Synergism; HL-60 Cells; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Mice; Neoplastic Stem Cells; Retinoids; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured

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

Therapy-related MDS and AML are complications of intensive chemotherapy regimens. Traditionally, patients exposed to topoisomerase II inhibitors are reported to develop secondary AML with abnormalities of chromosome 11q23. We evaluated the long-term hematologic toxicity of topoisomerase II-intensive high-dose mitoxantrone-based chemotherapy in 163 newly diagnosed acute leukemia patients treated over an 8 year period. Nine (5.5%) patients developed new cytogenetic abnormalities. Four patients developed MDS with progression to AML, three patients developed new abnormalities at the time of relapse, and three patients (including one of the former patients) had changes that were not associated with hematologic disease. The abnormalities most frequently involved chromosomes 7q, 20q, 1q, and 13q. Despite the use of topoisomerase II-intensive treatment, no patient developed an abnormality involving chromosome 11q23. Spontaneous resolution of some changes and prolonged persistence of others in the absence of hematologic disease indicates that some cytogenetic changes are not sufficient to promote leukemogenesis.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Chromosome Aberrations; Chromosomes, Human, Pair 11; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Cytarabine; Disease Progression; Disease-Free Survival; Enzyme Inhibitors; Etoposide; Female; Humans; Idarubicin; Incidence; Karyotyping; Leukemia, Myeloid; Life Tables; Male; Middle Aged; Mitoxantrone; Myelodysplastic Syndromes; Neoplasm Proteins; Neoplasms, Second Primary; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Randomized Controlled Trials as Topic; Remission Induction; Retrospective Studies; Topoisomerase II Inhibitors; Treatment Outcome; Tretinoin

Recent discoveries have identified key molecular events in the pathogenesis of acute promyelocytic leukemia (APL), caused by chromosomal rearrangements of the transcription factor RAR (resulting in a fusion protein with the product of other cellular genes, such as PML). Oligomerization of RAR, through a self-association domain present in PML, imposes an altered interaction with transcriptional co-regulators (NCoR/SMRT). NCoR/SMRT are responsible for recruitment of histone deacetylases (HDACs), which is required for transcriptional repression of PML-RAR target genes, and for the transforming potential of the fusion protein. Oligomerization and altered recruitment of HDACs are also responsible for transformation by the fusion protein AML1-ETO, extending these mechanisms to other forms of acute myeloid leukemias (AMLs) and suggesting that HDAC is a common target for myeloid leukemias. Strikingly, AML1-ETO expression blocks retinoic acid (RA) signaling in hematopoietic cells, suggesting that interference with the RA pathway (genetically altered in APL) by HDAC recruitment may be a common theme in AMLs. Treatment of APLs with RA, and of other AMLs with RA plus HDAC inhibitors (HDACi), results in myeloid differentiation. Thus, activation of the RA signaling pathway and inhibition of HDAC activity might represent a general strategy for the differentiation treatment of myeloid leukemias.

Topics: Acute Disease; Animals; Cell Differentiation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Tretinoin

Successful treatment of acute promyelocytic leukemia (APL) has identified several novel approaches to induce leukemic cell differentiation and selective apoptosis by overcoming the site-specific transcriptional repression by dominant fusion leukemogenic proteins characteristic of APL and other forms of acute myelogenous leukemia (AML). These therapeutic approaches include the use of site-specific ligands, receptors and cytokines, disruption of dominant fusion leukemogenic proteins, chromatin remodeling and combining the above with cytotoxic chemotherapy. With the exception of cytotoxic chemotherapy, the above therapeutic strategies do not significantly affect normal hematopoiesis and their combinations have been shown to be synergistic in inducing myeloid differentiation and apoptosis in several AML cell lines and in patients with APL. These approaches are, in general, non-cross resistant and should be well tolerated particularly in elderly patients with AML. Clinical studies which include biologic end points for differentiation induction, histone acetylation and selective apoptosis are presently in development to evaluate these strategies in the treatment of AML.

Topics: Acute Disease; Adult; Aged; Animals; Antineoplastic Agents; Apoptosis; Butyrates; Cell Differentiation; Cholecalciferol; Chromatin; Drug Design; Drug Synergism; Gene Expression Regulation, Leukemic; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Ligands; Mice; Middle Aged; Oncogene Proteins, Fusion; Phenylbutyrates; Transcription, Genetic; Translocation, Genetic; Tretinoin

This review briefly summarizes literature noteworthy in the field of adult acute leukemia published during 1999. The relationship between specific cytogenetic abnormalities and response to treatment was explored within a clinical framework. In particular, detailed analyses of the abnormalities seen in acute promyelocytic leukemia were examined. Two case reports of special interest were published: one shed light on the role of histone deacetylase inhibitors in combination with all-trans retinoic acid, and the other, on the role of granulocyte colony-stimulating factor in this disease. The clinical activity of arsenic was also reported and its mechanism of action explored. In acute lymphoblastic leukemia, attention was focused on occult translocations, and the importance of minimal residual disease was again emphasized. Lastly, results of early clinical trials using an anti-CD19 antibody were reported, with provocative results.

Topics: Acute Disease; Adolescent; Adult; Antibodies, Monoclonal; Antigens, CD19; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Child; Chromosome Aberrations; Clinical Trials as Topic; Combined Modality Therapy; DNA, Neoplasm; Drug Design; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Infant; Leukemia; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Middle Aged; Multicenter Studies as Topic; Neoplasm Proteins; Neoplasm, Residual; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Randomized Controlled Trials as Topic; Treatment Outcome; Tretinoin

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cytokines; Humans; Leukemia, Myeloid; Mice; Rats; Rats, Wistar; Tretinoin

Retinoic acid has antiproliferative and differentiative effects on many cell types. However, the molecular mechanisms involved in ATRA (all-trans retinoic acid) -dependent growth inhibition and cell differentiation are poorly understood. On the other hand, several different cytokine specific transcription factors such as signal transducers and activators of transcription (STAT) and interferon regulatory factors (IRF) are known to be instrumental in mediating differentiative, growth regulatory and antiproliferative effects in cells. The IRF family consists of six different proteins, of which IRF-1 has been demonstrated to have antiproliferative and tumor suppressive functions. We have shown that ATRA activates IRF-1 gene expression in several myeloid leukemia cell lines (HL-60, NB4, THP-1, U937), all of which respond to ATRA by growth inhibition. In addition, during ATRA-induced myeloid differentiation, gene expression of STAT1, STAT2, and p48 was upregulated. These proteins are involved in IFN-alpha specific signaling. ATRA-induced expression of IRF and/or STAT transcription factors may be one of the molecular mechanisms mediating growth inhibition by ATRA.

Topics: DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Interferon Regulatory Factor-1; Interferon Regulatory Factor-2; Leukemia, Myeloid; Phosphoproteins; Repressor Proteins; Signal Transduction; Transcription Factors; Tretinoin; Tumor Cells, Cultured

The bcl-2 gene encodes a mitochondrial protein that inhibits the onset of apoptosis induced by growth factor withdrawal or cytotoxic agents. Using quantitative flow cytometry and expressing bcl-2 levels as the number of molecules of equivalent soluble fluorochrome (MESF) per cell, we have shown that bcl-2 protein expression in the blast cells from patients with acute myeloblastic leukaemia (AML) is heterogeneous, but not related to FAB type. The blast cells from AML patients with the capacity to grow and survive autonomously in vitro were found to have higher bcl-2 MESF values than those that were dependent upon exogenous growth factors. We have previously reported that the blast cells from 70% of AML patients exhibit autonomous growth and autocrine growth factor production in vitro and that this has been shown to be an important indicator of poor prognosis in AML. High bcl-2 expression has also been associated with a low complete remission rate and poor survival in AML. In the patients whose blast cells exhibited autonomous growth, neutralisation of endogenous GM-CSF resulted in down-regulation of bcl-2 protein, whereas in blast cells from patients whose cells proliferated only in the presence of added growth factors, incorporation of recombinant human (rh) GM-CSF in the culture media resulted in up-regulation of bcl-2. Because CD34 positivity has been reported as another indicator of poor prognosis in AML, we compared bcl-2 expression in cases of CD34 positive AML, CD34 negative AML and CD34 positive normal bone marrow cells. Bcl-2 was found to be strongly expressed on the CD34+ normal bone marrow cells. The blast cells from CD34+ AML patients expressed significantly higher bcl-2 levels than CD34- AML patients. In five cases of CD34+ AML, the bcl-2 levels were determined on purified CD34+ and CD34- blast cell populations. The CD34+ blast cells were found to express significantly higher bcl-2 levels compared with the CD34-blast cells. Our data would suggest that quantification of bcl-2 in AML blast cell may be useful as a prognostic indicator in AML.

Topics: Acute Disease; Adult; Antigens, CD34; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Cell Division; Drug Resistance, Neoplasm; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Neoplasm Proteins; Neoplastic Stem Cells; Prognosis; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

Differentiating therapy is a new antineoplastic strategy which has received increasing attention due to the remarkable activity of the vitamin A derivative, all-trans retinoic acid (ATRA) in patients with acute promyelocytic leukemia (APL). Although it has been known for years that a variety of agents, including retinoids, could induce leukemic cells to differentiate in vitro, it was not until the initial report from Shanghai in 1988 that laboratory studies translated into clinical activity and benefit in patients. Since this initial report, a number of studies have confirmed that the majority of patients with both newly diagnosed and previously chemotherapy-treated patients with APL achieve complete remission (CR) with ATRA. In addition, the characteristic life-threatening coagulopathy resolves quickly. Several limitations to this approach have emerged, including the development of retinoid resistance, hyperleukocytosis and the retinoic acid syndrome, a constellation of findings including unexplained fever, fluid retention, pleuropericardial effusions and pulmonary infiltrates. Although ATRA is very effective in inducing CR, its benefits compared to conventional chemotherapy are only now being addressed. The first prospective randomized trial comparing ATRA plus chemotherapy to chemotherapy alone was terminated early because of an improved event-free survival for patients receiving ATRA. The benefit was attributable to a difference in relapse rate. A large, intergroup, prospective, randomized trial comparing conventional chemotherapy to ATRA for induction and ATRA to observation for maintenance has recently completed accrual and will provide insight into the emerging role of ATRA in patients with APL. ATRA represents the first example of a specific form of antileukemic therapy targeting a specific genetic abnormality and may serve as a paradigm for the development of differentiating therapy for patients with other hematologic malignancies.

Topics: Acute Disease; Cell Differentiation; Clinical Protocols; Clinical Trials, Phase II as Topic; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Randomized Controlled Trials as Topic; Translocation, Genetic; Tretinoin

Topics: Adult; Gingiva; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Leukemic Infiltration; Male; Tretinoin

A review of recent information on the expression and the ATRA-driven modulation of cell surface adhesion molecules of acute myelogenous leukemia blast cells is presented. Cytofluorometric studies on fresh blast cells have demonstrated that CD11a, CD11b CD11c, CD15, CD45RO and CD54 expression is significantly lower in acute promyelocytic leukemia (APL) than is acute myeloid leukemia of other subtypes (AML). In vitro treatment with ATRA dramatically modifies the adhesion phenotype of APL blast cells, promoting a consistently striking up-regulation of CD11b, CD11c, CD15, CD65, CD54, and CD38. Which is in general, poorly demonstrable in AML. The behaviour of CD15s is variable and fully independent from CD15 and CD65 in induction experiments, suggesting a differential enzyme regulation within the selectin ligand system. ATRA is capable, in both APL and AML, of producing a switch from the high- (RA) to the low- (RO) molecular weight isoform of CD54, Moreover, treatment with this retinoid exerts a negative regulation of the membrane expression of CD49e, CD58 and CD11a in APL as well as in AML. Of particular interest is the fact that the negative effect on CD1 1a expression generates an asynchronous phenotype in APL (CD11a-, CD11b+, CD15+), undetectable on normal maturing myeloid cells. In the last part of this review the possible implications of adhesion molecule modulation in the pathogenesis of ATRA syndrome are discussed.

Topics: Acute Disease; Cell Adhesion Molecules; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Tretinoin

Structurally and functionally altered retinoic acid receptors have been associated with rare human neoplasms: acute promyelocytic leukemia and hepatocellular carcinoma. Whereas the retinoic acid receptor beta (RARbeta) rearrangement in hepatocellular carcinoma is unique, in acute promyelocytic leukemia (APL), RARalpha fusion to the promyelocytic leukemia (PML) gene by the t(15;17) translocation is a general feature of the disease. APL is an important model in cancer biology because retinoic acid induces complete remissions in this malignancy, providing the first example of differentiation therapy and of an antineoplastic drug directly targeted at the underlying genetic lesion. The molecular basis of PML/RARalpha fusion leukemogenesis is discussed with respect to dominant negative inhibition of nuclear receptor and PML functions.

Topics: Animals; Carcinoma, Hepatocellular; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Humans; Leukemia, Myeloid; Liver Neoplasms; Mice; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

The hematopoietic developmental hierarchy originates with a rare population of lymphohematopoietic stem cells that are capable of extensive self-renewal as well as the continuous generation of more developmentally restricted progeny. The generation of mature blood cells from these pluripotent hematopoietic stem cells involves the highly regulated progression through successive stages involving commitment to a specific cell lineage, terminal differentiation of lineage-restricted progenitors, and growth arrest. Although the differentiation commitment of stem cells may be intrinsically determined, it is apparent that a wide variety of external and internal stimuli can influence and modulate lineage choice and differentiation. These factors cooperate with cellular transcription factors to activate or repress the expression of genes responsible for lineage choice, diverse mature phenotypes, and cell cycle progression. The extrinsic and genetic mechanisms that orchestrate the differentiation commitment and myeloid lineage restriction of pluripotent stem cells are of fundamental importance in the regulation of hematopoiesis. The elucidation of these mechanisms of normal myeloid differentiation has provided instrumental insights into the biology of leukemia and other hematopoietic disorders.

Topics: Animals; Blastocyst; Cell Differentiation; Hematopoiesis; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Interferons; Leukemia, Myeloid; Tretinoin

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cell Differentiation; DNA; Extremities; Gene Expression Regulation; Genes, Regulator; Leukemia, Myeloid; Molecular Sequence Data; Morphogenesis; Neoplasm Proteins; Receptors, Retinoic Acid; Sequence Homology, Nucleic Acid; Transcription, Genetic; Tretinoin

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Cytarabine; Daunorubicin; Drug Evaluation; Etoposide; Humans; Idarubicin; Leukemia, Myeloid; Middle Aged; Mitoxantrone; Remission Induction; Tretinoin

Topics: Animals; Cell Differentiation; Cell Division; Cholecalciferol; Gene Expression Regulation, Neoplastic; Granulocytes; Leukemia, Myeloid; Macrophages; Oncogenes; Peroxidase; Phorbol Esters; Tretinoin; Tumor Cells, Cultured

Existing data suggest that normal maturation can sometimes be re-established in leukemia. A number of differentiation-inducing substances have been reported; these include retinoic acid, vitamin D3 and cytokines such as differentiation-inducing factor, tumor necrosis factor and lymphotoxin. Different agents obviously act by separate mechanisms to provide terminal maturation.

Topics: Binding Sites; Cell Differentiation; Cholecalciferol; Humans; Leukemia, Myeloid; Lymphokines; Lymphotoxin-alpha; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Retinoids, synthetic and natural analogues of vitamin A, play fundamental roles both in directing the spatial organization of cells during the development of vertebrate limbs and in the maintenance of growth and differentiation of many adult tissues. They also block the phenotypic expression of cancer in vitro; inhibit growth and induce differentiation in many animal and human malignant cell types. They have proved beneficial in skin diseases, cancer prevention and in acute promyelocytic leukemia.

Topics: Animals; Cell Differentiation; Humans; Leukemia, Myeloid; Teratogens; Tretinoin; Tumor Cells, Cultured

Most drugs available for cancer chemotherapy exert their effects through cytodestruction. Although significant advances have been attained with these cytotoxic agents in several malignant diseases, response is often accompanied by significant morbidity and many common malignant tumours respond poorly to existing cytotoxic therapy. Development of chemotherapeutic agents with non-cytodestructive actions appears desirable. Considerable evidence exists which indicates that (a) the malignant state is not irreversible and represents a disease of altered maturation, and (b) some experimental tumour systems can be induced by chemical agents to differentiate to mature end-stage cells with no proliferative potential. Thus, it is conceivable that therapeutic agents can be developed which convert cancer cells to benign forms. To study the phenomenon of blocked maturation, squamous carcinoma SqCC/Y1 cells were employed in culture. Using this system it was possible to demonstrate that physiological levels of retinoic acid and epidermal growth factor were capable of preventing the differentiation of these malignant keratinocytes into a mature tissue-like structure. The terminal differentiation caused by certain antineoplastic agents was investigated in HL-60 promyelocytic leukaemia cells to provide information on the mechanism by which chemotherapeutic agents induce cells to by-pass a maturation block. The anthracyclines aclacinomycin A and marcellomycin were potent inhibitors of N-glycosidically linked glycoprotein biosynthesis and transferrin receptor activity, and active inducers of maturation; temporal studies suggested that the biochemical effects were associated with the differentiation process. 6-Thioguanine produced cytotoxicity in parental cells by forming analog nucleotide. In hypoxanthine-guanine phosphoribosyltransferase negative HL-60 cells the 6-thiopurine initiated maturation; this action was due to the free base (and possibly the deoxyribonucleoside), a finding which separated termination of proliferation due to cytotoxicity from that caused by maturation.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Leukemia, Myeloid; Models, Biological; Naphthacenes; Thioguanine; Tretinoin

Topics: Calcitriol; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Monokines; Phorbol Esters; Proteins; Tretinoin

Various methods of intensive chemotherapy have contributed to an improved survival in pediatric acute myeloid leukemia (AML). We here report the long-term results of the two consecutive trials of Tokyo Children's Cancer Study Group (TCCSG), incorporating repetitive use of high-dose cytarabine (HD-Ara-C) based combination chemotherapy in post-remission phase.. A total of 216 eligible children with newly diagnosed AML were treated in the two consecutive multi-center trials of TCCSG, M91-13 and M96-14, from August 1991 to September 1998. In M91-13 trial, patients received eight courses of intensive post-remission chemotherapy, including six HD-Ara-C containing courses, after remission-induction therapy. Autologous hematopoietic stem cell transplantation (HSCT) could be selected by physician's choice, and allogeneic HSCT was allocated if donor was available. In M96-14 trial, the last two HD-Ara-C courses were omitted from the chemotherapy arm.. The remission-induction rate was 88.8% and probability of 5-year Overall survival (OS) and event-free survival (EFS) were 62% (56-69% with 95% Confidence intervals (CIs)) and 56% (49-62%), respectively. Treatment-related mortality (TRM) was 7.8%. Among patients without Down syndrome (DS) or acute promyelocytic leukemia (APL), the presence of t(8;21) or inv(16) was a significant good prognostic factor both in the univariate and multivariate analyses. Children with DS (N = 10) and APL (N = 14) also showed a good survival exceeding 70% in 5 years.. These results suggest that repetitive use of HD-Ara-C was effective and safe for childhood AML. However, further optimization of AML therapy is required.

Topics: Acute Disease; Adolescent; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Combined Modality Therapy; Cytarabine; Disease-Free Survival; Down Syndrome; Doxorubicin; Drug Administration Schedule; Etoposide; Female; Hematopoietic Stem Cell Transplantation; Humans; Hydrocortisone; Infant; Infections; Japan; Kaplan-Meier Estimate; Leukemia, Myeloid; Male; Methotrexate; Mitoxantrone; Remission Induction; Survival Analysis; Transplantation, Autologous; Transplantation, Homologous; Treatment Outcome; Tretinoin; Vincristine

The survival of older patients with acute myeloid leukemia has not improved. Few clinical trials have been available for older patients who are not considered fit for an intensive chemotherapy approach.. Between December 1998 and November 2003, as part of National Cancer Research Institute Acute Myeloid Leukemia 14 Trial, 217 patients, who were deemed unfit for intensive chemotherapy were randomized to receive low-dose cytarabine (Ara-C) (20 mg twice daily for 10 days) or hydroxyurea with or without all-trans retinoic acid (ATRA).. Low-dose ara-C produced a better remission rate (18% vs 1%; odds ratio [OR], 0.15; 95% confidence interval [95% CI], 0.06-0.37; P = .00006) and better overall survival (OR, 0.60; 95% CI, 0.44-0.81; P = .0009), which was accounted for by the achievement of complete remission (CR) (duration of CR: 80 weeks vs 10 weeks for patients with no CR). Patients who had adverse cytogenetics did not benefit. ATRA had no effect. Toxicity scores or supportive care requirements did not differ between the treatment arms.. Older, less fit patients have a poor outcome, and few trials have been conducted in this patient group. Low-dose ara-C treatment was superior to best supportive care and hydroxyurea because it had greater success in achieving CR, and it could represent standard care against which new treatments may be compared in this patient group.

Topics: Acute Disease; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Female; Humans; Hydroxyurea; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Risk; Survival; Treatment Outcome; Tretinoin

Valproic acid (VPA) inhibits histone deacetylase activity and, synergizing with all-trans retinoic acid (ATRA), achieves differentiation induction of myeloid blast cells in vitro.. We used VPA in 58 patients with acute myeloid leukemia (AML) who were too old and/or medically unfit to receive intensive chemotherapy (32 AML secondary to myelodysplastic syndrome [MDS], 22 de novo AML, 4 AML secondary to myeloproliferative syndrome). VPA serum concentrations were 50-100 mug/mL. Thirty-one patients received VPA monotherapy. ATRA was added later in 13 patients who did not respond or who relapsed. Another 27 patients received VPA plus ATRA from the start. Median treatment duration was 93 days for VPA and 88 days for ATRA.. The response rate was only 5% according to International Working Group (IWG) criteria for AML but was 16% when IWG response criteria for MDS were used, which capture hematologic improvement and stabilization of the disease. These endpoints, which are not necessarily correlated with diminishing blast counts, are relevant for the patients' quality of life. Among 23 patients with a peripheral blast count > 5%, 6 (26%) showed a diminishing blast count, and 5 of these had a complete peripheral blast clearance.. Future trials should combine VPA with chemotherapy or demethylating agents.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Female; Histone Deacetylase Inhibitors; Humans; Leukemia, Myeloid; Male; Middle Aged; Treatment Outcome; Tretinoin; Valproic Acid

Topics: Acute Disease; Administration, Oral; Aged; Antineoplastic Combined Chemotherapy Protocols; Drug Administration Schedule; Etoposide; Female; Humans; Idarubicin; Injections, Intravenous; Leukemia, Myeloid; Male; Middle Aged; Recurrence; Remission Induction; Survival Rate; Treatment Outcome; Tretinoin

The authors investigated the efficacy and safety of the histone deacetylase inhibitors valproic acid (VPA) and all-trans retinoic acid (ATRA) as differentiation agents in a cohort of older, poor-risk patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).. Twenty older patients with recurrent or refractory AML or MDS were treated in a Phase II protocol with sequential VPA and ATRA therapy. VPA was started at a dose of 10 mg/kg per day and then escalated to achieve the serum concentration of 45-100 microg/mL. ATRA was added at 45 mg/square meters (sm) per day when VPA reached the target serum concentration. Only patients treated continuously for > or = 2 months were considered evaluable.. Hematologic improvement, according to World Health Organization criteria, was observed in 6 of 20 patients enrolled in the protocol but in 6 of 11 considered evaluable. In five patients, a major platelet response was observed, achieving platelet transfusion independence. Three of these five patients also exhibited a minor erythroid response. A sixth patient showed both a minor erythroid response and a platelet response. The median duration of response was 189 days (range, 63-550 days). No significant reduction in the blast count was observed. Grade 3 neurocortical toxicity was observed in four patients. Severe bone pain was experienced by 4 patients (2 Grade 4 and 2 Grade 3) and was associated with an increase in the peripheral blast cell count. Treatment with ATRA did not modify the response observed with VPA alone.. Differentiation therapy with VPA was of clinical benefit in approximately 30% of elderly patients with AML and MDS of the refractory anemia with excess of blast type with unfavorable prognostic features. A striking platelet transfusion independence lasting several months may be obtained in some patients, reducing the burden of palliative care and improving the quality of life.

Topics: Acute Disease; Aged; Female; Humans; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Platelet Count; Risk; Tretinoin; Valproic Acid

Valproic acid (VPA) inhibits histone deacetylase activity and induces differentiation of acute myeloid leukemia (AML) blasts in vitro. We observed clinical responses to VPA in patients with myelodysplastic syndrome (MDS) and AML. Here, we report follow-up data on 75 patients. Of these, 66 were started on VPA monotherapy, with later addition of all-trans retinoic acid (ATRA) in patients who did not respond or relapsed. Nine patients were treated with VPA + ATRA from the start. Median treatment duration was 4 months for VPA and 2 months for ATRA. Hematological improvement, according to international working group criteria for MDS, was observed in 18 patients (24%). Median response duration was 4 months. ATRA exerted no additional effect in patients receiving the combination from the start or benefited primary VPA nonresponders. However, of ten VPA responders who relapsed, four achieved a second response after addition of ATRA. Response rates were strongly dependent on disease type according to WHO classification. We found a response rate of 52% in MDS patients with a normal blast count (refractory sideroblastic anemia, refractory cytopenia with multilineage dysplasia, and refractory sideroblastic cytopenia with multilineage dysplasia). The response rate was 6% in refractory anemia with excess blasts (I + II), 16% in AML, and 0% in chronic myelomonocytic leukemia. Bone marrow blast count was the only variable that predicted responses. We conclude that VPA is clinically useful in low-risk MDS. For patients with high-risk MDS, VPA may be combined with chemotherapy or demethylating drugs. If patients relapse after an initial response to VPA, ATRA has the potential to induce a prolonged second response.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Remission Induction; Treatment Outcome; Tretinoin; Valproic Acid

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials as Topic; Humans; Leukemia, Myeloid; Prognosis; Transplantation, Homologous; Tretinoin

The purpose of our study was (i) to evaluate the impact of all-trans retinoic acid (ATRA) given as adjunct to chemotherapy and (ii) to compare second consolidation vs maintenance therapy in elderly patients with acute myeloid leukemia (AML). A total of 242 patients aged >or=61 years (median, 66.6 years) with AML were randomly assigned to ATRA beginning on day +3 after the initiation of chemotherapy (ATRA-arm, n=122) or no ATRA (standard-arm, n=120) in combination with induction and first consolidation therapy. A total of 61 patients in complete remission (CR) were randomly assigned to second intense consolidation (n=31) or 1-year oral maintenance therapy (n=30). After induction therapy the intention-to-treat analysis revealed a significant difference in CR rates between the ATRA- and the standard-arm (52 vs 39%; P=0.05). Event-free (EFS) and overall survival (OS) were significantly better in the ATRA-compared to the standard-arm (P=0.03 and 0.01, respectively). OS after second randomization was significantly better for patients assigned to intensive consolidation therapy (P<0.001). The multivariate model for survival revealed lactate dehydrogenase, cytogenetic risk group, age, and first and second randomization as prognostic variables. In conclusion, the addition of ATRA to induction and consolidation therapy may improve CR rate, EFS and OS in elderly patients with AML.

Topics: Acute Disease; Aged; Aged, 80 and over; Anemia, Refractory, with Excess of Blasts; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Cytarabine; Etoposide; Humans; Idarubicin; Leukemia, Myeloid; Middle Aged; Prognosis; Remission Induction; Survival Rate; Transplantation, Homologous; Tretinoin

All trans retinoic acid has shown a remarkable effectiveness in acute promyelocytic leukemia. These results have encouraged studies of treatment with ATRA in other acute myeloid leukemia subtypes.. In order to evaluate toxicity and antileukemic efficacy of all ATRA in patients with relapsed or refractory non promyelocytic AML, 95 patients (median age, 58 years; range, 20 to 80 years), with unclassified AML according to the FAB classification or secondary AML at diagnosis, or refractory or relapsing AML, received induction therapy with Idarubicin, 10 mg/m(2)/day, for 3 days and cytarabine, 1000 mg/m(2)/12 h, for 6 days, alone or combined, on a randomized basis, with ATRA, 45 mg/m(2)/day, from day 1 to complete remission. Patients in CR received maintenance therapy with 6 monthly courses combining Ida, 10 mg/m(2)/day, intravenously, on day 1 with Ara-C100 mg/m(2)/day, subcutaneously, from day 1 to day 5.. Results were evaluated after one induction course. Overall 54 patients (57%, 26 with ATRA and 28 without ATRA) achieved CR including five patients treated at time of initial diagnosis, seven previously resistant, 38 in first relapse and four in further relapse. Thirty patients (31%) had resistant disease and 11 (12%) died from toxicity. Median time for neutrophil recovery to 0.5 x 10(9)/l and platelets to 20 x 10(9)/l was 31 and 21 days respectively. Severe toxicity (WHO grade >or=3) included infections (37%), diarrhea (9%), bleeding (3%), vomiting (16%), hyperbilirubinemia (5%), mucositis (6%) and hypercreatininemia (2%). No ATRA syndrome was noted in the ATRA arm. Median overall survival for the entire cohort was 6.3 months and median disease-free survival was 4.7 months. There were no statistical differences in terms of CR, DFS, and OS between the two arms.. We conclude that ATRA in combination with Ida and Ara-C can be administered safely to high-risk AML patients. However, in this setting, ATRA did not offer any advantage when compared to chemotherapy alone.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Female; Humans; Idarubicin; Leukemia, Myeloid; Male; Middle Aged; Remission Induction; Salvage Therapy; Survival Analysis; Therapeutic Equivalency; Treatment Outcome; Tretinoin

To evaluate the efficiency of combination of interferon-alpha (INF) and all trans-retinoic acid (ATRA) as a treatment for maintaining remission in high risk group patients with acute myeloid leukemia (AML).. Three-day INF + ATRA course was administered every 3 months to 22 patients with AML from high risk group (impossibility of drug therapy during the first complete remission, resistant forms of AML, relapses, secondary AML, acute promyelocytic leukemia after attaining molecular remission).. INF + ATRA during remission maintained a long first complete remission (median 18 months) in patients with primary AML after small-volume drug therapy, led to long first and second complete remissions (median 12 months) in patients with resistant AML, and induced and maintained molecular remissions in patients with acute promyelocytic leukemia.

Topics: Acute Disease; Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Drug Therapy, Combination; Female; Humans; Interferon-alpha; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Recurrence; Remission Induction; Risk Factors; Time Factors; Tretinoin

To evaluate a regimen including high-dose mitoxantrone in previously untreated adults with AML, 45 patients aged 21-59 (median 41) were given cytarabine, 3 g/m2 days 1-5, mitoxantrone, 80 mg/m2 day 2 and etoposide, 150 mg/m2 days 1,3,5. Post-remission therapy consisted of 5 cycles combining the same agents at reduced doses. Complete remission was seen in 36 patients. The observed 3-year survival is 28%. Cytogenetic pattern and CD34 expression correlated with response and survival. Significant toxicity included myelosuppression, mucositis, diarrhea and hyperbilirubinemia. Ventricular ejection fraction was generally reduced, with clinical cardiac dysfunction in only 2 patients. This high-dose mitoxantrone combination can be administered to young adults with AML with tolerable toxicity and results comparable to those of other dose-intensive regimens.

Topics: Actuarial Analysis; Acute Disease; Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Combined Modality Therapy; Conjunctivitis; Cytarabine; Disease-Free Survival; Etoposide; Female; Gastrointestinal Diseases; Humans; Hyperbilirubinemia; Leukemia, Myeloid; Male; Methylprednisolone; Middle Aged; Mitoxantrone; Remission Induction; Risk; Stomatitis; Stroke Volume; Survival Analysis; Survival Rate; Treatment Outcome; Tretinoin; Ventricular Dysfunction, Left

Between May 1988 and March 1995, 359 children with acute myeloid leukaemia (AML) were treated in the MRC AML 10 trial. Three risk groups were identified based on cytogenetics and response to treatment. One hundred and twenty-five children relapsed--103 in the bone marrow only, 12 in the bone marrow combined with other sites, and six had isolated extramedullary relapses (site was not known in four cases). Eighty-seven children received further combination chemotherapy, one all-trans retinoic acid for acute promyelocytic leukaemia, and one a matched unrelated donor allograft in relapse, and 61 achieved a second remission. One patient with no details on reinduction therapy also achieved second remission. Treatment in second remission varied--44 children received a BMT (22 autografts, 12 matched unrelated donor allografts, 10 family donor allografts), and 17 were treated with chemotherapy alone. The overall survival rate for all children (treated and untreated) was 24% at 3 years, with a disease-free survival of 44% for those achieving a second remission. Length of first remission was the most important factor affecting response rates--children with a first remission of less than 1 year fared poorly (second remission rate 36%, 3 year survival 11%), whereas those with longer first remissions had a higher response rate (second remission rate 75%, 3 year survival 49%, P < 0.0001).

Topics: Acute Disease; Adolescent; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Child; Clinical Protocols; Disease-Free Survival; Follow-Up Studies; Humans; Leukemia, Myeloid; Recurrence; Survival Analysis; Transplantation, Autologous; Transplantation, Homologous; Tretinoin; United Kingdom

Preclinical data suggest that retinoids, eg, all-trans retinoic acid (ATRA), lower concentrations of antiapoptotic proteins such as bcl-2, possibly thereby improving the outcome of anti-acute myeloid leukemia (AML) chemotherapy. Granulocyte colony-stimulating factor (G-CSF) has been considered to be potentially synergistic with ATRA in this regard. Accordingly, we randomized 215 patients with newly diagnosed AML (153 patients) or high-risk myelodysplastic syndrome (MDS) (refractory anemia with excess blasts [RAEB] or RAEB-t, 62 patients) to receive fludarabine + ara-C + idarubicin (FAI) alone, FAI + ATRA, FAI + G-CSF, or FAI + ATRA + G-CSF. Eligibility required one of the following: age over 71 years, a history of abnormal blood counts before M.D. Anderson (MDA) presentation, secondary AML/MDS, failure to respond to one prior course of chemotherapy given outside MDA, or abnormal renal or hepatic function. For the two treatment arms containing ATRA, ATRA was given 2 days (day-2) before beginning and continued for 3 days after completion of FAI. For the two treatment arms including G-CSF, G-CSF began on day-1 and continued until neutrophil recovery. Patients with white blood cell (WBC) counts >50,000/microL began ATRA on day 1 and G-CSF on day 2. Events (death, failure to achieve complete remission [CR], or relapse from CR) have occurred in 77% of the 215 patients. Reflecting the poor prognosis of the patients entered, the CR rate was only 51%, median event-free survival (EFS) time once in CR was 36 weeks, and median survival time was 28 weeks. A Cox regression analysis indicated that, after accounting for patient prognostic variables, none of the three adjuvant treatment combinations (FAI + ATRA, FAI + G, FAI + ATRA + G) affected survival, EFS, or EFS once in CR compared with FAI. Similarly, there were no significant effects of either ATRA ignoring G-CSF, or of G-CSF ignoring ATRA. As previously found, a diagnosis of RAEB or RAEB-t rather than AML was insignificant. There were no indications that the effect of ATRA differed according to cytogenetic group, diagnosis (AML or MDS), or treatment schedule. Logistic regression analysis indicated that, after accounting for prognosis, addition of G-CSF +/- ATRA to FAI improved CR rate versus either FAI or FAI + ATRA, but G-CSF had no effect on the other outcomes. We conclude that addition of ATRA +/- G-CSF to FAI had no effect on CR rate, survival, EFS, or EFS in CR in poor prognosis, newly diagnosed AML or high-r

Topics: Acute Disease; Aged; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Cytarabine; Granulocyte Colony-Stimulating Factor; Humans; Idarubicin; Leukemia, Myeloid; Middle Aged; Myelodysplastic Syndromes; Probability; Prognosis; Survival Rate; Time Factors; Tretinoin; Vidarabine

We report a single institution sequential trial of two maintenance treatment regimens for patients with acute myelogenous leukemia (AML). A total of 175 consecutive patients with AML received initial remission induction therapy with high-dose cytosine arabinoside (ara-C) and glucocorticoids. For the initial 63 patients (group A), the control population, planned maintenance treatment was with conventional-dose ara-C given over 4 days for up to 18 months. The subsequent 107 patients (group B) had planned maintenance therapy of up to 6 courses of daunorubicin, ara-C and prednisone and daily cis-retinoic acid for up to two years. The presenting features of group A and B patients were similar as were the response to remission induction, 60 and 52%, respectively. Severe neurological toxicity was encountered once after high-dose ara-C; no drug-related deaths occurred during maintenance treatment. Median duration of remission for group B patients was 9.9 months compared with 5.5 for group A (p = 0.0685). Median survival duration for the two groups was similar, 9.1 months for group A and 10.4 for group B. Survival of patients in group B who attained a complete remission was significantly better than that of patients in group A (p = 0.0439). The studies confirm our initial experience with remission induction using single agent high-dose ara-C and suggest a positive role for maintenance therapy in AML.

Topics: Acute Disease; Adult; Age Factors; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Combined Modality Therapy; Cytarabine; Daunorubicin; Humans; Leukemia, Myeloid; Middle Aged; Prednisone; Survival Analysis; Tretinoin

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Cytarabine; Daunorubicin; Drug Evaluation; Etoposide; Humans; Idarubicin; Leukemia, Myeloid; Middle Aged; Mitoxantrone; Remission Induction; Tretinoin

The management of acute myeloid leukemia in the elderly (65 years and older) is unsatisfactory because of poor patient tolerance of standard myeloablative chemotherapy. The authors conducted a Phase II study to evaluate the effectiveness and toxicity of 13-cis-retinoic acid (CRA) in the therapy of elderly patients with acute myeloid leukemia (AML). Patients presenting with leukocyte counts less than 20,000/microliters were treated with CRA alone. Those with leukocyte counts of 20,000/microliters or greater were pretreated with hydroxyurea, followed by CRA. Twelve of 18 patients received at least 4 weeks of CRA and were thus considered evaluable for toxicity and response. No objective responses were observed. Cis-retinoic acid administration was well tolerated; only modest dermatologic, musculoskeletal, and gastrointestinal toxicity was observed. Alternative therapeutic strategies should be investigated in this subpopulation of AML patients.

Topics: Aged; Aged, 80 and over; Drug Evaluation; Humans; Hydroxyurea; Leukemia, Myeloid; Leukocyte Count; Pilot Projects; Survival Rate; Tretinoin

To investigate the clinical features of and therapeutic options for a rare morphology resembling APL with t (11;12) (p15;q13) in acute myeloid leukemia.. One case of APL-like acute leukemia with a t (11;12) (p15;q13) translocation is reported and related literature is retrospectively reviewed.. A rare acute myeloid leukemia with a t (11;12) (p15;q13) translocation was diagnosed by morphology, immunophenotyping, chromosome analysis, and fusion gene detection, without finding a classical t (15;17) (q24.1;q21.1) translocation and the PML-RARa fusion gene. The patient responded poorly to differentiation induction therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). In the three previous cases re-ported, poor results were obtained with ATRA and/or ATO therapy.. We reported a rare meaningful AML patient with t (11;12) (p15;q13). Standard AML regimens may be preferred. These APL-like leukemias may benefit from hematopoietic stem cell transplantation treatment. Further investigation involving more cases is needed to determine the role of t (11;12) (p15;q13) in AML and to find better therapy choices.

Topics: Acute Disease; Antineoplastic Agents; Arsenic Trioxide; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 12; Humans; Leukemia, Myeloid; Male; Oncogene Proteins, Fusion; Review Literature as Topic; Translocation, Genetic; Treatment Outcome; Tretinoin; Young Adult

All-trans retinoic acid (ATRA) is an effective drug for the induction therapy of acute promyelocytic leukemia. However, the treatment is associated with adverse events such as retinoic acid syndrome (RAS) in some patients, whose histologic characteristics included organ infiltration by leukemic cells. Matrix metalloproteinase 2 (MMP-2) is often upregulated in tumor cells and plays a role in tumor cell migration and invasion by degrading the extracellular matrix. In this study, we examined the possible modulatory effects of ATRA on MMP-2 expression and secretion in human myeloid leukemia cell line THP-1. The cells were treated with various concentrations of ATRA, and MMP-2 expression and secretion were examined. MMP-2 expression and secretion started to increase with ATRA concentration as low as 0.1 nM and gradually increased thereafter. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) alone could enhance MMP-2 secretion, and RAR or RXR antagonists alone could reverse ATRA-induced MMP-2 secretion. ATRA increased intracellular calcium ion levels, and a calcium-channel blocker inhibited ATRA-induced MMP-2 secretion. Dexamethasone suppressed ATRA-induced MMP-2 secretion. Our results suggest that ATRA enhances MMP-2 expression and secretion in human myeloid leukemia THP-1 cells in a calcium ion dependent manner through RAR/RXR signaling pathways, and this enhanced expression and secretion may be associated with the possible mechanisms of RAS.

Topics: Antineoplastic Agents; Cell Differentiation; Humans; Leukemia, Myeloid; Matrix Metalloproteinase 2; THP-1 Cells; Tretinoin; Tumor Cells, Cultured

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) is the first example of targeted therapy. In fact, the oncogenic fusion-protein (PML-RAR) typical of this leukemia contains the retinoid-nuclear-receptor RARα. PML-RAR is responsible for the differentiation block of the leukemic blast. Besides PML-RAR, two endogenous RARα proteins are present in APL blasts, i.e. RARα1 and RARα2. We developed different cell populations characterized by PML-RAR, RARα2 and RARα1 knock-down in the APL-derived NB4 cell-line. Unexpectedly, silencing of PML-RAR and RARα2 results in similar increases in the constitutive expression of several granulocytic differentiation markers. This is accompanied by enhanced expression of the same granulocytic markers upon exposure of the NB4 blasts to ATRA. Silencing of PML-RAR and RARα2 causes also similar perturbations in the whole genome gene-expression profiles of vehicle and ATRA treated NB4 cells. Unlike PML-RAR and RARα2, RARα1 knock-down blocks ATRA-dependent induction of several granulocytic differentiation markers. Many of the effects on myeloid differentiation are confirmed by over-expression of RARα2 in NB4 cells. RARα2 action on myeloid differentiation does not require the presence of PML-RAR, as it is recapitulated also upon knock-down in PML-RAR-negative HL-60 cells. Thus, relative to RARα1, PML-RAR and RARα2 exert opposite effects on APL-cell differentiation. These contrasting actions may be related to the fact that both PML-RAR and RARα2 interact with and inhibit the transcriptional activity of RARα1. The interaction surface is located in the carboxy-terminal domain containing the D/E/F regions and it is influenced by phosphorylation of Ser-369 of RARα1.

Topics: Acute Disease; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; COS Cells; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid; Oncogene Proteins, Fusion; Retinoic Acid Receptor alpha; RNA Interference; Tretinoin

All-trans-retinoic acid (ATRA), a naturally occurring metabolite of vitamin A, has been shown to have great potential as an antitumorigenic drug to treat acute leukaemia by promoting cancer cell differentiation. Cytochrome P450 oxidoreductase (POR) is the only obligate electron donor for all of the microsomal cytochrome P450 enzymes including CYP26A1 which is highly specific for ATRA metabolism and efficacy in human myeloid leukaemia cells. In this study, we aimed to investigate the effect of POR on ATRA efficacy and CYP26A1 expression in human myeloid leukaemia HL-60 cells.. Stably expressed POR and POR-RNAi HL-60 cell lines were established by transfecting POR overexpression or RNAi (RNA interference) vectors mediated by lentivirus. The protein expression of POR and CYP26A1 was examined by Western blot. The potential roles of POR on ATRA efficacy in HL-60 cells were explored by cell viability assay, cell cycle distribution, cellular differentiation and apoptosis analysis.. All-trans-retinoic acid treatment caused the expression of POR upregulation and CYP26A1 downregulation in dose- and time-dependent manners. POR overexpression decreased CYP26A1 expression in HL-60 cells. When POR gene was interfered, the downregulation of CYP26A1 expression by ATRA was abolished. In addition, POR overexpression in HL-60 cells significantly compromised ATRA-induced cell proliferation inhibition, cell cycle arrest, differentiation and apoptosis, whereas downregulation of POR significantly potentiated ATRA effects.. Our study therefore suggested that POR played an important role in regulating ATRA efficacy and CYP26A1 expression in HL-60 cells.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Cytochrome P-450 Enzyme System; HL-60 Cells; Humans; Inactivation, Metabolic; Leukemia, Myeloid; NADPH-Ferrihemoprotein Reductase; Retinoic Acid 4-Hydroxylase; Tretinoin

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of human acute promyelocytic leukemia (APL) HL60 cells, fresh human APL cells, and clinical remission in patients with APL. Retinoylation (acylation of proteins by RA) is a possible pathway for RA action. However, an understanding of the role that retinoylation plays in the actions of RA is lacking. In the current study, several retinoylated proteins were detected in RA-treated HL60 fractions following Mono Q anion exchange chromatography and analysis using two-dimensional polyacrylamide gel electrophoresis. One of the retinoylated proteins was identified as Rho-GDIβ (28kDa) by TOF-MS and co-migration with Rho-GDIβ (28kDa). Truncated Rho-GDIβ (23kDa, N∆19), a product of cleavage by caspase-3, was not retinoylated. RA covalently bound to the Thr2 residue in Rho-GDIβ (5kDa), which is the second product resulting from the cleavage of Rho-GDIβ (28kDa) by caspase-3. RA treatment increased the level of Rho-GDIβ (28kDa) and decreased the level of Rho-GDIβ (23kDa). RA did not induce caspase-3 activity or Rho-GDIβ mRNA expression. It is likely that retinoylation of Rho-GDIβ increases its metabolic stability.

Topics: Acylation; Amino Acid Sequence; Caspase 3; Cell Differentiation; Cell Line, Tumor; HL-60 Cells; Humans; Leukemia, Myeloid; rho Guanine Nucleotide Dissociation Inhibitor beta; RNA; RNA, Messenger; Tretinoin

Gap junctional intercellular communication (GJIC) is typically decreased in malignant tumors. Gap junction is not presented between hematopoietic cells but occurred in bone marrow stromal cells (BMSCs). Connexin 43 (Cx43) is the major gap junction (GJ) protein; our previous study revealed that Cx43 expression and GJIC were decreased in acute leukemic BMSCs. All-trans retinoic acid (ATRA) increases GJIC in a variety of cancer cells and has been used to treat acute promyelocytic leukemia, but the effects of ATRA on leukemic BMSCs is unknown. In this study, we evaluated the potential effects of ATRA on cell cycle, proliferation, and apoptosis of leukemic BMSCs. Effects of ATRA on Cx43 expression and GJIC were also examined.. Human BMSCs obtained from 25 patients with primary acute leukemia, and 10 normal healthy donors were cultured. Effects of ATRA on cell cycle, cell proliferation, and apoptosis were examined with or without co-treatment with amphotericin-B. Cx43 expression was examined at both the mRNA and protein expression levels. GJIC was examined by using a dye transfer assay and measuring the rate of fluorescence recovery after photobleaching (FRAP).. ATRA arrested the cell cycle progression, inhibited cell growth, and increased apoptosis in leukemic BMSCs. Both Cx43 expression and GJIC function were increased by ATRA treatment. Most of the observed effects mediated by ATRA were abolished by amphotericin-B pretreatment.. ATRA arrests cell cycle progression in leukemic BMSCs, likely due to upregulating Cx43 expression and enhancing GJIC function.

Topics: Acute Disease; Adult; Amphotericin B; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Communication; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Connexin 43; Female; Fluorescence Recovery After Photobleaching; Gap Junctions; Gene Expression; Humans; Leukemia, Myeloid; Male; Mesenchymal Stem Cells; Microscopy, Confocal; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38+ transfectants. In contrast, cells cultured for 48h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.

Topics: Adaptor Proteins, Signal Transducing; ADP-ribosyl Cyclase 1; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Class Ia Phosphatidylinositol 3-Kinase; Dasatinib; HL-60 Cells; Humans; Leukemia, Myeloid; Membrane Glycoproteins; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-cbl; Proto-Oncogene Proteins c-vav; Pyrimidines; src-Family Kinases; Thiazoles; Tretinoin

All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) are the classic drugs used for induction therapy of acute promyelocytic leukemia (APL). IL-3Ralpha (CD123) is a specific marker of acute myeloid leukemia stem cells (AML-LSCs). The over-expression of IL-3Ralpha in patients with AML is related to high white blood cells counts, high percentages of blast cells, and poor prognosis. Moreover, in some studies, IL-3Ralpha has been considered a new detection marker of minimal residual disease in the bone marrow from patients with APL. In contrast to ATRA, As2O3 reduces both mRNA and protein expression of IL-3Ralpha and inhibits the activity of PI3K/Akt after 24 h, 48 h, and 72 h of exposure. Furthermore, NB4 cells adhered to the human stroma cell line HS-5 cells were used as an in vitro model of APL cells in the bone marrow microenvironment. Our results demonstrate that adhesion to HS-5 cells up-regulated IL-3Ralpha protein expression and activated the downstream PI3K/Akt signaling pathway in NB4 cells. Compared with ATRA, As2O3 more potently inhibits proliferation of NB4 cells adhered to stroma cells.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Drug Resistance, Neoplasm; Humans; Interleukin-13 Receptor alpha1 Subunit; Leukemia, Myeloid; Neoplasm Proteins; Oncogene Protein v-akt; Oxides; Phosphatidylinositol 3-Kinases; RNA, Messenger; Signal Transduction; Tretinoin

Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47(phox) expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid's biological effects in WT HL60 cells.

Topics: ADP-ribosyl Cyclase 1; Antineoplastic Agents; CD11b Antigen; Cell Differentiation; Drug Resistance, Neoplasm; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid; Membrane Glycoproteins; NADPH Oxidases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Proto-Oncogene Proteins c-vav; Pyrimidines; Resting Phase, Cell Cycle; src-Family Kinases; Tretinoin

Unlike its cytotoxicity in p53-functional cell lines, Nutlin-1, the small-molecule inhibitor of murine double minute (MDM2), significantly enhanced the differentiation-inducing activity of all-trans retinoic acid (ATRA) in HL60 and NB4 cells (p53-nonfunctional) but not in U937 cells (p53 wild-type). Moreover, we demonstrated that the synergistic differentiation-inducing activity of Nutlin-1 combined with ATRA appeared in a p53-independent manner. In the present study, we found that ATRA could selectively induce expression of p-glycoprotein (p-gp) in HL60 and NB4 cells but not in U937 cells. Investigation of p-gp-ATPase activity showed that Nutlin-1 and ATRA were likely to act as p-gp transport substrates. Furthermore, Nutlin-1 enhanced the ability of ATRA to induce expression of the myeloid differentiation-related transcription factor C/EBPβ and to reduce expression of c-myc. Additionally, the expression of retinoic acid receptor α (RARα) was further reduced in cells treated with ATRA in combination with Nutlin-1. Taken together, the mechanisms of synergistic differentiation-inducing activity of Nutlin-1 combined with ATRA could be attributed to Nutlin-1 competitive binding to p-gp, leading to ATRA efflux inhibition, and then the differentiation pathways involved were therefore further activated. Nutlin-1 might be a useful adjuvant with ATRA for patients with retinoid-resistant leukemia induced by overexpression of p-gp.

Topics: Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding, Competitive; Biological Transport; CCAAT-Enhancer-Binding Protein-beta; Cell Differentiation; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid; Piperazines; Proto-Oncogene Proteins c-mdm2; Proto-Oncogene Proteins c-myc; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA Interference; Time Factors; Transfection; Tretinoin; Tumor Suppressor Protein p53; U937 Cells; Verapamil

All-trans-retinoic-acid (ATRA)-induced differentiation of human myeloid leukemia cells is characterized by persistent mitogen-activated protein kinase (MAPK) signaling. Fragmentary data suggests Src family kinase (SFK) inhibitors enhance differentiation, and thus have potential therapeutic value. The present study shows that SFK inhibitors PP2 and dasatinib enhance aspects of MAPK signaling and regulate a panel of differentiation markers, including CD11b and p47(phox). HL-60 and NB4 myeloid leukemia cells show accelerated ATRA-induced G1/0 arrest/differentiation with inhibitor co-treatment. We also identified components of a Lyn- and c-Raf-containing MAPK signaling complex augmented by the inhibitors. PP2 and dasatinib increased the ATRA-induced expression of Lyn and c-Raf (total and c-RafpS259) and their interaction. The Lyn-associated serine/threonine kinase, casein kinase II (CK2), also complexed with c-Raf and c-RafpS259, and the kinase suppressor of Ras 1 (KSR1) scaffold protein bound c-Raf, Lyn and extracellular signal-regulated kinase (ERK). c-Raf/ERK association was increased by the inhibitors, which is significant as ERK may cause c-Raf C-terminal domain (CTD) phosphorylation in a putative feedback mechanism. Consistent with this, inhibitor treatment caused more CTD phosphorylation. Lyn knockdown decreased c-Raf CTD and S259 phosphorylation. This is the first evidence suggesting SFK inhibitors enhance ATRA-induced differentiation through a possible feedback loop involving KSR1-scaffolded c-Raf and ERK complexed with Lyn and CK2.

Topics: Antineoplastic Agents; Blotting, Western; Cell Cycle; Cell Differentiation; Dasatinib; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Humans; Immunoprecipitation; Leukemia, Myeloid; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-raf; Pyrimidines; RNA, Small Interfering; Signal Transduction; src-Family Kinases; Thiazoles; Tretinoin; Tumor Cells, Cultured

This study was aimed to investigate the homeobox A9 (HOXA9) mRNA and protein expression in myeloid leukemia cell line HL-60 and effect of all-trans retinoic acid (ATRA 1 µmol/L) or arsenic trioxide (As2O3 1 µmol/L) on its expression, and to explore the pathogenesis of leukemia mediated by HOXA9 at mRNA and protein levels. The expression of HOXA9 mRNA and protein were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) and Western blot, respectively. HL-60 cells were divided into 3 group: normal control added with RPMI 1640 medium, ATRA group and As2O3 group. The results indicated that HOXA9 mRNA expression in each group showed a firstly increasing and then decreasing tendency, in which the expression of HOXA9 mRNA was observed at day 1, increased at day 2, and decreased at day 3; while HOXA9 protein expression in each group was observed at day 1, the expression of HOXA9 protein in control group and ATRA group showed firstly increase and then decrease. The expression of HOXA9 protein in As2O3 group showed a decreased tendency gradually. The expression of HOXA9 mRNA in ATRA groups at day 1-3 was higher than that in control group (P < 0.05). The differences of HOXA9 mRNA expression between As2O3 groups and the control group were not significant at day 1 and day 3, but was higher than that in control group at day 2 (P < 0.05). The expression of HOXA9 protein in ATRA group at day 1- day 3 was higher than that in control group (P < 0.05). Compared with control group, the expression of HOXA9 protein in As2O3 group was higher at day 1 (P < 0.05), lower at day 2 (P < 0.05), and no significant differences at day 3. It is concluded that HOXA9 mRNA and protein express in HL-60 cells. ATRA 1 µmol/L can up-regulate the expression of HOXA9 mRNA and protein in HL-60 cells. The mechanisms of treatment of leukemia by ATRA and As2O3 may be associated with the regulation of the HOXA9 mRNA or protein expression.

Topics: Arsenic Trioxide; Arsenicals; Cell Differentiation; HL-60 Cells; Homeodomain Proteins; Humans; Leukemia, Myeloid; Oxides; RNA, Messenger; Tretinoin

Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA(2) into the knockdown cells by stable transfection with a Prdx6-PLA(2) expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA(2) mutated in the Prdx active site; reintroduction of PLA(2) active site mutants, however, failed to restore the response. Thus, the PLA(2) activity of Prdx6-PLA(2) in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA(2) isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.

Topics: Cell Line, Tumor; Humans; Leukemia, Myeloid; NADPH Oxidases; Peroxiredoxin VI; Phospholipases A2; Superoxides; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Blotting, Western; Cell Differentiation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leukemia, Myeloid; Phosphorylation; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Clinical application of retinoic acids (RAs) and demethylation agents has proven to be effective in treating certain myeloid leukemia patients. However, the target genes that mediate these antileukemia activities are still poorly understood. In this study, we identified olfactomedin 4 (OLFM4), a myeloid-lineage-specific gene from the olfactomedin family, as a novel target gene for RAs and the demethylation agent, 5-aza-2'-deoxycytidine. We demonstrated that the retinoic acid receptor alpha/retinoic X receptor alpha heterodimer binds to a retinoic acid response-element (DR5) site in the OLFM4 promoter and mediates all-trans-retinoic acid (ATRA)-induced transactivation of the OLFM4 gene. OLFM4 overexpression in HL-60 cells led to growth inhibition, differentiation, and apoptosis, and potentiated ATRA induction of these effects. Conversely, down-regulation of endogenous OLFM4 in acute myeloid leukemia-193 cells compromised ATRA-induced growth inhibition, differentiation, and apoptosis. Overexpression of OLFM4 in HL-60 cells inhibited constitutive and ATRA-induced phosphorylation of the eukaryote initiation factor 4E-binding protein 1 (4E-BP1), whereas down-regulation of OLFM4 protein in acute myeloid leukemia-193 cells increased 4E-BP1 phosphorylation, suggesting that OLFM4 is a potent upstream inhibitor of 4E-BP1 phosphorylation/deactivation. Thus, our study demonstrates that OLFM4 plays an important role in myeloid leukemia cellular functions and induction of OLFM4-mediated effects may contribute to the therapeutic value of ATRA.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Apoptosis; Azacitidine; Blotting, Western; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Decitabine; DNA Methylation; Electrophoretic Mobility Shift Assay; Gene Expression; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; HL-60 Cells; Humans; Immunohistochemistry; Leukemia, Myeloid; Myeloid Cells; Phosphoproteins; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

The standard of care for promyelocytic leukemia includes use of the differentiating agent all-trans retinoic acid (RA) and chemotherapy. RA induces cell differentiation through retinoic acid receptor (RAR) transcription factors. Because redox mechanisms influence how readily transcription factors bind to DNA response elements (RARE), the impact of small molecule (E3330) inhibition of the redox regulatory protein, apurinic-apyrimidinic endonuclease/redox effector factor (APE1/Ref-1) on RAR DNA binding and function in RA-induced myeloid leukemia cell differentiation and apoptosis was investigated.. The redox function of APE1 was studied using the small molecule inhibitor E3330 in HL-60 and PLB acute myeloid leukemia cells. Electrophoretic mobility shift assays were employed to determine effect of inhibitor on APE1/Ref-1 redox signaling function. Trypan blue assays, Annexin-V/propidium iodide and CD11b staining, and real-time polymerase chain reaction analyses were employed to determine survival, apoptosis, and differentiation status of cells in culture.. RARα binds to its RARE in a redox-dependent manner mediated by APE1/Ref-1 redox regulation. Redox-dependent RAR-RARE binding is blocked by E3330, a small molecule redox inhibitor of APE1/Ref-1. Combination treatment of RA + E3330 results in a profound hypersensitivity of myeloid leukemia cells to RA-induced differentiation and apoptosis. Additionally, redox inhibition by E3330 results in enhanced RAR target gene, BLR-1, expression in myeloid leukemia cells.. The redox function of APE1/Ref-1 regulates RAR binding to its DNA RAREs influencing the response of myeloid leukemia cells to RA-induced differentiation. Targeting of APE1/Ref-1 redox function may allow manipulation of the retinoid response with therapeutic implications.

Topics: Apoptosis; Benzoquinones; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Leukemia, Myeloid; Oxidation-Reduction; Propionates; Receptors, CXCR5; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Tretinoin

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58muM, for NB4 and NB4-R2, respectively. alpha-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis.

Topics: alpha-Tocopherol; Antineoplastic Agents; Antioxidants; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromatography, High Pressure Liquid; Drug Therapy, Combination; Growth Inhibitors; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Lipid Peroxidation; Oxides; Tretinoin

N-Myc downstream-regulated gene 1 (NDRG1) protein has been shown to be up-regulated during leukemic cell differentiation induced by some differentiation-inducing agents such as all-trans retinoic acid (ATRA). However, the potential role of up-regulated NDRG1 in the event is greatly unknown. In this work, we show that inducible NDRG1 expression can drive leukemic U937 cells to undergo differentiation, while the knock-down of NDRG1 expression by specific small interfering RNA significantly antagonizes ATRA-induced differentiation of leukemic cells, proposing the role of NDRG1 in leukemic cell differentiation. Furthermore, our work shows that CCAAT/enhancer-binding protein beta (C/EBPbeta) and PU.1, which are important hematopoiesis-related transcription factors, may act as downstream effectors of NDRG1 in leukemic cell differentiation. Taking together, this study provides direct evidence for the role of NDRG1 protein in myeloid leukemic cell differentiation.

Topics: Antineoplastic Agents; CCAAT-Enhancer-Binding Protein-beta; Cell Cycle Proteins; Cell Differentiation; Gene Expression Regulation, Leukemic; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid; Neoplasm Proteins; Proto-Oncogene Proteins; Trans-Activators; Tretinoin; U937 Cells; Up-Regulation

The peptidyl-prolyl-isomerase Pin1 interacts with phosphorylated proteins, altering their conformation. The retinoic acid receptor RARalpha and the acute-promyelocytic-leukemia-specific counterpart PML-RARalpha directly interact with Pin1. Overexpression of Pin1 inhibits ligand-dependent activation of RARalpha and PML-RARalpha. Inhibition is relieved by Pin1-targeted short interfering RNAs and by pharmacologic inhibition of the catalytic activity of the protein. Mutants of Pin1 catalytically inactive or defective for client-protein-binding activity are incapable of inhibiting ligand-dependent RARalpha transcriptional activity. Functional inhibition of RARalpha and PML-RARalpha by Pin1 correlates with degradation of the nuclear receptors via the proteasome-dependent pathway. In the acute myelogenous leukemia cell lines HL-60 and NB4, Pin1 interacts with RARalpha in a constitutive fashion. Suppression of Pin1 by a specific short hairpin RNA in HL-60 or NB4 cells stabilizes RARalpha and PML-RARalpha, resulting in increased sensitivity to the cytodifferentiating and antiproliferative activities of all-trans retinoic acid. Treatment of the two cell lines and freshly isolated acute myelogenous leukemia blasts (M1 to M4) with ATRA and a pharmacologic inhibitor of Pin1 causes similar effects. Our results add a further layer of complexity to the regulation of nuclear retinoic acid receptors and suggest that Pin1 represents an important target for strategies aimed at increasing the therapeutic index of retinoids.

Topics: Acute Disease; Animals; Antineoplastic Agents; Chlorocebus aethiops; COS Cells; HL-60 Cells; Humans; Leukemia, Myeloid; NIMA-Interacting Peptidylprolyl Isomerase; Oncogene Proteins, Fusion; Peptidylprolyl Isomerase; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcriptional Activation; Transfection; Tretinoin

Transforming growth-interacting factor (TGIF) is a homeobox transcriptional repressor that has been implicated in holoprosencephaly and various types of cancer. TGIF is expressed in hematopoietic stem cells and modulates TGF-beta and retinoic acid (RA) signaling, both of which play an important role in hematopoiesis. We recently reported that TGIF's levels correlate inversely with survival in patients with acute myelogenous leukemia. Here we present the first direct evidence of a role for TGIF in myelopoiesis. We used short hairpin RNA interference to define the effects of TGIF knockdown on proliferation and differentiation of myeloid leukemia-derived cell lines. Decreased TGIF expression resulted in reduced proliferation and differentiation and lower expression of CEBPbeta, CEBPepsilon, PU.1 and RUNX1, key myeloid transcription factors. Furthermore, TGF-beta signaling was increased and RA signaling was decreased. Further insights into the molecular basis of TGIF's effects were provided by a genome-wide chromatin immunoprecipitation-based elucidation of TGIF target genes. Together, these data suggest that TGIF has an important role myelopoiesis and may regulate the balance between proliferation and differentiation. Reduced TGIF expression could tip the balance toward quiescence thus providing progenitor as well as hematopoietic stem cells protection from anti-cycle agents.

Topics: Animals; Apoptosis; Cell Cycle; Cell Differentiation; Cell Proliferation; Gene Knockdown Techniques; HL-60 Cells; Homeodomain Proteins; Humans; Leukemia, Myeloid; Mice; Myelopoiesis; Repressor Proteins; RNA Interference; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Tretinoin

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA is a potent anticancer agent that induces differentiation of leukemia cells as well as inhibiting cell growth. The current study examined HL60 proteins using anti-RA monoclonal antibodies (ARMAs) and found that some RA-binding proteins may be histones. These proteins eluted in the void volume fractions following Mono Q anion exchange chromatography and immunostained with ARMAs. These ARMAs showed specific binding to the proteins in a saturable manner that depended on antibody concentration. Certain of these proteins co-migrated with histones on one-dimensional polyacrylamide gel electrophoresis. It was also found that histones isolated from HL60 cells treated with RA also immunostained with ARMAs. These results indicate that basic proteins, including histones, may be bound to RA covalently in HL60 cells and that RA-binding histones may play significant roles in the transcriptional regulation of genes by RA.

Topics: Anion Exchange Resins; Antibodies, Monoclonal; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Histones; HL-60 Cells; Humans; Leukemia, Myeloid; Neoplasm Proteins; Protein Binding; Tretinoin

The clinical activities of all-trans retinoic acid in the treatment of acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia, have triggered extensive studies aimed at defining the mechanisms by which this compound induces differentiation of leukemic cells. Recent studies show that hypoxia-inducible factor-1 alpha (HIF-1 alpha) contributes to the differentiation of acute myeloid leukemia cells via transcriptional activity-independent mechanisms. We investigated whether all-trans retinoic acid affects HIF-1 alpha protein and whether this has a role in all-trans retinoic acid-induced differentiation.. The acute myeloid leukemia cell lines NB4 and U937 were treated with all-trans retinoic acid, and HIF-1 alpha/HIF-1 beta mRNA and proteins were measured respectively by real-time quantitative reverse transcriptase polymerase chain reaction and western blotting. To investigate the role of HIF-1 alpha in all-trans retinoic acid-induced differentiation, NB4 cells, U937 cells, U937 cells in which HIF-1 alpha was induced by the withdrawal of tetracycline and U937 cells with stable expression of specific short hairpin RNA against HIF-1 alpha, Runx1, C/EBP alpha and PU.1, were treated with all-trans retinoic acid and/or the hypoxiamimetic agent cobalt chloride (CoCl(2)). Cellular differentiation was evaluated by morphological criteria and myeloid differentiation antigens.. all-trans retinoic acid rapidly increased endogenous and inducible expressed or CoCl(2)-stabilized HIF-1 alpha protein in leukemic cells under normoxia. Importantly, suppression of HIF-1 alpha expression by specific short hairpin RNA partially but significantly inhibited all-trans retinoic acid-induced differentiation of the U937 cell line. Reciprocally, the differentiation induced by all-trans retinoic acid was significantly enhanced by conditional HIF-1 alpha induction and HIF-1 alpha-stabilizing CoCl(2) treatment. Furthermore, knock-down of PU.1, Runx1 and C/EBP alpha, three transcriptional factors crucial for normal hematopoiesis, greatly inhibited the differentiation cooperation of all-trans retinoic acid and HIF-1 alpha induction.. This work provides the first demonstration that HIF-1 alpha, a protein rapidly responsive to all-trans retinoic acid, plays a role in all-trans retinoic acid-induced differentiation of leukemic cells. These observations shed new light on the molecular mechanisms underlying all-trans retinoic acid-induced differentiation of acute myeloid leukemia cells.

Topics: CCAAT-Enhancer-Binding Protein-alpha; Cell Differentiation; Cell Line, Tumor; Copper; Core Binding Factor Alpha 2 Subunit; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leukemia, Myeloid; Proto-Oncogene Proteins; RNA Interference; RNA, Messenger; Trans-Activators; Tretinoin

Induction of differentiation is a new and promising approach to cancer therapy, well illustrated by the treatment of acute myeloid leukemia with all-trans retinoic acid (ATRA). Using combination of ATRA and chemotherapy, adverse effects such as retinoic acid syndrome have decreased, and long-term survival has improved. In this study, we demonstrated that the indeno[1,2-c]isoquinolines markedly enhanced differentiation of human myeloid leukemia HL-60 and NB4 cells when simultaneously combined with a low dose of ATRA. Of the tested compounds, 6-(4-methoxybenzyl)-2,11-dimethyl-6H,11H-indeno[1,2-c]isoquinolin-5-one (IIQ-16), an indeno[1,2-c]isoquinoline derivative, showed the highest differentiation-enhancing activity via a pathway involved with protein kinase C, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. The ability to enhance the differentiation potential of ATRA by IIQ-16 may improve outcomes in the therapy of acute promyelocytic leukemia.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Humans; Indenes; Isoquinolines; Leukemia, Myeloid; Molecular Structure; Structure-Activity Relationship; Tretinoin

Arsenic trioxide (As2O3) is highly efficacious in acute promyelocytic leukemia (APL). Aquaglyceroporin 9 (AQP9) is a transmembrane protein that may be involved in arsenic uptake. In 10 of 11 myeloid and lymphoid leukemia lines, quantitative polymerase chain reaction (Q-PCR) and Western blotting showed that AQP9 expression correlated positively with As2O3-induced cytotoxicity. As a proof-of-principle, transfection of EGFP-tagged AQP9 to the hepatoma line Hep3B, not expressing AQP9 and As2O3 insensitive, led to membrane AQP9 expression and increased As2O3-induced cytotoxicity. Similarly, the chronic myeloid leukemia line K562 expressed low levels of AQP9 and was As2O3 insensitive. The K562(EGFP-AQP9) transfectant accumulated significantly higher levels of intracellular arsenic than control K562(EGFP) when incubated with As2O3, resulting in significantly increased As2O3-induced cytotoxicity. Pretreatment of the myeloid leukemia line HL-60 with all-trans retinoic acid (ATRA) up-regulated AQP9, leading to a significantly increased arsenic uptake and As2O3-induced cytotoxicity on incubation with As2O3, which might explain the synergism between ATRA and As2O3. Therefore, AQP9 controlled arsenic transport and might determine As2O3 sensitivity. Q-PCR showed that primary APL cells expressed AQP9 significantly (2-3 logs) higher than other acute myeloid leukemias (AMLs), which might explain their exquisite As2O3 sensitivity. However, APL and AML with maturation expressed comparable AQP9 levels, suggesting that AQP9 expression was related to granulocytic maturation.

Topics: Acute Disease; Aquaporins; Arsenic Trioxide; Arsenicals; Cell Line, Tumor; Cell Proliferation; Gene Expression Profiling; Humans; K562 Cells; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Oxides; Point Mutation; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Tretinoin; Up-Regulation

Alteration of lineage-specific transcriptional programs for hematopoiesis causes differentiation block and promotes leukemia development. Here, we show that AML1/ETO, the most common translocation fusion product in acute myeloid leukemia (AML), counteracts the activity of retinoic acid (RA), a transcriptional regulator of myelopoiesis. AML1/ETO participates in a protein complex with the RA receptor alpha (RARalpha) at RA regulatory regions on RARbeta2, which is a key RA target gene mediating RA activity/resistance in cells. At these sites, AML1/ETO recruits histone deacetylase, DNA methyltransferase, and DNA-methyl-CpG binding activities that promote a repressed chromatin conformation. The link among AML1/ETO, heterochromatic RARbeta2 repression, RA resistance, and myeloid differentiation block is indicated by the ability of either siRNA-AML1/ETO or the DNA methylation inhibitor 5-azacytidine to revert these epigenetic alterations and to restore RA differentiation response in AML1/ETO blasts. Finally, RARbeta2 is commonly silenced by hypermethylation in primary AML blasts but not in normal hematopoietic precursors, thus suggesting a role for the epigenetic repression of the RA signaling pathway in myeloid leukemogenesis.

Topics: Acute Disease; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 2 Subunit; Gene Expression Regulation, Leukemic; Gene Silencing; Heterochromatin; Humans; Leukemia, Myeloid; Oncogene Proteins, Fusion; Protein Binding; Receptors, Retinoic Acid; Response Elements; Retinoid X Receptors; RUNX1 Translocation Partner 1 Protein; Signal Transduction; Transfection; Tretinoin; U937 Cells

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Arsenic Trioxide; Arsenicals; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Granulocytes; HL-60 Cells; Humans; Leukemia, Myeloid; Macrophages; Monocytes; Oxides; Protein Transport; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; RNA, Small Interfering; Tretinoin

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Topics: Acute Disease; Antigens, CD34; Antigens, Differentiation; Apoptosis Regulatory Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Line, Tumor; Chronic Disease; Death-Associated Protein Kinases; Gene Expression Profiling; Granulocytes; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Myeloid Cells; Myelopoiesis; Neutrophils; RNA, Small Interfering; Tretinoin; Up-Regulation

PU.1, a transcription factor of the ETS family, plays a pivotal role in normal hematopoiesis, and particularly in myeloid differentiation. Altered PU.1 function is possibly implicated in leukemogenesis, as PU.1 gene mutations were identified in some patients with acute myeloid leukemia (AML) and as several oncogenic products (AML1-ETO, promyelocytic leukemia-retinoic acid receptor alpha, FMS-like receptor tyrosine kinase 3 internal tandem duplication) are associated with PU.1 downregulation. To demonstrate directly a role of PU.1 in the blocked differentiation of leukemic blasts, we transduced cells from myeloid cell lines and primary blasts from AML patients with a lentivector encoding PU.1. In NB4 cells we obtained increases in PU.1 mRNA and protein, comparable to increases obtained with all-trans retinoic acid-stimulation. Transduced cells showed increased myelomonocytic surface antigen expression, decreased proliferation rates and increased apoptosis. Similar results were obtained in primary AML blasts from 12 patients. These phenotypic changes are characteristic of restored blast differentiation. PU.1 should therefore constitute an interesting target for therapeutic intervention in AML.

Topics: Adult; Aged; Apoptosis; Blast Crisis; CD13 Antigens; Cell Differentiation; Female; Genetic Vectors; Humans; Lentivirus; Leukemia, Myeloid; Male; Middle Aged; Proto-Oncogene Proteins; Trans-Activators; Tretinoin

Natural killer (NK) cells are potent effectors of innate antitumor defense and are currently exploited for immune-based therapy of human leukemia. However, malignant blood cells in acute myeloid leukemia (AML) display low levels of ligands for the activating immunoreceptor NKG2D and can thus evade NK immunosurveillance. We examined the possibility of up-regulating NKG2D-specific UL16-binding protein (ULBP) ligands using anti-neoplastic compounds with myeloid differentiation potential. Combinations of 5-aza-2'-deoxycytidine, trichostatin A, vitamin D3, bryostatin-1, and all-trans-retinoic acid, used together with myeloid growth factors and interferon-gamma, increased cell surface ULBP expression up to 10-fold in the AML cell line HL60 and in primary AML blasts. Up-regulation of ULBP ligands was associated with induction of myelomonocytic differentiation of AML cells. Higher ULBP expression increased NKG2D-dependent sensitivity of HL60 cells to NK-mediated killing. These findings identify NKG2D ligands as targets of leukemia differentiation therapy and suggest a clinical benefit in combining a pharmacological approach with NK cell-based immunotherapy in AML.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Bryostatins; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Cytotoxicity, Immunologic; Decitabine; Flow Cytometry; GPI-Linked Proteins; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Leukemia, Myeloid; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Up-Regulation

The tumor antigen preferentially expressed antigen of melanoma (PRAME) is frequently overexpressed in a wide variety of malignant diseases, including acute myeloid leukemia (AML). It was recently shown that PRAME can function as a repressor of retinoic acid signaling, as indicated by down-regulation of retinoic acid receptor-beta (RARB) and cyclin-dependent kinase inhibitor 1A (CDKN1A). Another study suggested that PRAME can induce caspase-independent apoptosis via down-regulation of heat shock 27-kDa protein 1 (HSPB1) and S100 calcium-binding protein A4 (S100A4). The transcriptional repression of PRAME depends on the formation of a complex with the enhancer of zeste homolog 2 (EZH2). To test whether these mechanisms play an important roll in AML, we analyzed the expression of PRAME, EZH2, RARB, HSPB1, S100A4, and CDKN1A by real-time polymerase chain reaction in primary leukemic cells from 52 children with AML. All genes were expressed in many patients, but the level of expression of the last four genes was not associated with either PRAME expression or PRAME and EZH2 co-expression. In conclusion, the above mechanisms do not seem to play a major role in the pathogenesis of AML; they could be neutralized by other pathways that affect the same targets.

Topics: Acute Disease; Adolescent; Adult; Antigens, Neoplasm; Biomarkers, Tumor; Child; Child, Preschool; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; Female; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Infant; Infant, Newborn; Leukemia, Myeloid; Male; Molecular Chaperones; Neoplasm Proteins; Polycomb Repressive Complex 2; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; S100 Calcium-Binding Protein A4; S100 Proteins; Signal Transduction; Transcription Factors; Tretinoin

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.

Topics: Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; HL-60 Cells; Humans; Leukemia, Myeloid; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Phospholipase D; Protein Kinases; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Steryl-Sulfatase; Transfection; Tretinoin

Dysregulation of hematopoietic cellular differentiation contributes to leukemogenesis. Unfortunately, relatively little is known about how cell differentiation is regulated. JWA (AF070523) is a novel all-trans retinoic acid (ATRA) responsible gene that initially isolated from ATRA-treated primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in the induction of differentiation of human myeloid leukemia cells. Our results showed that JWA was not only regulated by ATRA but also by several other differentiation inducers such as phorbol-12-myristate-13-acetate (TPA), arabinoside (Ara-C), and hemin, involved in the mechanisms of differentiation along different lineages of myeloid leukemia cells arrested at different stages of development. Generally, JWA was up-regulated by these inducers in a time-dependent manner. Inhibition of JWA by RNA interference decreased the induced cellular differentiation. However, in NB4 cells treated with ATRA, dissimilar with others, the expression of JWA was down-regulated, and the induced cellular differentiation could be enhanced by silencing of JWA. Collectively, JWA works as a potential critical molecule, associated with multi-directional differentiation of human myeloid leukemia cells. In NB4 cells, JWA may function as a lineage-restricted gene during differentiation along the monocyte/macrophage-like or granulocytic pathway.

Topics: Blotting, Western; Bone Marrow Cells; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Cytarabine; Gene Silencing; Granulocytes; Heat-Shock Proteins; Hemin; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; K562 Cells; Leukemia, Myeloid; Macrophages; Membrane Transport Proteins; Monocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; U937 Cells

Retinoic acid (RA), used as first-line therapy for acute promyelocytic leukemia (APL), exerts its antileukemic activity by inducing blast differentiation and activating tumor-selective TNF-related apoptosis-inducing ligand (TRAIL) signaling. To identify downstream mediators of RA signaling, we used retrovirus-mediated insertion mutagenesis in PLB985 leukemia cells and established the RA-resistant cell line WY-1. In PLB985, but not WY-1 cells, RA induced TRAIL and its DR4 and DR5 receptors. Knocking down TRAIL expression by RNA interference blocked RA-induced apoptosis. WY-1 cells are defective for RA-induced differentiation, G1 arrest and exhibit co-resistance to TRAIL. In WY-1 cells, a single virus copy is integrated into a novel RA-regulated gene termed RAM (retinoic acid modulator). RAM is expressed in the myelomonocytic lineage and extinguished by RA in PLB985, but not WY-1 cells. Whereas knocking down RAM expression by RNA interference promoted RA-induced differentiation and TRAIL-triggered apoptosis of PLB985 and WY-1 cells, overexpression of the predicted 109 amino-acid RAM open reading frame did not alter RA signaling in PLB985 cells. This indicates that, apart from encoding the putative RAM protein, RAM RNA may exert additional functions that are impaired by the retrovirus insertion. Our study demonstrates that RA induction of the TRAIL pathway is also operative in leukemia cells lacking an RARalpha oncofusion protein and identifies RAM as a novel RA-dependent modulator of myeloid differentiation and death.

Topics: Amino Acid Sequence; Apoptosis; Apoptosis Regulatory Proteins; Base Sequence; Cell Differentiation; Drug Resistance, Neoplasm; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid; Membrane Glycoproteins; Molecular Sequence Data; Mutagenesis, Insertional; rab GTP-Binding Proteins; rab27 GTP-Binding Proteins; Receptors, Retinoic Acid; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Retinoic Acid Receptor alpha; Retinoid X Receptors; RNA, Long Noncoding; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

Topics: Acute Disease; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cluster Analysis; Databases, Factual; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid; Meta-Analysis as Topic; Oligonucleotide Array Sequence Analysis; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Vitamin D; Vitamins

Among the topoisomerase (topo) II isozymes (alpha and beta), topo IIbeta has been suggested to regulate differentiation. In this study, we examined the role of topo IIbeta in all-trans retinoic acid (ATRA)-induced differentiation of myeloid leukemia cell lines. Inhibition of topo IIbeta activity or downregulation of protein expression enhanced ATRA-induced differentiation/growth arrest and apoptosis. ATRA-induced apoptosis in topo IIbeta-deficient cells involved activation of the caspase cascade and was rescued by ectopic expression of topo IIbeta. Gene expression profiling led to the identification of peroxiredoxin 2 (PRDX2) as a candidate gene that was downregulated in topo IIbeta-deficient cells. Reduced expression of PRDX2 validated at the mRNA and protein level, in topo IIbeta-deficient cells correlated with increased accumulation of reactive oxygen species (ROS) following ATRA-induced differentiation. Overexpression of PRDX2 in topo IIbeta-deficient cells led to reduced accumulation of ROS and partially reversed ATRA-induced apoptosis. These results support a role for topo IIbeta in survival of ATRA-differentiated myeloid leukemia cells. Reduced expression of topo IIbeta induces apoptosis in part by impairing the anti-oxidant capacity of the cell owing to downregulation of PRDX2. Thus, suppression of topo IIbeta and/or PRDX2 levels in myeloid leukemia cells provides a novel approach for improving ATRA-based differentiation therapy.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Division; Diketopiperazines; DNA Topoisomerases, Type II; DNA-Binding Proteins; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Leukemic; Heat-Shock Proteins; HL-60 Cells; Humans; Leukemia, Myeloid; Peroxidases; Peroxiredoxins; Piperazines; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Topoisomerase II Inhibitors; Tretinoin

Use of all-trans-retinoic acid (ATRA) in combinatorial differentiation therapy of acute promyelocytic leukemia (APL) results in exceptional cure rates. However, potent cell differentiation effects of ATRA are so far largely restricted to this disease and long-term survival rates in non-APL acute myelogeneous leukemia (AML) remain unacceptably poor, requiring development of novel therapeutic strategies. We demonstrate here that myelomonocytic growth factors (granulocyte colony-stimulating factor [G-CSF] and/or granulocyte macrophage colony-stimulating factor [GM-CSF]) potentiate differentiation effects of ATRA in different AML cell lines and primary cells from patients with myeloid leukemia. The ligand-dependent activities of endogenous and transiently expressed retinoic acid receptor alpha (RARalpha) isoforms can be potentiated by G/GM-CSF in U-937 cells and correlate with increased expression of ATRA-inducible RARalpha2 isoform. Specific inhibitors of mitogen mitogen-activated protein kinase (MAPK) (MEK)-1/-2 or p38 extracellular signal-related kinase (ERK) kinase diminish the ATRA as well as ATRA and G/GM-CSF-induced activation of the RARalpha proteins and decreased the differentiation-induced decline in cell numbers. Our data demonstrate that acting, at least in part, via the MAP kinase pathways, myelomonocytic growth factors enhance ATRA-dependent activation of the RARalpha isoforms and maturation of myeloid leukemia cells. These results suggest that combinatorial use of these agents may be effective in differentiation therapy of AML.

Topics: Animals; Cell Differentiation; Cell Division; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myeloid; MAP Kinase Signaling System; Mice; Protein Isoforms; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

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

Topics: Antineoplastic Agents; Core Binding Factor Alpha 2 Subunit; Drug Resistance, Neoplasm; Female; Humans; Karyotyping; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Middle Aged; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Tretinoin

The AML1/ETO and PML/RARalpha leukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARalpha and AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARalpha-N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis.

Topics: Acute Disease; Cell Differentiation; Cell Line, Tumor; Cholecalciferol; Core Binding Factor Alpha 2 Subunit; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; Neoplasm Proteins; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2; Oncogene Proteins, Fusion; Peptides; Protein Structure, Tertiary; Repressor Proteins; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Tretinoin

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, many cancer cells are resistant to apoptosis induction by TRAIL. The present study was designed to evaluate the sensitivity to TRAIL-induced apoptosis in acute myeloblastic leukemias (AML).. TRAIL/TRAIL receptor (TRAIL-R) expression and sensitivity to TRAIL-mediated apoptosis were explored in 79 AML patients, including 17 patients with acute promyelocytic leukemia (APL).. In non-APL AML we observed frequent expression of TRAIL decoy receptors (TRAIL-R3 and TRAIL-R4), while TRAIL-R1 and TRAIL-R2 expression was restricted to AML exhibiting monocytic features. Total leukemic blasts, as well as AML colony-forming units (AML-CFU), were invariably resistant to TRAIL-mediated apoptosis. APL express membrane-bound TRAIL on their surface and exhibit a pattern of TRAIL-R expression similar to that observed in the other types of AML. Before, during and after retinoic acid treatment APL cells are TRAIL-resistant. The induction of granulocytic maturation of APL cells by retinoic acid was associated with a marked decline of TRAIL expression.. The analysis of experimental APL models (i.e., U937 cells engineered to express PML/RAR-Eo and NB4 cells) provided evidence that PML/RAR-Eo expression was associated with downmodulation of TRAIL-R1 and with resistance to TRAIL-mediated apoptosis. We suggest that AML blasts, including APL blasts, are resistant to TRAIL-mediated apoptosis, a phenomenon seemingly related to the expression of TRAIL decoy receptors on these cells. Finally, APL blasts express membrane-bound TRAIL that could confer an immunologic privilege to these cells.

Topics: Acute Disease; Adolescent; Adult; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 8; Cell Differentiation; Cell Membrane; Cytarabine; Drug Resistance, Neoplasm; Etoposide; Female; GPI-Linked Proteins; Granulocytes; HL-60 Cells; Humans; Hydroxyurea; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Monocytes; Oncogene Proteins, Fusion; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Member 10c; Recombinant Fusion Proteins; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor Decoy Receptors; Tumor Stem Cell Assay; U937 Cells

We studied wild-type FLT3 mRNA expression at diagnosis in bone marrow samples from 85 patients with acute myeloid leukemia (AML), 23 of whom were in complete remission, and determined its utility as a marker for minimal residual disease (MRD). We conclude that FLT3 expression is of limited value as a prognostic marker and for MRD monitoring.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Marrow; Combined Modality Therapy; Cytarabine; Daunorubicin; Etoposide; Female; fms-Like Tyrosine Kinase 3; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid; Male; Middle Aged; Neoplasm Proteins; Neoplasm, Residual; Prognosis; Remission Induction; RNA, Messenger; RNA, Neoplasm; Survival Analysis; Tretinoin

Cytokinins are important purine derivatives that act as redifferentiation-inducing hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA) and kinetin are very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to IPA using cDNA microarrays and compared the results with those obtained with other inducers of differentiation, such as all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 (VD3) and cotylenin A (CN-A). Many genes were up-regulated, and only a small fraction were down-regulated, upon exposure to the inducers. IPA and CN-A, but not ATRA or VD3, immediately induced the expression of mRNA for the calcium-binding protein S100P. The up-regulation of S100P was confirmed at the protein expression level. We also examined the expression of other S100 proteins, including S100A8, S100A9 and S100A12, and found that IPA preferentially up-regulated S100P at the early stages of differentiation. IPA-induced differentiation of HL-60 cells was suppressed by treatment with antisense oligonucleotides against S100P, suggesting that S100P plays an important role in cell differentiation.

Topics: Adenine; Calcitriol; Calcium-Binding Proteins; Cell Differentiation; Cell Line, Tumor; Cytokinins; Diterpenes; DNA, Complementary; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Isopentenyladenosine; Leukemia, Myeloid; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Tretinoin; U937 Cells; Up-Regulation

Most acute promyelocytic leukaemia (APL) patients suffer from disordered haemostasis. APL can be treated successfully in most instances by all-trans retinoic acid (ATRA) therapy, which induces endpoint maturation of the leukaemic promyelocytes with the characteristic t(15;17). Annexin II (AnII), a profibrinolytic protein, has been implicated in the bleeding manifestation seen in APL. Our group has shown previously that high levels of AnII are expressed on other acute myeloid leukaemia subtypes that are sometimes associated with disordered haemostasis, albeit less frequently than APL. This study examined the effects of ATRA on AnII expression and cell differentiation, on myeloid leukaemia cell lines to determine whether a regulatory influence on AnII may contribute to the return of haemostatic stability in APL following treatment. The results confirmed that AnII expression in the APL cell line (NB4) was significantly downregulated in response to ATRA (P < 0.01), with associated morphological and immunophenotypical evidence of myeloid differentiation. ATRA also downregulated AnII expression on other myeloid cell lines, albeit to a lesser extent than observed on NB4 cells. The results provide evidence that ATRA may resolve the hyperfibrinolysis in APL by downregulation of AnII expression.

Topics: Analysis of Variance; Annexin A2; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Fibrinolysis; Flow Cytometry; Hemostasis; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

The retinoic acid receptor alpha (RARA) gene is disrupted by PML/RARA fusion in acute promyelocytic leukemia (APL). The P2 promoter of RARA, controlling the RARalpha2 isoform, contains an RA-responsive element and may be targeted in APL. To test whether aberrant methylation of P2 was involved, 47 APL at diagnosis, 16 APL at first relapse, 50 acute myeloid leukemia (AML) and 22 acute lymphoblastic leukemia (ALL) were tested by methylation-specific polymerase chain reaction. RARA P2 methylation was highly associated with APL (APL: 25/63 vs AML/ALL: 2/75, P<0.0001). P2 methylation occurred at similar frequencies in APL at diagnosis and relapse, suggesting it was an initiating leukemogenic event. In the APL line NB4, RARalpha2 was not expressed, with the untranslocated RARA shown to be P2 methylated. 5-Azacytadine treatment of NB4 led to progressive P2 demethylation and re-expression of RARalpha2, confirming that RARA methylation collaborated with PML/RARA in totally suppressing RARalpha. In APL, RARA P2 methylation was unrelated to gender, age, presenting leukocyte counts and additional cytogenetic aberrations. For APL patients receiving all-trans retinoic acid for induction, P2 methylation did not affect the complete remission rates and survivals. RARA is the first myeloid-specific transcription factor shown to be dysregulated by both translocation and aberrant methylation.

Topics: Acute Disease; Biological Transport; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recurrence; Remission Induction; Retinoic Acid Receptor alpha; Survival Rate; Tretinoin

Translocation (9;11) is the most common t(11q23) in acute myeloid leukemia (AML). A considerable number of patients with this cytogenetic abnormality relapse and die of their disease. We evaluated the clinical significance of minimal residual disease (MRD) monitoring in t(9;11)(p22;q23)-positive AML patients using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) analysis.. We identified 34 newly diagnosed patients with t(9;11)(p22;q23)-positive AML treated within three multicenter trials of the AML Study Group. MRD could be investigated by RQ-PCR in 19 patients during and after therapy. Because of the relatively low sensitivity of the RQ-PCR (10(-3) to 10(-4) at the cellular level), samples from RQ-PCR-negative patients were also analyzed by nested polymerase chain reaction (nPCR; sensitivity 10-4 to 10-5 at the cellular level).. RQ-PCR monitoring revealed two groups of patients: group 1 (n=11) had negative RQ-PCR in all samples collected in hematologic complete remission whereas group 2 (n=8) had at least one positive RQ-PCR in samples collected in complete remission during therapy. Group 1 had a significantly lower cumulative incidence of relapse (p=0.004) and better overall survival (p=0.003) compared to group 2. nPCR did not add information to that gained from RQ-PCR. Molecular relapse was detected in two patients by RQ-PCR four and six weeks, respectively before hematologic relapse occurred. Quantitative MLL-AF9 levels at diagnosis or during and after therapy had no prognostic impact.. Early achievement of sustained RQ-PCR negativity appears to be a prerequisite for long-term hematologic complete remission in t(9;11)-positive AML. Furthermore, RQ-PCR might be useful for early detection of relapse. Additional patients need to be studied to corroborate these findings.

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 9; Cohort Studies; Combined Modality Therapy; Cytarabine; Etoposide; Humans; Idarubicin; Leukemia, Myeloid; Middle Aged; Mitoxantrone; Multicenter Studies as Topic; Myeloid-Lymphoid Leukemia Protein; Neoplasm, Residual; Oncogene Proteins, Fusion; Peripheral Blood Stem Cell Transplantation; Polymerase Chain Reaction; Prognosis; Randomized Controlled Trials as Topic; Recurrence; Remission Induction; Survival Analysis; Translocation, Genetic; Treatment Outcome; Tretinoin

The phosphatidylinositol 3-kinase (PI3K)/AKT protein kinase pathway is involved in cell growth, proliferation, and apoptosis. The functional activation of PI3K/AKT provides survival signals and blockade of this pathway may facilitate cell death. Downstream targets of PI3K-AKT include the proapoptotic protein BAD, caspase-9, NF-kappaB, and Forkhead. We have previously reported that BAD is constitutively phosphorylated in primary acute myeloid leukemia (AML) cells, a post-transcriptional modification, which inactivates its proapoptotic function. In this study, we tested the hypothesis that the inhibition of PI3K by LY294002 results in the dephosphorylation of AKT and BAD, and thus promote leukemia cell apoptosis. We investigated the effects of LY294002 in megakaryocytic leukemia-derived MO7E cells, primary AML and normal bone marrow progenitor cells. In MO7E cells, LY294002 reduced AKT kinase activity, induced dephosphorylation of AKT and BAD, and increased apoptosis. Concomitant inhibition of mitogen-activated protein kinase signaling or combination with all-trans retinoic acid further enhanced apoptosis of leukemic cells. In primary AML samples, clonogenic cell growth was significantly reduced. Normal hematopoietic progenitors were less affected, suggesting preferential targeting of leukemia cells. In conclusion, the data suggest that the inhibition of the PI3K/AKT signaling pathway restores apoptosis in AML and may be explored as a novel target for molecular therapeutics in AML.

Topics: Apoptosis; bcl-Associated Death Protein; Carrier Proteins; Cell Line, Tumor; Chromones; Drug Interactions; Humans; Leukemia, Myeloid; MAP Kinase Signaling System; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Tretinoin

Insulin-like growth factor binding protein-3 (IGFBP-3) can cause growth suppressive and proapoptotic effects on retinoids in many types of cancer cells. However, the expression and effects of IGFBP-3 in myeloid leukemia cells have not been elucidated. In this study, we found no IGFBP-3 expression in the human myeloid leukemia cell lines either at baseline or after stimulation with all-trans retinoic acid (ATRA). Human recombinant IGFBP-3 induced growth arrest and apoptosis of HL-60 and NB4 cells. We have previously identified RXR alpha as a nuclear receptor for IGFBP-3 and have proceeded to examine further the role of this interaction in leukemia cell lines. In signaling assays, IGFBP-3 potently suppressed RAR- and VDR-mediated signaling while enhancing RXR signaling. Interestingly, when IGFBP-3 was administered to these cells in combination with an RAR-selective ligand, the ability of these retinoids to induce differentiation was blunted. On the other hand, IGFBP-3 enhanced the effect of an RXR-selective ligand to induce differentiation of HL-60 and NB4 cells. Further studies showed that IGFBP-3 down-regulated (at the transcriptional level) the retinoid-induced expression of C/EBP epsilon in NB4 cells. Taken together, these results indicate that IGFBP-3 has antiproliferative activity against myeloid leukemia cells; while it enhances signaling through RXR/RXR, it blunts signaling by activated RAR/RXR.

Topics: Alitretinoin; Apoptosis; Calcitriol; Cell Division; Dose-Response Relationship, Drug; Drug Interactions; Fatty Acids, Unsaturated; Gene Expression; HL-60 Cells; Humans; Insulin-Like Growth Factor Binding Protein 3; Leukemia, Myeloid; Protease Inhibitors; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Signal Transduction; Tetrahydronaphthalenes; Transcription Factors; Tretinoin; U937 Cells

To study the effect of oxidized low density lipoprotein (ox-LDL) on ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages.. After exposing the cultured THP-1 macrophages to ox-LDL for different periods, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and protein level were determined by reverse trancriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. The cholesterol level in THP-1 macrophage foam cells was detected by high performance liquid chromatography.. ox-LDL elevated ABCA1 in both protein and mRNA levels and increased apolipoprotein (apo) A-I-mediated cholesterol efflux in a time- and dose-dependent manner. 22(R)-hydroxycholesterol and 9-cis-retinoic acid did significantly increase cholesterol efflux in THP-1 macrophage foam cells (P<0.05), respectively. Both of them further promoted cholesterol efflux (P<0.01). As expected, liver X receptor (LXR) agonist decreased content of esterified cholesterol in the macrophage foam cells compared with control, whereas only a slight decrease of free cholesterol was observed. LXR activity was slightly increased by oxidized LDL by 12 % at 12 h compared with 6 h. However, LXR activity was increased about 1.8 times at 24 h, and oxidized LDL further increased LXR activity by about 2.6 times at 48 h.. ABCA1 gene expression was markedly increased in cholesterol-loaded cells as a result of activation of LXR/RXR. ABCA1 plays an important role in the homeostasis of cholesterol in the macrophages.

Topics: Alitretinoin; Apolipoprotein A-I; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Cholesterol; DNA-Binding Proteins; Foam Cells; Humans; Hydroxycholesterols; Leukemia, Myeloid; Lipoproteins, LDL; Liver X Receptors; Macrophages; Orphan Nuclear Receptors; Oxidation-Reduction; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Acute leukemia patients with MLL (mixed linage leukemia) rearrangements tend to respond poorly to conventional therapies. We examined differentiation of human myeloid leukemia cells displaying the MLL-AF9 gene, using several differentiation agents. When MOLM-14 cells were treated with all-trans retinoic acid (ATRA) or 1beta,25-dihydroxyvitamin D3, significant induced differentiation was observed. Trichostatin A (TSA), an inhibitor of histone deacetylase, demonstrated enhance effects with ATRA in regard to growth inhibition and differentiation induction in MOLM-14 cells. Pretreatment with TSA before exposure to ATRA displayed increased effect. Based on these findings, combined treatment with ATRA and TSA may be clinically useful in therapy for acute leukemia displaying MLL-AF9 fusion gene.

Topics: Acute Disease; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 9; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Granulocytes; Humans; Hydroxamic Acids; Leukemia, Myeloid; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Translocation, Genetic; Tretinoin; Up-Regulation

Inhibition of phosphodiesterase IV by N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (piclamilast) enhances the myeloid differentiation induced by all-trans-retinoic acid (ATRA), retinoic acid receptor alpha (RARalpha), or retinoic acid receptor X agonists in NB4 and other retinoid-sensitive myeloid leukemia cell types. ATRA-resistant NB4.R2 cells are also partially responsive to the action of piclamilast and retinoic acid receptor X agonists. Treatment of NB4 cells with piclamilast or ATRA results in activation of the cAMP signaling pathway and nuclear translocation of cAMP-dependent protein kinase. This causes a transitory increase in cAMP-responsive element-binding protein phosphorylation, which is followed by down-modulation of the system. ATRA + piclamilast have no additive effects on the modulation of the cAMP pathway, and the combination has complex effects on cAMP-regulated genes. Piclamilast potentiates the ligand-dependent transactivation and degradation of RARalpha through a cAMP-dependent protein kinase-dependent phosphorylation. Enhanced transactivation is also observed in the case of PML-RARalpha. In NB4 cells, increased transactivation is likely to be at the basis of enhanced myeloid maturation and enhanced expression of many retinoid-dependent genes. Piclamilast and/or ATRA exert major effects on the expression of cEBP and STAT1, two types of transcription factors involved in myeloid maturation. Induction and activation of STAT1 correlates directly with enhanced cytodifferentiation. Finally, ERK and the cAMP target protein, Epac, do not participate in the maturation program activated by ATRA + piclamilast. Initial in vivo studies conducted in severe combined immunodeficiency mice transplanted with NB4 leukemia cells indicate that the enhancing effect of piclamilast on ATRA-induced myeloid maturation translates into a therapeutic benefit.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Benzamides; Cell Differentiation; Cell Line, Tumor; Cyclic Nucleotide Phosphodiesterases, Type 4; Drug Synergism; Drug Therapy, Combination; Humans; Leukemia, Myeloid; Mice; Mice, SCID; Neoplasm Transplantation; Pyridines; Retinoids; Second Messenger Systems; Transcription Factors; Transplantation, Heterologous; Treatment Outcome; Tretinoin; Tumor Cells, Cultured

Exogenous expression of the transcription factor Scl (Tal1) in WEHI-3B D+ myelomonocytic leukemia cells interferes with their capacity to respond to all-trans retinoic acid (ATRA) induced differentiation; combination of ATRA with LiCl, however, circumvents the inhibition of differentiation produced by Scl. To gain information on the possible involvement of this transcription factor in the non-responsiveness of acute myelocytic leukemia (AML) patients to ATRA, we compared the endogenous expression levels of Scl and its transcription complex partners [i.e., Rbtn1 (LMO1), Rbtn2 (LMO2), Ldb1, and GATA family proteins] in leukemic blast cells from patients with AML and acute promyelocytic leukemia (APL), and determined the effects of lithium chloride alone or in combination with ATRA on the capacity of blast cells to differentiate during short-term ex vivo culture. Levels of Scl, Rbtn2, GATA1, and Ldb1 expression were comparable in AML and APL blasts, while the levels of expression of Rbtn1, GATA2, and GATA3 were absent or markedly lower in APL cells. Differentiation markers (cell surface myeloid antigens CD11b, CD15, CD14, and CD33) were also analyzed in blast cells. ATRA produced changes in at least one surface antigen differentiation marker in 89% of patient blasts, while LiCl caused such changes in 72% of the leukemic cells of patients. The combination of LiCl and ATRA induced the differentiation of leukemic blasts from 94% of patients. Although the expression of the transcription factors did not act as individual predictors of responsiveness or non-responsiveness to the inducers of differentiation, ATRA or ATRA plus LiCl, the addition of LiCl to ATRA increased the differentiation response over that of ATRA alone in a number of leukemic samples. These findings suggest that the combination of LiCl and ATRA may produce some clinical benefit in the treatment of the myeloid leukemias.

Topics: Acute Disease; Adult; Aged; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow Cells; Cell Differentiation; DNA Primers; DNA-Binding Proteins; Female; Flow Cytometry; Humans; Leukemia, Myeloid; Lithium Chloride; Male; Middle Aged; Proto-Oncogene Proteins; T-Cell Acute Lymphocytic Leukemia Protein 1; Transcription Factors; Tretinoin

CCAAT/enhancer binding proteins (C/EBPs) are a family of factors that regulate cell growth and differentiation. These factors, particularly C/EBPalpha and C/EBPepsilon, have important roles in normal myelopoiesis. In addition, loss of C/EBP activity appears to have a role in the pathogenesis of myeloid disorders including acute myeloid leukemia (AML). Acute promyelocytic leukemia (APL) is a subtype of AML in which a role for C/EBPs has been postulated. In almost all cases of APL, a promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) fusion protein is expressed as a result of a t(15;17)(q22;q12) chromosomal translocation. PML-RARalpha inhibits expression of C/EBPepsilon, whereas all-trans retinoic acid (tRA), a differentiating agent to which APL is particularly susceptible, induces C/EBPepsilon expression. PML-RARalpha may also inhibit C/EBPalpha activity. Thus, the effects of PML-RARalpha on C/EBPs may contribute to both the development of leukemia and the unique sensitivity of APL to tRA. We tested the hypothesis that increasing the activity of C/EBPs would revert the leukemic phenotype. C/EBPalpha and C/EBPepsilon were introduced into the FDC-P1 myeloid cell line and into leukemic cells from PML-RARA transgenic mice. C/EBP factors suppressed growth and induced partial differentiation in vitro. In vivo, enhanced expression of C/EBPs prolonged survival. By using a tamoxifen-responsive version of C/EBPepsilon, we observed that C/EBPepsilon could mimic the effect of tRA, driving neutrophilic differentiation in leukemic animals. Our results support the hypothesis that induction of C/EBP activity is a critical effect of tRA in APL. Furthermore, our findings suggest that targeted modulation of C/EBP activities could provide a new approach to therapy of AML.

Topics: Acute Disease; Animals; Antineoplastic Agents; CCAAT-Enhancer-Binding Protein-alpha; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Division; Humans; Leukemia, Myeloid; Mice; Mice, Transgenic; Phenotype; Transduction, Genetic; Tretinoin; Tumor Cells, Cultured

Therapeutic nucleoside analogue 3-deazauridine (DU) exerts cytotoxic activity against cancer cells by disruption of DNA synthesis resulting in cell death. The present study evaluates whether DU alone at doses 2.5-15 microM or in combination with all trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) is effective against myelogenous leukemia. The data of this study indicate that DU induces dose-dependent cell death by apoptosis in myeloid leukemia cell lines HL-60, NB4, HEL and K562 as demonstrated by cell staining or flow cytometry and agarose gel electrophoresis. 24h-treatment with DU produced dose-dependent HL-60 cell growth inhibition and dose-independent S phase arrest that was not reversed upon removal of higher doses of DU (10-15 microM). Exposition to nontoxic dose of DU (2.5 microM) for 24h followed by RA or dbcAMP and 96 h-cotreatment with DU significantly enhanced RA- but not dbcAMP-mediated granulocytic differentiation. Cell maturation was paralleled with an increase in the proportion of cells in G1 or G2+M phase. We conclude that, depending on the dose or the sequence of administration with RA, an inhibitor of DNA replication, DU triggers a process of either differentiation or apoptosis in myeloid leukemia cells.

Topics: 3-Deazauridine; Apoptosis; Bucladesine; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; Granulocyte Precursor Cells; Granulocytes; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Tretinoin

Retinoic acid induces clinical remission in acute promyelocytic leukemia (APL) by triggering differentiation of leukemia promyelocytes. Here, we have characterized a gene encoding a member of the immunoglobulin superfamily, among novel retinoic acid-induced genes identified in APL cells. This protein, which was named JAML (junctional adhesion molecule-like), contains 2 extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic tail. JAML mRNA is expressed in hematopoietic tissues and is prominently expressed in granulocytes. The fact that JAML protein is localized at the cell plasma membrane in the areas of cell-cell contacts, whereas it is not detected at free cell borders, suggests that JAML is engaged in homophilic interactions. Furthermore, a conserved dimerization motif among JAM members was shown to be important for JAML localization at the cell membrane. Finally, exogenous expression of JAML in myeloid leukemia cells resulted in enhanced cell adhesion to endothelial cells. Altogether, our results point to JAML as a novel member of the JAM family expressed on leukocytes with a possible role in leukocyte transmigration.

Topics: Base Sequence; Cell Adhesion; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Endothelium, Vascular; Gene Expression Regulation; Humans; Junctional Adhesion Molecules; Leukemia, Myeloid; Leukocytes; Molecular Sequence Data; RNA, Messenger; Sequence Analysis; Tretinoin; Tumor Cells, Cultured

Topics: Acute Disease; Adult; Antineoplastic Combined Chemotherapy Protocols; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 9; Female; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Neoplasms, Second Primary; Translocation, Genetic; Tretinoin

The characterisation of leukemic cell autofluorescence during differentiation, induced by 12-O-tetradecanoylphorbol 13-acetate and all-trans retinoic acid, was performed by autofluorescence microspectroscopy and multispectral imaging autofluorescence microscopy. We have found that a dependence exists between the cell autofluorescence pattern and the degree of cell differentiation. When cells differentiate, their autofluorescence emission changes, following the morphological and functional rearrangement of cell structures. A decrease in emission intensity and a different distribution of endogenous fluorophores are observed. Thus, autofluorescence monitoring on living cells is a potentially useful tool for in vitro study of the differentiation processes. Furthermore, different maturation steps can be distinguished on the basis of the cell fluorescence pattern, leading the way to future application of the technique in diagnostics.

Topics: Cell Differentiation; Cell Line, Tumor; Fluorescence; Histocytochemistry; HL-60 Cells; Humans; Immunohistochemistry; In Vitro Techniques; Leukemia, Myeloid; Microscopy, Fluorescence; Photobiology; Tetradecanoylphorbol Acetate; Thymidine; Tretinoin

Topics: Acute Disease; Adult; Dermatologic Agents; Female; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Keratosis; Leukemia, Myeloid; Nipples; Treatment Outcome; Tretinoin

Interactions between hemopoietic cells and the stromal microenvironment or immunoreactive cells are mediated by specific cell surface receptors. The expression of those molecules may alter the adhesive qualities (mobility and homing) as well as immune response behavior of leukemic blasts. L-Selectin (CD62L) is suggested to play a role in the redistribution and homing of hemopoietic progenitor cells to the bone marrow (BM). Down-regulation of L-selectin is responsible for mobilization of blasts from the BM into the circulation and ligation of L-selectin stimulates proliferation of progenitor cells. This could have an influence on the process of leukemia.. We have studied the expression of L-selectin on mononuclear BM cells of 36 acute myeloid leukemia (AML) patients at first diagnosis by FACS analysis using a directly fluorescein isothiocyanate conjugated antibody (clone DRE G56).. On average the patients presented with 88% blasts in the BM. The expression tended to be higher in primary (p) AML compared with secondary (s) AML. L-Selectin was very heterogenously expressed in all FAB groups. Highest expression was found in cases with AML-M4 with four of nine cases presenting with an inv(16) karyotype. Separating our patient cohort in cytogenetic risk groups we could detect a significantly higher expression of L-selectin in cases with a 'good risk' karyotype and a very low expression in cases with a 'bad risk' karyotype (P = 0.037). Comparing patients who achieved remission after double induction therapy (responders) with patients who showed persisting disease (non-responders) we found a higher percentage of L-selectin+ cases or cells in the responder group than in the non-responder group, although the differences were not significant because of only five cases in the 'non-responder' group. Evaluating cut-off points greatest differences in relapse-free survival probabilities were found in patients who presented with > or = 30% L-selectin+ BM cells compared with cases with < 30%: 86% of cases with > or = 30% L-selectin+ cells were still in remission after a mean follow up time of only 8 months compared with only 46% in the group with < 30% L-selectin+ cells.. We can conclude that (i) expression of L-selectin on AML blasts is variable. This reveals the great diversitiy of immunophenotypes in AML and might contribute to identify individual blast phenotypes in order to detect minimal residual disease in remission. (ii) Low L-selectin expression correlates with a bad cytogenetic risk, with a lower probability to achieve remission and with a shorter relapse-free survival time. This might reflect a decreased homing of the blasts to the BM as well as an impaired cytotoxic T-cell reaction against leukemic cells. The expression of L-selectin on leukemic blasts might be influenced by different cytokine therapies (e.g. with interferon alpha) and this might result in an altered hematologic reconstitution after cytotoxic therapies as well as in an altered immunologic recognition of blasts.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Cells; Chromosome Aberrations; Cytarabine; Daunorubicin; Disease-Free Survival; Female; Humans; L-Selectin; Leukemia, Myeloid; Male; Middle Aged; Mitoxantrone; Neoplasm Proteins; Neoplastic Stem Cells; Remission Induction; Risk; Thioguanine; Treatment Outcome; Tretinoin

All-trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase-9 (MMP-9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL-21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP-9 were much higher in NB4 cells than in PL-21 cells. Stimulation with ATRA significantly increased MMP-9 levels approximately three- to fivefold in both PL-21 and NB4-conditioned media. MMP-9 mRNA levels increased in ATRA-treated cells and was almost in parallel with the increase in MMP-9 activity, suggesting that ATRA induced MMP-9 by activating its gene expression. ATRA can induce interleukin 8 (IL-8) in APL cells. IL-8, chemokine for neutrophils and a potent inducer of MMP-9, was also induced by ATRA in PL-21 cells. However, recombinant IL-8 did not induce MMP-9 expression. In addition, a neutralizing antibody against IL-8 did not inhibit ATRA-induced MMP-9 expression in either cell type. These observations suggest that ATRA can induce both MMP-9 and IL-8, but IL-8 is not involved in ATRA-induced MMP-9 expression. As MMP-9 can truncate and activate IL-8, simultaneous induction of MMP-9 and IL-8 by ATRA could activate leucocytes excessively, causing the hyper-inflammatory events in retinoic acid syndrome.

Topics: Antineoplastic Agents; Humans; Interleukin-8; Leukemia, Myeloid; Matrix Metalloproteinase 9; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti-apoptotic genes Bcl-2 and Bcl-X(L) have been implicated in cell cycle regulation, we investigated the expression of these genes in non-proliferating (Q) and proliferating (P) AML and normal CD34+ progenitor cells. Using reverse transcription polymerase chain reaction, Bcl-X(L) and Bcl-2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl-XS and Bax expression was found. Furthermore, the Bcl-X(L)/X(S) but not the Bcl-2/Bax ratio was higher in Q AML compared with normal CD34+ Q cells (P = 0.001). An inverse correlation between Bcl-2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all-trans retinoic acid (ATRA) reduced Bcl-2 and Bcl-X(L) expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara-C). These findings demonstrate overexpression of the anti-apoptotic proteins Bcl-X(L) and Bcl-2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl-2 and proliferation suggests a role for Bcl-2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antigens, CD34; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; bcl-X Protein; beta 2-Microglobulin; Bone Marrow Cells; Cell Division; Cytarabine; Down-Regulation; fas Receptor; Flow Cytometry; Genes, bcl-2; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Tretinoin

An exploratory trial was conducted to evaluate toxicity and potential therapeutic role of all trans-retinoic acid (ATRA) given long-term together with chemotherapy and G-CSF to adult patients with acute myelogenous leukemia (AML).. ATRA was administered orally at 45 mg/m(2)/day on days 1-14 and 25 mg/m(2)/day on days 15-28 of two standard cycles (idarubicin, etoposide, cytarabine, G-CSF) and of up to three high-dose courses (cytarabine, G-CSF). The results obtained in 19 patients enrolled in the ATRA trial were compared with those from 29 comparable cases treated with the same schedule without ATRA, according to patient risk class and an in vitro study.. ATRA was administered for a median of 52 days to the patients selected for study who achieved a remission. ATRA-related toxicity was mostly non-severe apart from high incidence of headache in conjunction with high-dose cytarabine. Complete remission (CR) rate after cycle 1 (54%), kinetics of hematological recovery, postremission treatment realization, disease-free survival (DFS 37.5% at three years) and overall survival (30% at three years) were not different between ATRA-treated and untreated patients. The only significant prognostic factor was the patient risk class, as defined by cytogenetics and other clinical criteria: DFS rate was 57% at three years in standard-risk cases compared to only 19% in the high-risk group, with no influx by ATRA in either category. The in vitro study, in patients with a definite clinical response, failed to document any inhibitory or pro-apoptotic effect of ATRA on AML blast cells.. As a consequence to these results, the pilot ATRA phase was closed. This study does not suggest a significant role for the present ATRA schedule as an adjunct to standard antileukemic therapy in adult AML.

Topics: Acute Disease; Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cytarabine; Disease-Free Survival; Etoposide; Female; Filgrastim; Granulocyte Colony-Stimulating Factor; Headache; Humans; Idarubicin; Leukemia, Myeloid; Life Tables; Male; Middle Aged; Neoplastic Stem Cells; Patient Compliance; Pilot Projects; Recombinant Proteins; Remission Induction; Survival Analysis; Treatment Failure; Tretinoin

Enhancing the pharmacologic activity of all-trans retinoic acid (ATRA) is potentially useful in the management of acute promyelocytic leukemia (APL) and other types of myeloid leukemia. In this report, we identify a novel class of experimental agents selectively potentiating the cytodifferentiating activity of ATRA and synthetic retinoic acid receptor alpha agonists in APL and other myeloid leukemia cell lines. These agents have a bis-indolic structure (BISINDS), and ST1346 is the prototypical compound of the series. Gene-profiling experiments and determination of the level of expression of myeloid-associated markers indicate that ST1346 stimulates many aspects of the granulocytic maturation process set in motion by ATRA. Stimulation of the cytodifferentiating activity of ATRA by ST1346 enhances the efficacy of the retinoid in vivo, as demonstrated in the APL model of the severe combined immunodeficiency (SCID) mouse receiving transplants of NB4 cells. Although the molecular mechanisms underlying the ATRA-potentiating action of ST1346 and congeners have not been completely clarified, bis-indols are not ligands and do not exert any direct effect on the ATRA-dependent transactivation of nuclear receptors. However, ST1346 inhibits the down-regulation of cyclic adenosine monophosphate (cAMP)-dependent CREB transcriptional complexes and enhances the level of expression of signal transducers and activators of transcription-1 (STAT1), 2 putative molecular determinants of the differentiation process activated by ATRA in APL cells. More importantly, ST1346 relieves the down-regulation of Jun N-terminal kinases (JNK) afforded by ATRA. In addition, a specific JNK inhibitor blocks the enhancing effect of ST1346 on ATRA-induced maturation of NB4 cells. This demonstrates an important role for the mitogen-activated protein kinase in the molecular mechanisms underlying the pharmacologic activity of the bis-indol.

Topics: Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Division; Drug Synergism; Drug Therapy, Combination; Gene Expression; Gene Expression Profiling; Humans; Indoles; Leukemia, Myeloid; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Structure-Activity Relationship; Transcription Factors; Tretinoin; Tumor Cells, Cultured

PTPases are key signaling molecules and targets for developing novel therapeutics. We have studied the in vitro biological activity of PTPase inhibitor sodium stibogluconate (SS) on differentiation and proliferation of myeloid leukemia cell lines (NB4, HL-60 and U937). SS (250 microg/ml, 6 days) induced 87% of NB4 cells to reduce nitroblue tetrazolium (NBT), in comparison to the 90% induced by ATRA (1 microM, 6 days). SS treatment of NB4 cells resulted in an increase of CD11b expression and of a morphologically more mature population, coincident with growth arrest at S phase and increased cell death. The effect of SS on NB4 differentiation was irreversible and required continuous drug exposure. SS (400 microg/ml, 6 days) induced 60% and 55% of NBT-positive cells in HL-60 and U937 cell lines, which were augmented in the presence of GM-CSF (25 ng/ml) to levels (85% and 81%, respectively) comparable to those induced by ATRA. SS induced increased tyrosine phosphorylation of cellular proteins in the AML cell lines and inactivated SHP-1 PTPase in NB4 cells, consistent with SS functioning as a PTPase inhibitor in the leukemia cells. These results provide the first evidence of an anti-leukemia activity of SS as a PTPase inhibitor.

Topics: Acute Disease; Antimony Sodium Gluconate; Antineoplastic Agents; Antiprotozoal Agents; Cell Cycle; Cell Differentiation; Cell Division; Gene Expression Regulation, Leukemic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid; Nitroblue Tetrazolium; Oxidation-Reduction; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Tretinoin; Tumor Cells, Cultured; Tyrosine

Resistance to chemotherapy-induced apoptosis and a multidrug-resistance phenotype is the major problem in the treatment of acute myeloid leukemia (AML).. We recently demonstrated that the coexpression of at least two proteins, including P glycoprotein, multidrug resistance-related protein, bcl-2 (flow cytometry), p53 (luminometric immunoassay), and heat shock protein 27 (Western blotting), was predictive for response to induction therapy in de novo AML comparing leukemic blasts of 20 responders with 20 nonresponders. After long-term follow-up, we now present our evaluation on the prognostic significance of these proteins in leukemic blasts of 124 untreated AML patients with regard to the probability of remission (PoR) and overall survival (OS).. Analyzing leukemic blasts obtained from bone marrow samples, we found that no single protein significantly correlated with PoR or OS. In contrast, the coexpression of at least two of these proteins was predictive for reduced OS in univariate as well as multivariate analysis. Although we could not identify any particular protein combination predictive for reduced OS, those patients with no or only one protein expressed in their leukemic blasts had a survival probability of 48% in contrast to 24% in those patients with the coexpression of two or more proteins. Among the clinical markers, only response to chemotherapy had a significant effect on OS and age was of prognostic relevance for PoR.. We conclude that overexpression of only one protein possibly involved in resistance, is not sufficient to influence the prognosis for long-term survival in AML, whereas the expression of more than one protein is predictive for reduced OS. Protein combination seems to be individually different, and targeting only one protein in further clinical trials may not help to overcome multifactorial resistance.

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cytarabine; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Follow-Up Studies; Gene Expression; Genes, bcl-2; Genes, MDR; Genes, p53; Heat-Shock Proteins; Humans; Idarubicin; Leukemia, Myeloid; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Prognosis; Proto-Oncogene Proteins c-bcl-2; Remission Induction; Survival Rate; Treatment Outcome; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Acute myeloid leukemia (AML)-associated chromosomal translocations result in formation of chimeric transcription factors, such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, of which the components are involved in regulation of transcription by chromatin modeling through histone acetylation/deacetylation. The leukemic differentiation block is attributed to deregulated transcription caused by these chimeric fusion proteins, which aberrantly recruit histone-deacetylase (HDAC) activity. One essential differentiation pathway blocked by the leukemic fusion proteins is the vitamin (Vit) D(3) signaling. Here we investigated the mechanisms by which the leukemic fusion proteins interfere with VitD(3)-induced differentiation. The VitD(3)-receptor (VDR) is, like the retinoid receptors RAR, retinoid X receptor, and the thyroid hormone receptor (TR), a ligand-inducible transcription factor. In the absence of ligand, the transcriptional activity of TR and RAR is silenced by recruitment of HDAC activity through binding to corepressors. In the presence of ligand, TR and RAR activate transcription by releasing HDAC activity and by recruiting histone-acetyltransferase activity. Here we report that VDR binds corepressors in a ligand-dependent manner and that inhibition of HDAC activity increases VitD(3) sensitivity of HL-60 cells. Nevertheless, the inhibition of HDAC activity is unable to overcome the block of VitD(3)-induced differentiation caused by PLZF/RARalpha expression. Here we demonstrate that the expression of the translocation products PML/RARalpha and PLZF/RARalpha impairs the localization of VDR in the nucleus by binding to VDR. Furthermore, the overexpression of VDR in U937 cells expressing AML-related translocation products completely abolishes the block of VitD(3)-induced differentiation. Taken together these data indicate that the AML-associated translocation products block differentiation not only by interfering with chromatin-modeling but also by sequestering factors involved in the differentiation signaling pathways, such as VDR in the VitD(3)-induced differentiation.

Topics: Cell Differentiation; Cholecalciferol; Core Binding Factor Alpha 2 Subunit; Histone Deacetylase Inhibitors; Histone Deacetylases; HL-60 Cells; Humans; Leukemia, Myeloid; Neoplasm Proteins; Oncogene Proteins, Fusion; Protein Structure, Tertiary; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RUNX1 Translocation Partner 1 Protein; Signal Transduction; Transcription Factors; Transfection; Translocation, Genetic; Tretinoin

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 microM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.

Topics: Antigens, CD; Antineoplastic Agents; Apoptosis; Butadienes; Cell Differentiation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Granulocytes; Humans; Leukemia, Myeloid; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Monocytes; Nitriles; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Signal Transduction; Tetradecanoylphorbol Acetate; Tretinoin

All-trans retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia. Because ATRA has effects (increase in apoptosis, suppression of bcl-2), it has also been used for the treatment of other French-American-British (FAB) subtypes of acute myelogenous leukemia (AML). To find out the in vivo and in vitro effects of ATRA in AML, we analyzed 37 patients with de novo AML. Twenty-seven patients received ATRA before remission-induction (RI) treatment (ATRA group). Results were compared to a control group (10 patients) that received induction without ATRA during the same time period. Bone marrow or peripheral blood samples were collected from all patients on d 0 and 4. The immunphenotype, myeloperoxidase (MPO), reac tion and the efflux uptake of rhodamine 123 (Rh123) were analyzed on myeloblasts in these samples. In the myeloblasts from patients treated with ATRA, the uptake of Rh123 was increased significantly (p = 0.026) from d 0 to d 4, and all other parameters remained unaltered. ATRA administration increased the complete remission (CR) rate (88%, 22/25 vs 55%, 5/9) significantly (p = 0.042). Logistic regression analysis revealed that ATRA administration was the important factor in CR, among other potential factors including age, white blood count, bcl-2 expression, and the uptake and efflux of Rh123 (p = 0.05). Estimated disease-free survival and overall survival were similar between these two groups (43% vs 37.5% and 51.2% vs 37.5%, respectively). In conclusion, ATRA treatment prior to RI treatment may improve the CR rate in patients with de novo AML, which seems to be related to its beneficial effect on multidrug resistance.

Topics: Acute Disease; Adolescent; Adult; Aged; Antigens, CD; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Case-Control Studies; Cytarabine; Drug Resistance, Multiple; Etoposide; Female; Fluorescent Dyes; Humans; Idarubicin; Immunophenotyping; Leukemia, Myeloid; Leukocyte Count; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Remission Induction; Rhodamine 123; Treatment Outcome; Tretinoin

In this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients.

Topics: Acute Disease; Antigens, CD; Antigens, CD34; Cell Differentiation; Enzyme Inhibitors; Estrenes; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Granulocytes; Hematopoietic Stem Cells; Humans; Interleukin-3; Isoenzymes; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Phospholipase C beta; Protein Isoforms; Pyrrolidinones; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases

A natural product, resveratrol (3,4,40-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other food products, is known as a cancer chemopreventive agent. We studied the in vitro biological activity of this compound by examining its effect on proliferation and differentiation in myeloid leukemia cell lines (HL-60, NB4, U937,THP-1, ML-1, Kasumi-1) and fresh samples from 17 patients with acute myeloid leukemia. Resveratrol (20 microM, 4 days) alone inhibited the growth in liquid culture of each of the 6 cell lines. Resveratrol (10 microM) enhanced the expression of adhesion molecules (CD11a, CD11b, CD18, CD54) in each of the cell lines except for Kasumi-1. Moreover, resveratrol (25 microM, 4 days) induced 37% of U937 cells to produce superoxide as measured by the ability to reduce nitroblue tetrazolium (NBT). The combination of resveratrol (10 microM) and all-trans-retinoic acid (ATRA) (50 nM, 4 days) induced 95% of the NB4 cells to become NBT-positive, whereas <1% and 12% of the cells became positive for NBT after a similar exposure to either resveratrol or ATRA alone, respectively. In U937 cells exposed to resveratrol (25 microM, 3 days), the binding activity of nuclear factor-kappaB (NFkappaB) protein was suppressed. Eight of 19 samples of fresh acute leukemia cells reduced NBT after exposure to resveratrol (20 microM, 4 days). Taken together, these findings show that resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells.

Topics: Antineoplastic Agents, Phytogenic; Cell Adhesion Molecules; Cell Differentiation; Cell Division; Drug Synergism; Humans; Leukemia, Myeloid; NF-kappa B; Resveratrol; Stilbenes; Superoxides; Tretinoin; Tumor Cells, Cultured; Vitis

To clarify the characteristics of de novo acute myeloid leukemia (AML) among the elderly, we reviewed 112 patients over 60 years old (median age 72 years) who were treated at hospitals in Nagasaki Prefecture with a population of 1.5 million between 1987 and 1994. Reclassification of morphological diagnosis revealed that the proportion of M3 was lower but that of M6 and the incidence of cases with trilineage dysplasia (TLD), known as poor prognostic features, were higher in the elderly than in patients less than 60 years old. Similarly, chromosomal data showed a lower frequency of favorable karyotypes such as t(8;21) and t(15;17) in the elderly. The overall survival of all 112 patients was 10.3% at 5 years. Multivariate analysis indicated that good performance status (PS), low WBC at diagnosis, standard dose multi-drug chemotherapy and all-trans retinoic acid (ATRA) treatment for M3 patients, and morphological findings without TLD were significantly correlated with longer survival. Most of the long-term survivors were found among those who received standard dose therapy in this series, although no consensus has been established how to treat elderly AML patients. We propose that a prospective controlled trial is necessary to confirm the role of standard dose chemotherapy for elderly patients with de novo AML.

Topics: Acute Disease; Aged; Aged, 80 and over; Aging; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Female; Humans; Karyotyping; Leukemia, Myeloid; Male; Mercaptopurine; Middle Aged; Prognosis; Treatment Outcome; Tretinoin

Some pyrimidine analogues have been found to induce differentiation of several human myeloid leukemia cells. Newly synthesized heterocyclic pyrimidine derivatives promote neurite outgrowth and survival in neuronal cell lines. In this study, the growth-inhibiting and differentiation-inducing effects of these pyrimidine derivatives on human myeloid leukemia cells were examined.. Several myeloid leukemia cells were cultured with novel heterocyclic pyrimidine derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphologic changes, expression of CD11b, lysozyme activity, and hemoglobin production.. MS-430 (2-piperidino-5,6-dihydro-7-methyl-6-oxo (7H) pyrrolo [2,3-d] pyrimidine maleate) effectively induced HL-60 cells into mature granulocytes. MS-430 activated the mitogen-activated protein kinase (MAPK) of the cells before causing granulocytic differentiation. MAPK activation was necessary for MS-430-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MS-430. MS-430 also induced monocytic differentiation of THP-1, P39/Tsu, and P31/Fuj leukemia cells, but did not affect erythroid differentiation of K562 or HEL cells.. MS-430 potently induces differentiation of some myelomonocytic leukemia cells. This novel synthesized pyrimidine compound shows promise as a therapeutic agent for treatment of leukemia and as a neurotrophic drug.

Topics: Calcitriol; Cell Differentiation; Cell Division; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Granulocytes; HL-60 Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Mitogen-Activated Protein Kinases; Monocytes; Nitroblue Tetrazolium; Pyrimidines; Tretinoin; Tumor Cells, Cultured

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Histocytochemistry; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Male; Mercaptopurine; Methotrexate; Testicular Neoplasms; Tretinoin

Regulators that play an important role in the differentiation and development of plants or invertebrates may also affect the differentiation of human leukaemia cells through a common signal transduction system, and might be clinically useful for treating acute myeloid leukaemia. Cotylenin A has been isolated as a plant growth regulator. We examined the effects of cotylenin A on the differentiation of several myelogenous leukaemia cells, and found that cotylenin A is a potent and novel inducer of the monocytic differentiation of human myeloid leukaemia cells. Cotylenin A induced the functional and morphological differentiation of myeloblastic and promyelocytic leukaemia cells, but did not effectively induce the differentiation of monocytoid leukaemia cells. Cotylenin A-induced differentiation was not affected by several inhibitors of signal transduction, suggesting that this inducer exhibits a unique mode of action.

Topics: Androstadienes; Antineoplastic Agents; Biomarkers; Cell Differentiation; Diterpenes; Enzyme Inhibitors; Flavonoids; HL-60 Cells; Humans; Indoles; Interferon-gamma; Leukemia, Myeloid; Lipopolysaccharide Receptors; Maleimides; Monocytes; Naphthalenes; Plant Growth Regulators; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; U937 Cells; Virulence Factors, Bordetella; Wortmannin

The active form of vitamin D(3), 1,25(OH)(2)D(3), inhibits proliferation and induces differentiation of a variety of malignant cells. A new class of vitamin D(3) analogs, having 2 identical side chains attached to carbon-20, was synthesized and the anticancer effects evaluated. Four analogs were evaluated for their ability to inhibit growth of myeloid leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer cells. All 4 analogs inhibited growth in a dose-dependent manner. Most effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3) (Gemini-19-nor), which has 2 side chains and removal of the C-19. Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more potent than 1,25(OH)(2)D(3) in inhibiting 50% clonal growth (ED(50)) of NB4, HL-60, MCF-7, and LNCaP cells, respectively. Gemini-19-nor (10(-8) M) strongly induced expression of CD11b and CD14 on HL-60 cells (90%); in contrast, 1,25(OH)(2)D(3) (10(-8) M) stimulated only 50% expression. Annexin V assay showed that Gemini-19-nor and 1,25(OH)(2)D(3) induced apoptosis in a dose-dependent fashion. Gemini-19-nor (10(-8) M, 4 days) caused apoptosis in approximately 20% of cells, whereas 1,25(OH)(2)D(3) at the same concentration did not induce apoptosis. Gemini-19-nor increased in HL-60 both the proportion of cells in the G(1)/G(0) phase and expression level of p27(kip1). Moreover, Gemini-19-nor stimulated expression of the potential tumor suppressor, PTEN. Furthermore, other inducers of differentiation, all-trans-retinoic acid and 12-O-tetradecanoylphorbol 13-acetate, increased PTEN expression in HL-60. In summary, Gemini-19-nor strongly inhibited clonal proliferation in various types of cancer cells, especially NB4 cells, suggesting that further studies to explore its anticancer potential are warranted. In addition, PTEN expression appears to parallel terminal differentiation of myeloid cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcitriol; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid; Male; Microtubule-Associated Proteins; Neoplasm Proteins; Phosphoric Monoester Hydrolases; Prostatic Neoplasms; PTEN Phosphohydrolase; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

Although all-trans retinoic acid (ATRA) can restore the differentiation capacity of leukemic promyelocytes, early leukemic myeloblasts are conversely not responsive to ATRA induced granulocytic differentiation. To assess whether this resistance to ATRA is related to an impaired function of the Retinoic Acid Receptor alpha (RARalpha), we performed an analysis of RARalpha expression and transactivation activity, in several myeloid leukemic cell lines, representative of different types of spontaneous acute myeloid leukemias. Our results indicate that a functionally active RARalpha nuclear receptor is expressed in all the analyzed cell lines, regardless of their differentiation capacity following exposure to ATRA. The observation that ATRA treatment is able to induce the expression of retinoic acid target genes, in late- but not in early-myeloblastic leukemic cells, raises the possibility that the differentiation block of these cells is achieved through a chromatin mediated mechanism. Acetylation is apparently not involved in this process, since the histone deacetylase inhibitor trichostatin A, is not able to restore the differentiation capacity of early leukemic myeloblasts. Further investigation is needed to clarify whether myeloid transcription factors, distinct to RARalpha, play a role in the resistance of these cells to ATRA treatment.

Topics: Alkaline Phosphatase; Blotting, Northern; Blotting, Western; Cell Differentiation; Cell Nucleus; Dimerization; DNA; Enhancer Elements, Genetic; Flow Cytometry; Gene Expression; Genetic Vectors; Humans; Hydroxamic Acids; Leukemia, Myeloid; Macrophage-1 Antigen; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Differentiating myeloid cells may become resistant to various apoptotic stimuli. In the present study, dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA) were found to modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis in a time-dependent manner. During the early stages of differentiation, DMSO treatment increased the response of HL-60 cells to tumor necrosis factor alpha; (TNF-alpha), but enhanced responsiveness was lost during later differentiation stages. In contrast, ATRA treatment induced resistance to TNF-alpha-induced apoptosis. HL-60 cells were resistant to Fas-mediated apoptosis but were sensitized by culturing in serum-free conditions. Similar to its effect on TNF-alpha sensitivity, DMSO pretreatment augmented the response to Fas-mediated signaling, which coincided with increased expression of Fas on DMSO-pretreated cells. However, during the later stages of DMSO-induced differentiation, sensitivity to anti-Fas antibody-induced apoptosis declined significantly, although Fas expression was still elevated. The reduced sensitivity to anti-Fas treatment partially correlated with increased Fas-associated phosphatase-1 mRNA expression. Thus, regardless of either Fas up-regulation or potentiation of TNF-alpha-mediated apoptosis during early DMSO-induced differentiation, a slow increase in resistance to apoptosis mediated through these death receptors occurs during DMSO-induced differentiation, which contrasts with the rapid induction of resistance following treatment with ATRA.

Topics: Antibodies, Monoclonal; Antigens, CD; Apoptosis; Carrier Proteins; Cell Differentiation; Culture Media, Serum-Free; Dimethyl Sulfoxide; fas Receptor; HL-60 Cells; Humans; Kinetics; Leukemia, Myeloid; Protein Phosphatase 1; Protein Tyrosine Phosphatase, Non-Receptor Type 13; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type II; RNA, Messenger; Tretinoin; Tumor Necrosis Factor-alpha; Up-Regulation

Cotylenin A, which has a diterpenoid tricarbocyclic skeleton, has been isolated as a plant growth regulator, has been shown to affect several physiological processes of higher plants and have differentiation-inducing activity in several myeloid leukaemia cell lines. We examined the effect of cotylenin A on the differentiation of leukaemic cells that were freshly isolated from acute myeloid leukaemia (AML) patients in primary culture. Cotylenin A significantly stimulated both functional and morphological differentiation of leukaemia cells in 9 out of 12 cases. This differentiation-inducing activity was more potent than those of all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3 (VD3). Treatment with a combination of cotylenin A and VD3 was more effective than cotylenin A or VD3 alone at inducing the monocytic differentiation of AML cells.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Calcitriol; Cell Differentiation; Diterpenes; Drug Synergism; Female; Humans; Lectins, C-Type; Leukemia, Myeloid; Lipopolysaccharide Receptors; Macrophage-1 Antigen; Male; Middle Aged; Plant Growth Regulators; Statistics, Nonparametric; Tretinoin; Tumor Cells, Cultured

Carnosic acid, the polyphenolic diterpene derived from rosemary, is a strong dietary antioxidant that exhibits antimutagenic properties in bacteria and anticarcinogenic activity in various cell and animal models. In the present study, we show that carnosic acid (2.5-10 microM) inhibits proliferation of HL-60 and U937 human myeloid leukemia cells (half-maximal inhibitory concentration = 6-7 microM) without induction of apoptotic or necrotic cell death. Growth arrest occurred concomitantly with a transient cell cycle block in the G1 phase, which was accompanied by an increase in the immunodetectable levels of the universal cyclin-dependent kinase inhibitors p21WAFI and p27Kipl. Carnosic acid caused only a marginal induction of differentiation, as monitored by the capacity to generate superoxide radicals and the expression of cell surface antigens (CD11b and CD14) and receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. However, at low concentrations, this polyphenol substantially augmented (100- to 1,000-fold) the differentiating effects of 1,25-dihydroxyvitamin D3 and all-trans retinoic acid. Furthermore, such combinations of carnosic acid and any of these differentiation inducers synergistically inhibited proliferation and cell cycle progression. These results indicate that carnosic acid is capable of antiproliferative action in leukemic cells and can cooperate with other natural anticancer compounds in growth-inhibitory and differentiating effects.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; Calcitriol; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Diterpenes; Drug Interactions; Flow Cytometry; G1 Phase; HL-60 Cells; Humans; Leukemia; Leukemia, Myeloid; Plant Extracts; Rosmarinus; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; U937 Cells

The differentiation-inducing effect of clinically applicable analogs of deoxyadenosine on myelomonocytic leukemia cells was examined. Monocytoid leukemia cells were more sensitive to the analogs than were erythroid or myeloid leukemia cells based on the inhibition of cell growth and induction of cell differentiation. Monocytoid leukemia cells were highly sensitive to combined treatment with 2'-deoxycoformycin (dCF) and 9-beta-D-arabinofuranosyladenine (Ara A) for inducing cell differentiation. Ara A induced the differentiation of monocytoid leukemia U937 and THP-1 cells at concentrations which were 1/1000-10000 of that at which it induced the differentiation of other cell lines in the presence of dCF. In combination with a low concentration of 1alpha,25-dihydroxyvitamin D3 (VD3), the induction of the monocytic differentiation was greater in monoblastic U937 cells. Adenosine deaminase-resistant analogs such as fludarabine (FLU) and cladribine (CdA) also induced the differentiation of human myelomonocytic leukemia cells, and these analogs synergistically enhanced the differentiation induced by all-trans retinoic acid (ATRA) or VD3. CdA was the most potent analog for inducing the differentiation of myeloid leukemia NB4 and HL-60 cells in the presence or absence of ATRA. These findings indicate that dCF + Ara A and CdA may be effective for the therapy of acute monocytoid and myeloid leukemia, respectively.

Topics: Acute Disease; Adenosine Deaminase Inhibitors; Antimetabolites, Antineoplastic; Calcitriol; Cell Differentiation; Cladribine; Deoxyadenosines; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Monocytes; Neoplasm Proteins; Neoplastic Stem Cells; Pentostatin; Tretinoin; Tumor Cells, Cultured; U937 Cells; Vidarabine

All-trans retinoic acid (ATRA) induces complete remission in acute promyelocytic leukemia (APL or M3). In this study we measured the effect of retinoids alone and in combination with daunorubicin (DNR) on cell growth and apoptosis in blast cells from patients with non-M3 AML. Cells from 21 patients were incubated in 0.2 microM daunorubicin for 1 h or in 1 microM ATRA or 9-cis-RA continuously and in the combinations of DNR with both retinoids. Cell toxicity and apoptosis were analyzed after 96 h. Both ATRA and 9-cis-RA reduced the viability significantly to 86 and 84%, respectively (P = 0.003 for ATRA and 0.02 for 9-cis-RA). The expression of CD34 correlated to a higher sensitivity to ATRA (P = 0.003). When retinoids were added to DNR the mean decrease in viability was 11 percentage points with ATRA (P = 0.003) and nine percentage points with 9-cis-RA (P = 0.02). Apoptosis was induced by both retinoids and the percentage of apoptotic cells was increased from 16% in the controls to 24% with ATRA (P = 0.03) and to 26% with 9-cis-RA (P = 0.04). When the retinoids were added to DNR the apoptotic rate increased from 41% with DNR alone to 51% with ATRA (P = 0.01) and to 49% with 9-cis-RA (P = 0.03). We conclude that ATRA and RA exert a slight but clear cytotoxic and apoptotic effect on AML blast cells after 96 h incubation and that retinoids can have an additive or synergistic effects on cell toxicity when added to daunorubicin.

Topics: Acute Disease; Adenosine Triphosphate; Adult; Aged; Aged, 80 and over; Alitretinoin; Antibiotics, Antineoplastic; Antigens, CD34; Antineoplastic Agents; Apoptosis; Daunorubicin; Drug Synergism; Female; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Male; Middle Aged; Neoplastic Stem Cells; Tretinoin; Tumor Cells, Cultured

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all-trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto-oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA-resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA-sensitive and -resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.

Topics: Acute Disease; Cell Differentiation; Heterogeneous-Nuclear Ribonucleoproteins; HL-60 Cells; Humans; Leukemia, Myeloid; Proto-Oncogene Mas; Ribonucleoproteins; RNA-Binding Protein FUS; Tretinoin

Topics: Acute Disease; Aged; Antineoplastic Agents; Female; Fever; Humans; Leukemia, Myeloid; Respiratory Distress Syndrome; Tretinoin

Alterations in the response of leukaemic cells to apoptosis-inducing stimuli may account for resistance to chemotherapy and treatment failure, either by disruption of the apoptotic pathway itself or by altered DNA repair; quiescent cells and those with disrupted cell-cycle checkpoints may also display decreased apoptosis. Quiescence can be induced by the differentiation of myeloid cells, and this led us to investigate whether the modulation of drug-induced apoptosis associated with differentiation might be a model for quiescence-associated resistance generally. We have demonstrated that resistance to idarubicin-induced apoptosis increased with greater duration of incubation of HL60 and U937 cells with ATRA and 1,25(OH)2 D3 and that this protective effect correlated with the degree of G0/G1 accumulation. In addition, the cytoprotective effects held for other classes of cytotoxic drugs with different mechanisms of action to idarubicin. Prolonged exposure to idarubicin or vinblastine was associated with diminution of the protective effect and re-entry of cells into cycle. The full cytoprotective effect was restored by resupplementation with ATRA or 1,25(OH)2 D3 during exposure to idarubicin, with concomitant persistence of G0/G1 accumulation. Differentiating agents prevented the accumulation of leukaemic cells at the G2/M checkpoint in response to low concentrations of idarubicin. Understanding how differentiating agents modulate these cell-cycle checkpoints, and how quiescent cells evade apoptosis, may allow the development of therapeutic strategies to limit such apoptosis-inhibiting effects and maximise cell kill from chemotherapy.

Topics: Acute Disease; Apoptosis; Calcitriol; Cell Cycle; Cell Differentiation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; G1 Phase; HL-60 Cells; Humans; Idarubicin; Leukemia, Myeloid; Neoplastic Stem Cells; Resting Phase, Cell Cycle; Tretinoin; U937 Cells; Vinblastine

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Topics: Cell Transformation, Neoplastic; Core Binding Factor Alpha 2 Subunit; Histone Deacetylases; Humans; Leukemia; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Oncogene Proteins, Fusion; Peptide Fragments; Protein Binding; Protein Structure, Quaternary; Repressor Proteins; Response Elements; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Transcription, Genetic; Tretinoin

The role of the p53 pathway in apoptosis induced by all-trans-retinoic acid (ATRA) was studied in 5 human acute myeloid leukaemia (AML) cell lines, OU-AML-3, -4, -5, -7 and -8, previously established and characterized by the authors. Although all the cell lines have a wild-type (wt) p53 gene, the protein is in a mutant conformation detectable by the anti-p53 antibody PAb 240. Exposure of the cell lines to 1.0 microM ATRA for 72 h caused induction of apoptosis detectable by morphology and the annexin V assay. The number of apoptotic cells according to the annexin V assay varied from 16 +/- 8% (OU-AML-7) to 61 +/- 4% (OU-AML-3) in ATRA-treated cells, while it was 7 +/- 6% in control cells. Western blotting and flow cytometry showed down-regulation of the p53 protein by ATRA. The conformation of p53 remained unchanged, being detectable in flow cytometry by PAb 240, but not by PAb 1620 (an antibody which only detects p53 in wt conformation). At the same time bcl-2 was down-regulated as shown by Western blotting and flow cytometry, while no induction of bax was observed by ATRA. On the basis of these results, ATRA-induced apoptosis in these AML cell lines is independent of the p53 pathway, although it is associated with the down-regulation of bcl-2.

Topics: Acute Disease; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Flow Cytometry; Gene Expression Regulation, Leukemic; Genes, bcl-2; Genes, p53; Humans; Leukemia, Myeloid; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We reported previously that treatment with all-trans retinoic acid (ATRA) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces differentiation of human myeloblastic leukemia ML-1 cells to granulocytes, whereas treatment with ATRA alone induces practically no differentiation of these cells. To investigate the mechanism of the synergistic effect of these factors, we examined the effect of GM-CSF on retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in ML-1 cells. We reveal that GM-CSF induces the expression of RAR alpha mRNA and protein and stimulates the binding of nuclear proteins to direct repeat 5, a consensus sequence with high affinity for RAR-RXR heterodimers. Furthermore, expression of CD38 mRNA mediated through RAR alpha is induced synergistically by treatment with ATRA + GM-CSF. These results suggest that GM-CSF stimulates transcriptional activity mediated via RAR alpha in ML-1 cells. The induction of RAR alpha by GM-CSF may therefore be a mechanism for stimulation by GM-CSF. The induction of RAR alpha by GM-CSF was also detected in other myeloid leukemia cell lines (THP-1 and KG-1) that showed a synergistic effect similar to that seen in ML-1 cells in response to ATRA + GM-CSF. We also found that GM-CSF induced the expression of RAR alpha in blood cells obtained from patients with acute myeloid leukemia. This activity of GM-CSF may serve as a useful adjunct to differentiation therapy for retinoic acid-nonresponsive leukemias.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Antigens, CD; Antigens, Differentiation; Antineoplastic Agents; Cell Differentiation; Drug Synergism; Gene Expression; Gene Expression Regulation, Leukemic; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Humans; Leukemia, Myeloid; Membrane Glycoproteins; NAD+ Nucleosidase; Nuclear Proteins; Protein Binding; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Retinoid X Receptors; RNA, Messenger; Stimulation, Chemical; Transcription Factors; Tretinoin; Tumor Cells, Cultured

De novo acute myeloid leukemia (AML) with dysplastic features in erythroblasts, granulocytes and megakaryocytes, similar to those in myelodysplastic syndrome (MDS) has been described as AML with trilineage dysplasia (AML-TLD) since 1987. Several reports have suggested that AML-TLD is a subtype of de novo AML in adults and has a poor clinical outcome when treated by conventional chemotherapy. It is not certain whether allogeneic bone marrow transplantation (BMT) brings a favorable outcome for AML-TLD. To evaluate the therapeutic efficacy of allogeneic BMT for AML-TLD, we investigated the clinical data and outcomes of conventional chemotherapy and allogeneic BMT for 118 patients with de novo AML. These patients were registered consecutively for the Japan Adult Leukemia Study Group (JALSG) protocols at our institutes. We treated 28 AML-TLD patients and 90 AML-nonTLD patients with conventional chemotherapeutic protocols. AML-TLD patients did not have a significantly different complete remission (CR) rate (75.0% and 88.4% P = 0.1234), but had a significantly higher relapse rate than AML-nonTLD patients (94.1% and 49.3%, P= 0.0007). The outcome of chemotherapy for AML-TLD was significantly worse than that for AML-nonTLD. The overall survival (OS) and leukemia-free survival (LFS) at 6 years were 9.4% and 0% in AML-TLD group, and 51.9% (P= 0.0017) and 46.3% (P< 0.0001) in AML-nonTLD group, respectively. Meanwhile, among the patients who underwent allogeneic BMT, five of eight AML-TLD patients and eight of 14 AML-nonTLD patients were alive, and three and five patients survived more than 3 years, respectively. These results suggest that allogeneic BMT can improve the outcome for AML-TLD, which is poor when conventional chemotherapy is given alone. Allogeneic BMT before relapse may be the best therapeutic strategy for AML-TLD patients under 50 years of age if a donor is available.

Topics: Adolescent; Adult; Age Factors; Aged; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Transplantation; Cell Lineage; Combined Modality Therapy; Cytarabine; Daunorubicin; Disease-Free Survival; Female; Humans; Japan; Leukemia, Myeloid; Life Tables; Male; Middle Aged; Neoplastic Stem Cells; Remission Induction; Retrospective Studies; Survival Analysis; Transplantation Conditioning; Transplantation, Homologous; Treatment Outcome; Tretinoin

Folate receptor (FR) type beta is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-beta expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-beta expression in vitro in myeloid leukemia cells from patient marrow. FR-beta was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D(3), or transforming growth factor beta. ATRA did not induce FR-beta expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-beta expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-beta promoter-luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-beta promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)alpha, RARbeta, or RARgamma, it appears that the retinoid effect on FR-beta expression could be mediated by ligand binding to RARs alpha, beta, or gamma, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARalpha and RARgamma selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-beta expression. Thus, blocks in the RARalpha-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-beta expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-beta expression in myeloid leukemia cells refractory to retinoid differentiation therapy.

Topics: Antineoplastic Agents; Bone Marrow Cells; Carrier Proteins; Cell Differentiation; Cell Division; DNA, Recombinant; Folate Receptors, GPI-Anchored; Gene Expression Regulation; Glycosylphosphatidylinositols; Humans; Leukemia, Myeloid; Neoplasm Proteins; Phosphatidylinositol Diacylglycerol-Lyase; Promoter Regions, Genetic; Protein Isoforms; Receptors, Cell Surface; Receptors, Retinoic Acid; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured; Type C Phospholipases; Up-Regulation

Inhibitors of the protease of human immunodeficiency virus type 1 (HIV-1) may inhibit cytoplasmic retinoic acid-binding proteins, cytochrome P450 isoforms, as well as P-glycoproteins. These features of the protease inhibitors might enhance the activity of retinoids. To explore this hypothesis, myeloid leukemia cells were cultured with all-trans retinoic acid (ATRA) either alone or in combination with the HIV-1 protease inhibitors indinavir, ritonavir, and saquinavir. Consistent with the hypothesis, the HIV-1 protease inhibitors enhanced the ability of ATRA to inhibit growth and induce differentiation of HL-60 and NB4 myeloid leukemia cells, as measured by expression of CD11b and CD66b cell surface antigens, as well as reduction of nitroblue tetrazolium. Growth of ATRA-resistant UF-1 cells was also inhibited when cultured with the combination of ATRA and indinavir. Moreover, indinavir enhanced the ability of ATRA to induce expression of the myeloid differentiation-related transcription factor C/EBPepsilon messenger RNA in NB4 cells by 9.5-fold. Taken together, the results show that HIV-1 protease inhibitors enhance the antiproliferative and differentiating effects of ATRA on myeloid leukemia cells. An HIV-1 protease inhibitor might be a useful adjuvant with ATRA for patients with acute promyelocytic leukemia and possibly retinoid-resistant cancers.

Topics: CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; HIV Protease Inhibitors; HL-60 Cells; Humans; Immunophenotyping; Indinavir; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Ritonavir; RNA, Messenger; Saquinavir; Tretinoin; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDACIs) have been used to focus on the effects of inducing gene expression through the acetylation of histones which results in chromatin remodeling. The study explored whether HDACIs could induce the expression of costimulatory/adhesion molecules on acute myeloid leukemia (AML) cells, thereby effectively inducing tumor immunity. The expression of CD80, CD86, human leukocyte antigen (HLA)-DR, HLA-ABC, and intracellular adhesion molecule-1 (ICAM-1) was tested in human AML cell lines after the addition of HDACI, sodium butyrate (SB). Generally, increased expression of CD86 was observed by SB treatment in a majority of cell lines, and ICAM-1 was expressed in fewer cell lines. Essentially the same results were obtained using other HDACIs such as FR901228, trichostatin A, and trapoxin A. Quantitation of transcripts of CD86 accompanied with RNA synthesis inhibition assay and nuclear run-on assay revealed that SB up-regulates the CD86 expression transcriptionally. Furthermore, chromatin immunoprecipitation experiments showed that HDACI treatment caused remarkable acetylation on histone H3 and H4 at CD86 promoter chromatin in vivo. In 30 clinical AML samples, CD86 expression was significantly increased (P <.001) by SB treatment, and the expression of HLA-DR and ICAM-1 was moderately increased (P <.05) by SB treatment. Finally, the allogeneic mixed leukocyte reaction (allo-MLR) against HL60 cells pretreated with SB was enhanced 4-fold compared with allo-MLR obtained with non-treated HL60 cells. These results suggest that the immunotherapeutic use of HDACIs may become a novel tool for treatment of AML. (Blood. 2000;96:3847-3856)

Topics: Acetylation; Acute Disease; Antigens, CD; B7-2 Antigen; Butyric Acid; Cell Adhesion Molecules; Cell Differentiation; Chromatin; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Immunophenotyping; Intercellular Adhesion Molecule-1; Interferon-gamma; Leukemia, Myeloid; Lymphocyte Culture Test, Mixed; Membrane Glycoproteins; Promoter Regions, Genetic; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Activation of phospholipase D (PLD) occurs in response to various stimuli and results from the activity of two isozymes, hPLD1 and hPLD2. PLD activity appears to be involved in several myeloid cell processes during their development and activation, including proliferation of myeloblasts in the bone marrow and secretion, phagocytosis and NADPH oxidase activation, essential functions of differentiated neutrophils. The present work studies PLD characteristics, activity and both isozyme expression during maturation and differentiation of myeloid cells by using three different systems: leukemic myeloblasts at different stages of maturation, terminally differentiated neutrophils ex vivo and four human myeloid cell lines, NB4, HL-60, PLB 985 and U937, induced to differentiate with alltrans retinoic acid (ATRA), a cyclic adenosine monophosphate (cAMP) analogue or both agents together. HL-60, a bipotential cell line has also been differentiated along the granulocytic pathway with DMSO and the monocytic pathway with 1,25-dihydroxy vitamin D3. In all these systems, PLD activity increases with maturation and differentiation whatever the inducer used and the granulocytic or monocytic pathways. Increase in basal activity which reflects the expression during development of both hPLD1 and hPLD2 appears to be mainly related to the former isozyme expression. Association of PLD characteristic changes with maturation and differentiation was also confirmed using two NB4 clones resistant to these processes. Comparison between PLD characteristics in myeloblasts during maturation and differentiation ex vivo and in vitro in the different cell lines demonstrated that NB4 induced to differentiate with ATRA represents the best model for further studies on the specific roles of each PLD isoform in various functions of differentiated myeloid cells.

Topics: Cell Differentiation; Cell Line; Cyclic AMP; Humans; Isoenzymes; Leukemia, Myeloid; Phospholipase D; Tretinoin

CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels.

Topics: Blotting, Northern; Blotting, Western; Burkitt Lymphoma; CCCTC-Binding Factor; Cell Differentiation; Cytarabine; Dimethyl Sulfoxide; DNA-Binding Proteins; Down-Regulation; Erythrocytes; Gene Expression Regulation; Humans; Isoelectric Focusing; Leukemia, Myeloid; Leukocytes; Megakaryocytes; Phosphorylation; Proto-Oncogene Proteins c-myc; Repressor Proteins; RNA Processing, Post-Transcriptional; Staurosporine; Tetradecanoylphorbol Acetate; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis-RA) have an important role in many aspects of proliferation and differentiation of hematopoietic cells. They exert their effects by binding to retinoic acid receptors (RARs) and/or retinoid X receptors (RXRs). We studied the effects of novel retinoids on proliferation and differentiation of HL-60 and NB4 myeloid leukemic cells, as well as acute promyelocytic leukemia (APL) cells from patients. RXR-selective SR11345 (Retinoid C) had little ability to inhibit the clonal growth and to induce the differentiation of either HL-60 or NB4 cells. However, SR11276 (Retinoid E), which activated both the RAR and RXR classes, and SR11278 (Retinoid D), which activated the RAR subtypes alpha, beta, and gamma, could inhibit clonal growth of both cell types, as well as leukemic cells from APL patients. The combination of ATRA and either SR11276 or SR11278 additively inhibited APL cell proliferation. SR11302 (Retinoid A), with reported anti-AP-1 activity and no activation of RARs and RXR and SR11363 (Retinoid B), which selectively activated RARbeta and gamma, were inactive. The clonal proliferation of both HL-60 and NB4 cells that were pulse-exposed to 10(-9) mol/L ATRA, SR11276, SR11278, or SR11345 for 3 days, washed, and plated in methylcellulose culture were inhibited by 0%, 51%, 21%, and 1% for HL-60 cells and 43%, 41%, 35%, and 1% for NB4, respectively, compared with nontreated control cells. When the HL-60 cells were pulse-exposed to 10(-9) mol/L of either SR11278 or SR11276, plus 10(-9) mol/L ATRA for 3 days, colony numbers were reduced by 46% and 64%, respectively. Induction of leukemic cell differentiation as determined by the nitroblue tetrazolium (NBT) assay showed that the combination of 10(-7) mol/L of either SR11278 or SR11276 with 10(-7) mol/L ATRA had additive effects on HL-60 cells, NB4 cells, and fresh APL cells. Induction of CD11b expression on both HL-60 and NB4 cells occurs during their differentiation. Expression of this antigen was synergistically augmented by the combination of either 10(-7) to 10(-8) mol/L SR11278 or 10(-7) to 10(-9) mol/L SR11276 with 10(-9) mol/L ATRA compared with either analog alone in HL-60 cells. Expression of the novel myeloid specific transcription factor C/EBPepsilon was increased by SR11278 and SR11276 in both the HL-60 and NB4 cell lines. We conclude that retinoids or combination of retinoids with specificities for both RAR and RXR may marked

Topics: CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Division; Dimerization; DNA-Binding Proteins; Gene Expression; Genes, Reporter; HL-60 Cells; Humans; Leukemia, Myeloid; Nuclear Proteins; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Blockage in myeloid differentiation characterizes acute myeloid leukemia (AML); the stage of the blockage defines distinct AML subtypes (AML1/2 to AML5). Differentiation therapy in AML has recently raised interest because the survival of AML3 patients has been greatly improved using the differentiating agent retinoic acid. However, this molecule is ineffective in other AML subtypes. The CD44 surface antigen, on leukemic blasts from most AML patients, is involved in myeloid differentiation. Here, we report that ligation of CD44 with specific anti-CD44 monoclonal antibodies or with hyaluronan, its natural ligand, can reverse myeloid differentiation blockage in AML1/2 to AML5 subtypes. The differentiation of AML blasts was evidenced by the ability to produce oxidative bursts, the expression of lineage antigens and cytological modifications, all specific to normal differentiated myeloid cells. These results indicate new possibilities for the development of CD44-targeted differentiation therapy in the AML1/2 to AML5 subtypes.

Topics: Acute Disease; Antibodies, Monoclonal; Bone Marrow; Cell Differentiation; Dose-Response Relationship, Drug; Granulocyte Colony-Stimulating Factor; Granulocytes; Humans; Hyaluronan Receptors; Hyaluronic Acid; Leukemia, Myeloid; Lewis X Antigen; Lipopolysaccharide Receptors; Macrophage Colony-Stimulating Factor; Monocytes; Neoplasm Proteins; Oncogene Proteins, Fusion; Respiratory Burst; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Lodgement, proliferation, and migration of leukemic cells within bone marrow (BM) microenvironment involves adhesion of these cells to the BM extracellular matrix molecules fibronectin and laminin. The 67-kDa laminin receptor (67LR) is a nonintegrin protein with high affinity for laminin, which plays a critical role in basement membrane invasion and metastasis of cancer cells. By Western blotting, we documented that 67LR was strongly expressed in myelomonocytic THP1 and histiocytic U937 cells and was weakly expressed in promyelocytic HL-60 cells. In HL-60 cells, 67LR expression almost disappeared after retinoic-induced granulocytic differentiation, whereas it strongly increased after phorbol ester-induced monocytic differentiation. We did not detect 67LR expression in normal BM hematopoietic cells, in precursor-B acute lymphoblastic leukemia, in chronic lymphocytic leukemia, or in chronic myeloid leukemia in chronic phase. By contrast, we detected enhanced 67LR expression in 40% of 53 de novo acute myeloid leukemias (AMLs), which frequently exhibited monocytic or myelomonocytic morphology and expressed CD14 and CD11a (P < 0.05). Using a colorimetric assay, we found that the expression pattern of this receptor corresponded to a higher adhesion to laminin; the adhesion was specific because in vitro addition to laminin-coated wells of recombinant 37-kDa laminin receptor precursor (37LRP), which is the cytoplasmic precursor containing both laminin-binding domains of cell surface 67LR, significantly reduced laminin binding of AML cells. The expression of 67LR on AML cell surface did not correlate with other differentiation and integrin antigens such as CD7, CD13, CD33, CD34, CD11b, CD11c, CD49d, CD49e, CD45RA, and CD45RO. In contrast with 67LR behavior in solid tumors, no statistically significant difference was found between 67LR expression and any hematological characteristic of the disease at diagnosis, nor between 67LR expression and outcome of the disease as measured by complete remission rate, disease-free survival, or overall survival. In conclusion, our results indicate that 67LR expression mediates specific adhesion to laminin and that the detection of this molecule may be a valuable addition to other lineage-associated antigens in identifying monocytic-oriented AML.

Topics: Acute Disease; Adolescent; Adult; Aged; Antigens, CD; Blotting, Western; Cell Adhesion; Cell Differentiation; Cells, Cultured; Female; HL-60 Cells; Humans; Immunophenotyping; Laminin; Leukemia, Myeloid; Male; Middle Aged; Monocytes; Prognosis; Receptors, Laminin; Survival Rate; Tetradecanoylphorbol Acetate; Tretinoin

We have shown previously that granulocytic maturation and differentiation occurred when HL-60 cells and leukemia cells from a patient with acute promyelocytic leukemia (APL) were exposed to all-trans retinoic acid (ATRA) after treatment with a noncytotoxic concentration of vincristine (VCR), suggesting that VCR might have synergistic action with ATRA in the treatment of APL. Leukemic cells obtained from 24 patients with AML were exposed to 20 nM VCR for 1 h, followed by 1 microM ATRA for 6 days. Changes in the expression of myeloid leukocyte antigens were observed using flow cytometry. Differentiation phenotype as determined by the decrease or increase in maturation cell marker was observed in three samples treated with VCR alone, four samples treated with RA alone, and two samples treated with the combination of VCR and RA. The results suggest that treatment using VCR and ATRA may be effective in the differentiation therapy of AML.

Topics: Acute Disease; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Flow Cytometry; Gene Expression; HL-60 Cells; HLA Antigens; Humans; Leukemia, Myeloid; Middle Aged; Phenotype; Tretinoin; Tumor Cells, Cultured; Vincristine

P39/Tsugane is a myelomonocytoid cell line derived from a patient with myelodysplastic syndrome (MDS). The cells readily undergo apoptosis in response to various agents, and the cell line has been suggested as a useful model to study apoptosis in MDS. The aims of the present study were to assess differentiation and apoptosis induced with all-trans retinoic acid (ATRA) and etoposide, to characterize the mode of apoptosis in these two model systems, and to assess the influence of granulocyte colony-stimulating factor (G-CSF), which in combination with erythropoietin has been shown to inhibit apoptosis in MDS. ATRA induced differentiation and apoptosis in a concentration- and time-dependent manner. Differentiated cells were partially rescued (by 50%) from apoptosis with G-CSF. Etoposide induced apoptosis in a concentration- and time-dependent manner, but no signs of preceding maturation or G-CSF rescue were detected. ATRA- and etoposide-induced apoptosis were both mediated through the caspase pathway and were partially blocked with the general caspase inhibitor zVAD-fmk. Simultaneous treatment with G-CSF and zVAD-fmk additively blocked ATRA-induced apoptosis. However, the two pathways differed in terms of substrate cleavage during apoptosis. ATRA-induced apoptosis caused actin cleavage, which was not affected by G-CSF, and Bcl-2 downregulation. Etoposide induced a caspase-dependent cleavage of Bcl-2, while actin remained intact. The Fas system did not seem to play a major role in any of these apoptotic pathways. Our results may provide new tools to study the mechanisms of apoptosis in MDS.

Topics: Actins; Acute Disease; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Blast Crisis; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Cytoskeleton; Erythropoietin; Etoposide; fas Receptor; Granulocyte Colony-Stimulating Factor; Humans; Leukemia, Myeloid; Myelodysplastic Syndromes; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tretinoin; Tumor Cells, Cultured

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

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

Despite preliminary evidence of clinical activity of the putative differentiating agent sodium phenylbutyrate (PB) in the treatment of myeloid neoplasms, it has proven difficult to maintain therapeutic levels of PB above 0.5 mM, well below the ED50 of 1-2 mM. We have studied the impact of combining PB with all-trans retinoic acid (ATRA) on the ML-1 myeloid leukemia cell line. ATRA augmented PB-induced differentiation, cell-cycle arrest, and apoptosis. ATRA augmented PB induction of the myelomonocytic marker CD11b at all doses of ATRA tested (0.0025-1 microM). Although ATRA did not significantly affect the ED50 of PB, the combination of ATRA (1 microM) and PB (0.5 mM) augmented PB-induced CD11b expression eight-fold. Compared to PB alone, this combination of ATRA and PB induced greater cell cycle arrest (S-phase 14% vs 38%; G0/G1-phase cells 72% vs 52%) and greater apoptosis (24% vs 16% by TUNEL assay). Treatment with ATRA (0.5 microM) in combination with PB (0.5 mM) led to significantly greater inhibition of colony formation (4.8% vs 48% inhibition). ATRA combined synergistically with PB to augment CD11b expression and inhibit colony formation. This combination also showed significant interaction in terms of S-phase inhibition. However, this interaction varied as a function of ATRA concentration: antagonistic at low concentrations of ATRA, synergistic at higher concentrations of ATRA. These data suggest that retinoids may significantly augment the cytostatic and differentiating activity of PB, leading to increased potency of the latter drug at clinically achievable doses.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Drug Synergism; Humans; Leukemia, Myeloid; Phenylbutyrates; Tretinoin; Tumor Cells, Cultured

Acute myeloid leukaemia (AML) is a heterogeneous malignant disease in which disease progression at the level of CD34 positive cells has a major impact in drug resistance and relapse. The multi-drug resistance (MDR1) gene product, P-glycoprotein is expressed mainly in CD34 positive AML cells and Bcl-2 is expressed simultaneously with several putative drug resistance parameters in these cells. Bcl-2 over-expression is associated with CD34 positivity, poor response to chemotherapy and reduced overall survival in AML patients. Recently, all-trans retinoic acid (RA) has been reported to enhance cytarabine-induced apoptosis and downregulate Bcl-2 in several human myeloid leukaemia CD34 negative cells. The two CD34 positive human myeloid leukaemia cell lines: KG1 and KGla have the unique feature of expressing significant functional P-glycoprotein. Thus, the efficacy of RA in enhancing cytrabine- and fludarabine-induced apoptosis and overcoming the resistance was examined in both KG1 (CD34+CD7-) and KGla (CD34+CD7+) human myeloid leukaemia cells in the present study. Both cytarabine and fludarabine induced a dose dependent increase in the number of apoptotic cells in both CD34 positive cell types. Interestingly, the cytarabine-induced apoptosis was significantly more than fludarabine-induced apoptosis in both cell types. All-trans RA alone failed to induce apoptosis or inhibit proliferation of either of the two human CD34 positive leukaemia cell types. However, RA enhanced cytarabine- or fludarabine-induced apoptosis and inhibition of proliferation in KG1 CD34+CD7- but not in KGla CD34+CD7+ myeloid leukaemia cells. As single agents, RA, cytarabine and fludarabine reduced Bcl-2 expression in a dose dependent manner in both cell types. Using a quantitative ELISA assay, the Bcl-2 protein concentration was reduced by 86 or 100%, after 72 h of treatment with 10 microM cytarabine or fludarabine, respectively, in both CD34 positive leukaemia cell types. The addition of RA to cytarabine enhanced its induced reduction of Bcl-2 in KG1 CD34+CD7- but not in KGla CD34+CD7+ human myeloid leukaemia cells. Meanwhile, RA failed to augment fludarabine-induced reduction of Bcl-2 in both cell types. In conclusion, the present results suggest a potential role for the combination of RA and cytarabine in the treatment of refractory and/or relapsed AML patients with CD34+CD7- but not CD34+CD7+ blast cells.

Topics: Antigens, CD34; Antimetabolites, Antineoplastic; Apoptosis; Cytarabine; Humans; Leukemia, Myeloid; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured; Vidarabine

Peroxisome proliferator activated receptor gamma (PPARgamma) plays a central role in the process of adipocyte differentiation. This receptor and its heterodimeric partner, retinoid X receptor alpha (RXRalpha), form a DNA-binding complex that regulates transcription of adipocyte-specific genes. Troglitazone, an antidiabetic drug, has recently been identified as a synthetic ligand for PPARgamma. We studied the effects of troglitazone on proliferation and differentiation of normal and malignant hematopoietic cells. Expression of PPARgamma was easily detectable by Western blot analyses in all five myeloid leukemia cell lines. Troglitazone alone (10-5 M) did not induce differentiation in any of the cell lines; however, this compound suppressed the clonal growth (10-75% of inhibition) of all five myeloid leukemia cell lines. Myelomonocytic U937 cells, which were the most responsive to the growth suppressing effects of troglitazone, were arrested in the G1 phase of the cell cycle when cultured with this compound. Simultaneous treatment of myeloid leukemia cell lines with both troglitazone and a ligand that specifically binds either RXR (LG100268), or retinoic acid receptors (RAR, ATRA, ALART1550), or both (9-cis RA) resulted in additive suppression of clonal growth. In summary, our studies showed that troglitazone when combined with a retinoid was a moderately potent inhibitor of clonogenic growth of acute myeloid leukemia cells.

Topics: Alitretinoin; Antineoplastic Agents; Antioxidants; Cell Cycle; Cell Differentiation; Cell Division; Chromans; DNA-Binding Proteins; HL-60 Cells; Humans; Leukemia, Myeloid; Receptors, Cytoplasmic and Nuclear; Superoxides; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin; Troglitazone; Tumor Cells, Cultured; U937 Cells

Among the three major mitogen-activated protein kinase (MAPK) cascades--the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway--retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to activate ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCgamma and PI-3 kinase activation, or the delta205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.

Topics: Cell Differentiation; Enzyme Activation; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, Myeloid; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Tretinoin

The expression of Bcl-2 family members was examined in normal and leukemic hematopoietic cells. Immature hematopoietic progenitor cells (CD34+/33-/13-) did not express Bcl-2 but Bcl-XL, the majority of CD34 cells expressed Bcl-2, Bcl-XL and BAD, and normal promyelocytes (CD34-/33+) lacked expression of both Bcl-2 and Bcl-XL, while leukemic CD34+progenitors and promyelocytes expressed these anti-apoptotic proteins. In AML, Bcl-2 expression was higher on CD34+ than on all AML cells, however, expression of Bcl-2 or Bcl-XL did not predict achievement of complete remission. Surprisingly, low Bcl-2 content was associated with poor survival in a group of patients with poor prognosis cytogenetics. The anti-apoptotic BAD protein was found to be expressed in AML, but was phosphorylated in 41/42 samples. Phosphorylation was found at both sites, Ser 112 and Ser 136. During induction chemotherapy, Bcl-2 levels of CD34 cells increased significantly. In the context of evidence for small numbers of leukemic CD34+ cells expressing very high levels of Bcl-2 prior to therapy, this finding is interpreted as a survival advantage of Bcl-2 overexpressing progenitors and rapid elimination of cells with low Bcl-2. Bcl-2 and Bcl-XL were both expressed in minimal residual disease cells. Downregulation of Bcl-2 mRNA and protein was observed by ATRA and the combination of Ara-C, followed by ATRA, resulted in markedly increased cytotoxicity in HL-60 cells, as compared to Ara-C alone or ATRA followed by Ara-C. Implications of these findings for the development of new therapeutic strategies for AML are discussed.

Topics: Acute Disease; Antigens, CD34; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Carrier Proteins; Cytarabine; Down-Regulation; Flow Cytometry; Gene Expression; Genes, bcl-2; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Neoplasm, Residual; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Survival Rate; Tretinoin; Tumor Cells, Cultured

To explore the relationship among intercellular -SH, caspase, retinoic acid (RA) and arsenic trioxide(As2O3)-induced apoptosis.. The in vitro effect of different thiols compounds, RA and caspase inhibitors on As2O3-induced apoptosis in NB4 and HL-60 cells was studied.. 1. NAC completely blocked, BSO potentiated while MTG, BAL had no effect on As2O3-induced apoptosis. 2. Z-VAD.fmk blocked while Y-VAD.fmk had no effect on As2O3-induced apoptosis. 3. RA and As2O3 showed synergism in HL-60 cells, while showed antagonism in NB4 cell.. 1. As2O3 binds with intracellular -SH, changes signal transduction, selectively activates caspase and causes apoptosis. 2. The regulating effect of RA on As2O3-induced apoptosis depends on cell types.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Caspase Inhibitors; HL-60 Cells; Humans; Leukemia, Myeloid; Oligopeptides; Oxides; Tretinoin; Tumor Cells, Cultured

The novel uracil analog, 6-chloro-5-(2-propenyl)uracil (TI90), inhibited the growth of myeloid leukemia cells and induced morphologic and functional differentiation of the cells. Although TI90 was a weak inducer of differentiation, it greatly enhanced the growth inhibition and differentiation of the leukemia cells previously induced by 1alpha,25-dihydroxyvitamin D3 (VD3) or all-trans retinoic acid (ATRA). TI90 cooperated with VD3 much more effectively than with ATRA in inhibiting cell growth and inducing differentiation. It also decreased the effective concentration of VD3 to the 10(-10) M level. On the other hand, there was no significant synergy between VD3 and the other uracil analogs. TI90 did not affect VD3 metabolism or the number and affinity of VD3 receptors (VDR) in HL-60 cells. Signals from VD3 are predominantly mediated by VDR and the ligand-activated binding of VDR to vitamin D-responsive element (VDRE) as a heterodimer with the retinoid X receptor (RXR). According to the results of a gel shift assay, TI90 enhanced the intensity of the retarded band with synthetic VDRE oligomer in the presence of VD3, suggesting that TI90 increases the number of phosphorylated receptors by inhibiting phosphatase activity, and also stimulates the formation of a functional complex of VDR with RXR.

Topics: Antimetabolites, Antineoplastic; Calcitriol; Cell Differentiation; Cell Nucleus; DNA-Binding Proteins; Growth Inhibitors; HL-60 Cells; Humans; Leukemia, Myeloid; Nitroblue Tetrazolium; Oxidoreductases; Receptors, Calcitriol; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Uracil

Autologous peripheral blood haemopoietic stem cells (PBSC) were harvested from 30 patients with de novo acute leukaemia, 29 of whom had entered remission following standard chemotherapy. Correlation of CD34+ cells/kg to CFU-GM/kg in the harvests was good (correlation coefficient = 0.72, P < 0.001). We demonstrated significant associations between the CFU-GM content of the harvest and the following: time to platelets >50 x 10(9)/l post final induction course (P < 0.001), days to harvest from day 1 of intensification/mobilization (correlation coefficient = -0.73, P < 0.001), platelets >20 x 10(9)/l at time of harvest (P = 0.02), time to WBC >1.0 x 10(9)/l post intensification/mobilization (correlation coefficient = -0.70, P < 0.001), and WBC on day of harvest (correlation coefficient = 0.60, P < 0.001). In contrast, we found no relationship between the CFU-GM content of the harvest and patient age up to 65 years, presence of absence of coexistent features of trilineage myelodysplasia at diagnosis, number of induction courses to remission or total number of courses of chemotherapy prior to intensification/mobilization. Haemopoietic recovery after reinfusion of PBSC was highly correlated to the number of CFU-GM infused (neutrophils >0.5 x 10(9)/l rs = -0.72, P = 0.001; platelets >20 x 10(9)/l unsupported rs = -0.71, P = 0.001). Our results show that the number of induction courses received, and thus exposure to cytotoxic agents received, made no significant difference to subsequent CFU-GM harvest content. We collected superior harvests from those patients with faster platelet recovery following mobilization therapy. We also found that faster platelet recovery following the final induction therapy was a better predictor of the CFU-GM harvest following mobilization than was the neutrophil recovery following final induction.

Topics: Acute Disease; Adolescent; Adult; Antineoplastic Agents, Alkylating; Busulfan; Cyclophosphamide; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Leukocyte Count; Male; Middle Aged; Neutrophils; Platelet Count; Tretinoin

RAS mutations arise at high frequency in human malignancy and have been shown to play a role in the disruption of both normal differentiation and proliferation. In addition, RAS influences a number of intracellular signaling pathways, which impinge on proteins that regulate programmed cell death. In this study, we have examined whether this oncogene can influence the activation of the apoptotic process induced by a range of therapeutic agents used to treat leukemia, and we have identified the downstream targets of RAS mediating the observed changes in sensitivity. Using myeloid leukemia cells (P39) retrovirally transduced with mutant H-RAS, we found that the influence of this oncogene was highly dependent on the inducer used: whereas RAS had no significant effect on spontaneous apoptosis or on the response to the cytotoxic drugs (doxorubicin or 1-beta-arabinofuranosylcytosine), P39-RAS cells showed a strongly augmented response to all-trans-retinoic acid (ATRA) in both the induction of apoptosis and differentiation. Because, under some circumstances, RAF has been associated with promoting apoptosis, we examined whether the activation of this kinase by mutant RAS could be responsible for the augmented response to ATRA. However, constitutive activation of RAF did not alter the apoptotic sensitivity of these cells, making it unlikely that RAS promotes apoptosis by stimulating this kinase. Nor did we find that BCL-2 was differentially down-regulated in P39-RAS cells. Rather, we found that the activation of protein kinase C (PKC) by low-dose phorbol ester could almost entirely recapitulate transformation by RAS, in terms of promoting both apoptosis and differentiation after treatment with ATRA. Moreover, the RAS-induced phenotype could be completely abolished by a specific inhibition of PKC under conditions that had no effect on the response of control cells. In conclusion, we have shown that mutant RAS promotes differentiation-associated cell death in P39 cells by stimulating the activity of PKC, which is itself an important regulator of myeloid differentiation. PKC activation, in turn, powerfully synergizes with the PKC-independent action of ATRA. This work identifies a possible explanation for the ability of this oncogene to promote myeloid differentiation of hematopoietic cells. Clinically, it raises the possibility that although leukemias expressing mutant RAS may not show an altered response to cytotoxic agents, they may show enhanced sensitivity to diff

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cytarabine; DNA Primers; Dose-Response Relationship, Drug; Doxorubicin; Humans; Leukemia, Myeloid; Mice; Mutation; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Transfection; Tretinoin; Tumor Cells, Cultured

The WEHI-3B D+ leukemia is a near-diploid differentiation-competent cell line that undergoes myeloid differentiation in response to retinoic acid. WEHI-3B D- cells, derived from WEHI-3B D+ cells, are near tetraploid and not responsive to the differentiation-inducing properties of the retinoid. To gain information on mechanisms that regulate the maturation of these two cell lines, several multiploid cell lines have been established through fusion of WEHI-3B D+ and WEHI-3B D- cells. Studies with the multiploid cell lines have shown that (a) the cellular growth rate decreases with increased DNA ploidy; (b) near-tetraploid D+/+ cells, obtained by fusing WEHI-3B D+ with WEHI-3B D+ cells, remain differentiation-competent, demonstrating that no direct relationship exists between differentiation competency and DNA ploidy; and (c) near-hexaploid D +/- and D -/+ cells, formed by fusion of WEHI-3B D+ with WEHI-3B D- cells, do not respond to differentiation inducers, suggesting the inhibition of the differentiation machinery of WEHI-3B D+ cells by components from maturation-incompetent WEHI-3B D- cells. The scl transcription factor gene is expressed in WEHI-3B D- cells and is absent in WEHI-3B D+ cells. Overexpression of scl by transfection of scl cDNA in WEHI-3B D+ cells markedly decreased the capacity of retinoic acid to induce differentiation, suggesting that scl functions as a repressor of differentiation in WEHI-3B cell lines.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Division; Cell Fusion; Cell Size; Cholecalciferol; Diploidy; DNA-Binding Proteins; Gene Expression; Granulocyte Colony-Stimulating Factor; Leukemia, Myeloid; Lithium Chloride; Mice; Polyploidy; Proto-Oncogene Proteins; RNA, Messenger; RNA, Neoplasm; T-Cell Acute Lymphocytic Leukemia Protein 1; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces HL-60 cells to differentiate terminally into mature granulocytes, which subsequently die by apoptosis. The biological effects of RA are mediated by two distinct families of transcription factors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs form heterodimers and regulate retinoid-mediated gene expression. We have recently developed a novel RAR-selective antagonist (ER27191) which prevents RAR activation by retinoids. Using this RAR-selective antagonist, and RXR and RAR agonist, we demonstrate the RAR-mediated signaling pathway is important for differentiation and apoptosis of myeloid leukemic cells. Simple activation of RXRs is not sufficient to induce apoptosis of the cells. Interestingly, the combination of the RAR-selective antagonist and 9-cis RA resulted in partial differentiation and apoptosis of HL-60 and NB4 cells, whereas the RAR antagonist completely blocked all-trans RA-induced differentiation and apoptosis of the cells. Additional experiments showed that levels of BCL-2 protein decreased during differentiation of myeloid leukemic cells. Furthermore, HL-60 cells transduced with a bcl-2 expression vector showed the same differentiation response to retinoids as did parental HL-60 cells even though apoptosis was inhibited in these bcl-2-transduced cells, suggesting that differentiation and apoptosis are regulated independently in myeloid leukemic cells.

Topics: Alitretinoin; Anthracenes; Apoptosis; Cell Differentiation; Cell Division; Drug Resistance; HL-60 Cells; Humans; Leukemia, Myeloid; Leukocytes; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Signal Transduction; Tretinoin

Bufalin, a cardiotonic steroid isolated from the Chinese toad venom preparation Chan'su, has differentiation-inducing activity in several myeloid leukemia cell lines. We examined the effect of bufalin on differentiation of leukemic cells from acute myeloid leukemia (AML) patients in primary culture. Bufalin significantly stimulated functional and morphologic differentiation of leukemia cells in four of 20 cases, suggesting that bufalin alone is only a modest inducer of differentiation of AML cells in primary culture. In contrast, acute promyelocytic leukemia (APL) cells showed synergistic differentiation after treatment with all-trans retinoic acid (RA) and bufalin. In some cases, bufalin restored RA sensitivity to previously resistant APL cells. The effective concentration of bufalin for differentiation-inducing activity in APL cells was lower than for its cardiac action. Combined treatment with bufalin and RA may be more effective than RA alone in differentiation therapy of APL.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Bufanolides; Cardiotonic Agents; Cell Differentiation; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Tretinoin; Tumor Cells, Cultured

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myelo

Topics: Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Drug Resistance, Neoplasm; G1 Phase; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid; Microtubule-Associated Proteins; Molecular Structure; Neoplasm Proteins; Nitroblue Tetrazolium; Oxidation-Reduction; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vitamin D

9-cis-Retinoic acid (RA) and peroxisome proliferator activated receptor gamma (PPARgamma) regulates cellular growth and differentiation. In THP-1 cells, a human monocytic leukemia cell line, RA markedly induced PPARgamma1 RNA, nuclear PPARgamma1 protein and suppressed cell growth. The PPARgamma ligand, BRL49653 enhanced RA's growth suppression ability. With BRL49653 alone, THP-1 cell growth was only marginally suppressed. Cell cycle analysis revealed the G1 phase cell population was significantly increased when cells were treated with both ligands. RA induced growth suppression did not differentiate the THP-1 cells to macrophages. Phorbol ester (PMA) induced differentiation of cells to macrophage also induced PPARgamma1 expression, however when RA is given either simultaneously or sequentially to these cells, no further increase in expression of the nuclear receptor was observed. Overall, these data suggest RA induction of PPARgamma1 may block cell growth and may have application for the treatment of proliferative diseases.

Topics: Alitretinoin; Antineoplastic Agents; Cell Division; Humans; Isomerism; Leukemia, Myeloid; Receptors, Cytoplasmic and Nuclear; Transcription Factors; Tretinoin; Tumor Cells, Cultured

In the present study we investigated the effects of various doses of gamma-irradiation, followed by induction of granulocytic differentiation with all-trans-retinoic acid (ATRA), on proliferative rate, differentiation capability and oxidative metabolism of leukaemic cells from two different myeloid leukaemia cell lines, HL-60 and PLB-985. Regarding the effects of such combined treatment on the proliferative capabilities of HL-60 and PLB-985 cell lines, we showed that their growth kinetics were similar after 2 Gy gamma-irradiation combined with ATRA. However, with doses >2 Gy, the behaviour of the cell lines differed largely. Indeed, HL-60 appeared to be more radiosensitive than PLB-985 regarding cell viability and proliferation. Besides, whatever dose of irradiation (2, 5 or 10 Gy) was applied, ATRA was still able to induce differentiation of HL-60 and PLB-985 into granulocytes that retained the capacity to produce superoxide anion. The results of these in vitro studies suggest that leukaemia cell lines retain their ability to respond to ATRA, a granulocytic-differentiating inducer following high doses of irradiation. This may have implications for the use of radiation therapy in combination with ATRA for the treatment of extramedullary infiltrations of myeloid leukaemias in humans.

Topics: Cell Differentiation; Cell Division; Combined Modality Therapy; Gamma Rays; HL-60 Cells; Humans; Leukemia, Myeloid; Tretinoin

Mer-NF8054X is a new type of steroid whose structure has been established as 11-oxo-18, 22-cycloergosta-6, 8(14)-diene-3beta, 5beta, 9beta, 23S-tetraol (an 18, 22-cycloergostane), which has been reported to have antifungal activity against Aspergillus fumigatus. However, other biological activities are unknown. Herein, we reported that Mer-NF8054X inhibited cell growth of HL60 human leukemia cells, when used either singly or in combination with retinoic acid (RA). In addition, Mer-NF8054X alone induced differentiation and apoptosis of HL60 cells. The induction of differentiation of HL60 cells by Mer-NF8054X was synergistic in combination with RA. On the other hand, Emesterone A, an analogue of Mer-NF8054X which is missing a hydroxy residue from the third position, showed much lower activity than Mer-NF8054X on the inhibition of cell growth and the induction of cell differentiation and apoptosis. However, Emesterone B, an analogue of Emesterone A which is missing a hydroxy residue from the fifth position, showed higher activity than Emesterone A but lower activity than Mer-NF8054X when examined for the inhibition of cell growth and the induction of cell differentiation and apoptosis. These results suggested that Mer-NF8054X and its analogs may be a new type of differentiation inducing agent. The hydroxy residue at the third position or fifth position in Mer-NF8054X may be necessary, but not essential, for inhibition of growth and induction of both differentiation and apoptosis of HL60 cells. In addition, Mer-NF8054X enhanced the differentiation of HL60 cells induced by RA. Based on these results, Mer-NF8054X may have utility in the clinic in combination with RA for leukemia patients.

Topics: Antineoplastic Agents; Apoptosis; Ascomycota; Aspergillus; Cell Differentiation; Cell Division; Cell Size; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Fenretinide; HL-60 Cells; Humans; Ketosteroids; Leukemia, Myeloid; Macrophages; Monocytes; Tretinoin

Topics: Antineoplastic Combined Chemotherapy Protocols; Humans; Interferon alpha-2; Interferon-alpha; Leukemia, Myeloid; Recombinant Proteins; Sarcoma; Treatment Outcome; Tretinoin

In a liquid culture system, all-trans retinoic acid (ATRA), alone and in combination with dihydroxylated vitamin D3 (D3) or alpha interferon (alphaIFN) at concentrations achievable in vivo, could significantly suppress the maintenance of non-promyelocytic myeloid leukemia clonogenic cells (CFU-L) in 9/20, 9/18 and 7/11 cases, respectively. That suppression was counteracted only slightly by the addition of 'stem cell factor', a cytokine which promotes CFU-L expansion in vitro. Differentiated cells slightly increased in 5/17 cases only, suggesting the prevalence of anti-proliferative rather than differentiating mechanisms. The present results extend our previous ones and suggest the possible therapeutical value of ATRA+D3 or alphaIFN, even in cases of non-promyelocytic myeloid leukemia.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Cell Differentiation; Cholecalciferol; Humans; Interferon alpha-2; Interferon-alpha; Leukemia, Myeloid; Middle Aged; Recombinant Proteins; Stem Cell Factor; Tretinoin; Tumor Cells, Cultured

The human DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/ref-1) is a multifunctional protein in the DNA base excision repair (BER) pathway that is responsible for repair of apurinic/apyrimidinic (AP) sites in DNA. DNA repair and programmed cell death both function using different mechanisms to protect the organism from the consequences of extensive cellular damage; however, little is known about the relationship of the DNA BER repair pathway to apoptosis. We have determined the relationship of a BER DNA repair enzyme, APE, to apoptosis using the myeloid leukemia cell line HL-60, which can be induced to differentiate down the granulocytic or monocytic/ macrophage pathway. Treatment of HL-60 cells with retinoic acid/DMSO (granulocytic) or phorbol 12-myristate 13-acetate (monocytic) results in apoptosis and in down-regulation of APE expression at both the RNA and protein levels. Moreover, double-labeling experiments using APE immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay for apoptosis demonstrate that individual cells undergoing apoptosis lose expression of APE regardless of their state of differentiation. Blocking apoptosis by overexpression of the bcl-2 proto-oncogene in HL-60 cells or by a bcr-abl-related mechanism in K562 cells and subsequent differentiation results in morphological differentiation but no loss of APE expression. These studies establish that down-regulation of APE expression is associated with programmed cell death in cells of the myeloid lineage.

Topics: Apoptosis; Cell Differentiation; Deoxyribonuclease IV (Phage T4-Induced); DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Down-Regulation; HL-60 Cells; Humans; Leukemia, Myeloid; Lyases; Proto-Oncogene Mas; Proto-Oncogene Proteins c-bcl-2; Tretinoin

The TAL-1 gene specifies a basic helix-loop-helix domain (bHLH) transcription factor, which heterodimerizes with E2A gene family proteins. tal-1 protein is abnormally expressed in the majority of T-cell acute lymphoblastic leukemias (T-ALLs). tal-1 is expressed and plays a significant role in normal erythropoietic differentiation and maturation, while its expression in early myeloid differentiation is abruptly shut off at the level of late progenitors/early differentiated precursors (G. L. Condorelli, L. Vitelli, M. Valtieri, I. Marta, E. Montesoro, V. Lulli, R. Baer, and C. Peschle, Blood 86:164-175, 1995). We show that in late myeloid progenitors (the phenotypically normal murine 32D cell line) and early leukemic precursors (the human HL-60 promyelocytic leukemia cell line) ectopic tal-1 expression induces (i) a proliferative effect under suboptimal culture conditions (i.e., low growth factor and serum concentrations respectively), via an antiapoptotic effect in 32D cells or increased DNA synthesis in HL-60 cells, and (ii) a total or marked inhibitory effect on differentiation, respectively, on granulocyte colony-stimulating factor-induced granulopoiesis in 32D cells or retinoic acid- and vitamin D3-induced granulo- and monocytopoiesis in HL-60 cells. Furthermore, experiments with 32D temperature-sensitive p53 cells indicate that aberrant tal-1 expression at the permissive temperature does not exert a proliferative effect but causes p53-mediated apoptosis, i.e., the tal-1 proliferative effect depends on the integrity of the cell cycle checkpoints of the host cell, as observed for c-myc and other oncogenes. tal-1 mutant experiments indicate that ectopic tal-1 effects are mediated by both the DNA-binding and the heterodimerization domains, while the N-terminally truncated tal-1 variant (M3) expressed in T-ALL malignant cells mimics the effects of the wild-type protein. Altogether, our results (i) indicate proliferative and antidifferentiative effects of ectopic tal-1 expression, (ii) shed light on the underlying mechanisms (i.e., requirement for the integrity of the tal-1 bHLH domain and cell cycle checkpoints in the host cell, particularly p53), and (iii) provide new experimental models to further investigate these mechanisms.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Division; Cholecalciferol; DNA-Binding Proteins; Granulocyte Colony-Stimulating Factor; Helix-Loop-Helix Motifs; HL-60 Cells; Humans; Interleukin-3; Leukemia, Myeloid; Phenotype; Proto-Oncogene Proteins; T-Cell Acute Lymphocytic Leukemia Protein 1; Transcription Factors; Tretinoin

Treatment of human promyelocytic leukemia HL-60 cells with 10 muM curcumin for 48 h inhibited cellular proliferation and induced small increases in differentiation (100-200%) as measured by the proportion of cells that reduced nitroblue tetrazolium (NBT) and expressed Mac-1. Synergistic induction of differentiation as measured by the above markers was observed when 1-10 muM curcumin was combined with 10-100 nM all-trans retinoic acid (RA) or with 100 nM 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3). Cell morphology and flow cytometric studies (with the monocytic surface antigen CD14) indicated that combinations of RA and curcumin stimulated differentiation predominantly to granulocytes whereas combinations of vitamin D3 and curcumin stimulated differentiation predominantly to monocytes. Studies on cell cycle kinetics indicated that treatment of HL-60 cells with a combination of RA and curcumin for 48 or 96 h reduced the proportion of cells in the S phase of the cell cycle and increased the proportion of cells in the G0/G1 phase of the cell cycle to a greater extent than occurred for cells treated with either compound alone. Combinations of vitamin D3 and curcumin did not alter cell cycle kinetics to a greater extent than was observed for either compound alone. Combinations of RA and curcumin or vitamin D3 and curcumin inhibited the proliferation of HL-60 cells to a greater extent than was observed for either compound alone. The results indicate that curcumin is a weak stimulator of differentiation in HL-60 cells and that is has synergistic effects when combined with RA or vitamin D3. Combinations of curcumin and RA have a particularly potent inhibitory effect on the proliferation of HL-60 cells.

Topics: Antigens, Differentiation, Myelomonocytic; Calcitriol; Cell Cycle; Cell Differentiation; Cell Division; Curcumin; Dose-Response Relationship, Drug; Drug Interactions; HL-60 Cells; Humans; Leukemia, Myeloid; Lipopolysaccharide Receptors; Macrophage-1 Antigen; Oxidation-Reduction; Tretinoin

Phorbol 12-myristate 13-acetate (PMA), N,N'-hexamethylenebisacetamide (HMBA) and retinoic acid induce cell differentiation in U-937 promonocytic cells. This report examines the effects of these agents on DNA topoisomerase I activity. A decrease in enzyme activity could be detected as early as 30 min after treatment with all three differentiating compounds and lasted at least 48 h. No alteration in the levels of DNA topoisomerase I transcript or protein was observed during these treatments. The results might be explained by post-translational events that render DNA topoisomerase type I less active.

Topics: Acetamides; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; DNA Topoisomerases, Type I; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Myeloid; Tetradecanoylphorbol Acetate; Transcription, Genetic; 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

IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha.

Topics: Acute-Phase Proteins; Blotting, Northern; Blotting, Western; DNA-Binding Proteins; Gene Expression Regulation; GTP-Binding Proteins; Humans; Interferon-alpha; Interferon-gamma; Interferon-Stimulated Gene Factor 3; Interferon-Stimulated Gene Factor 3, gamma Subunit; Leukemia, Myeloid; Myxovirus Resistance Proteins; Neoplasm Proteins; Proteins; RNA, Messenger; Signal Transduction; STAT1 Transcription Factor; STAT2 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Transcription Factors; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

Management of prostate cancer that either is detectable by prostate specific antigen (PSA) measurements after curative intent or has spread outside of its capsule is a serious problem. Innovative, nontoxic approaches to the disease are required. One approach might be therapy with retinoids. Retinoid activities are mediated by two distinct families of transcription factors: the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 that usually mediates cellular proliferative signals. The RARs require heterodimerization with RXRs. RXRs can form either heterodimers or homodimers; and the latter can bind to DNA response elements that are distinct from those bound by the RAR/RXR heterodimers.. A series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers or RAR/RXR heterodimers, or that selectively inhibit AP-1 activity without activating transcription were evaluated for their ability to inhibit clonal growth of three human prostate cancer cell lines (PC-3, DU-145, and LNCaP).. Several notable findings were: 1) RXR-selective retinoids, such as SR11246, were able to inhibit the clonal growth of prostate cancer cells. In contrast, SR11246 had little effect on clonal growth of myeloid leukemic cells. 2) RAR-selective retinoids also inhibited clonal growth of prostate cancer cells. 3) The retinoid (SR11238) with potent anti-AP-1 activity had no effect on the clonal growth of prostate cancer cells.. This study shows that both RXR- and RAR-selective retinoids are worthy of further study and may be candidates for future clinical trials in prostate cancer.

Topics: Antineoplastic Agents; Cell Division; Dimerization; Humans; Kinetics; Leukemia, Myeloid; Male; Molecular Structure; Prostatic Neoplasms; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Structure-Activity Relationship; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

2'-Deoxycoformycin (dCF), a specific and potent inhibitor of adenosine deaminase, has demonstrated significant antitumor effect on lymphoid malignancies. The drug induced functional and morphologic differentiation of myeloid leukemia cells in combination with 2'-deoxyadenosine (dAd), but not dCF alone. NB4, a cell line derived from a patient with t(15; 17) acute promyelocytic leukemia (APL) underwent granulocytic differentiation when treated with all-trans retinoic acid (ATRA) or dCF plus dAd, but not with cytosine arabinoside. Pre-exposure of NB4 cells to ATRA greatly potentiated differentiation induced by dCF plus dAd, but pretreatment with dCF plus dAd before exposure to ATRA was less effective. Differentiation of NB4 cells was effectively induced by clinically applicable concentrations of dCF in combination with dAd. These findings may provide useful information about induction of differentiation in vivo.

Topics: Antibiotics, Antineoplastic; Cell Differentiation; Cytarabine; Deoxyadenosines; Drug Combinations; Growth Inhibitors; HL-60 Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Pentostatin; Tretinoin; Tumor Cells, Cultured

The block of differentiation in myeloid leukaemia can be overcome by treatment with a variety of agents including cytokines. Interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF) induce macrophage differentiation and growth arrest through activation of the Janus kinase (Jak)/signal transducers and activators of transcription (Stat) signal pathway in murine M1 myeloid leukaemia cells. Treatment of various other myeloid leukaemia lines with LIF or IL-6 did not lead to induction of differentiation. Several defects in the cytokine triggered Jak/Stat signal pathway were striking in these lines. They expressed a decreased or undetectable amount of at least one of the components of the specific cytokine receptor complexes. Three lines contained a constitutively activated Jak/Stat signal cascade and in two of them, lines C and BMC-63, this cascade was inducible by treatment with IL-6, despite of a very low density of IL-6-receptors. Apart from the cytokine receptors, additional components of the Jak/Stat signal cascade were altered in these lines. Expression and activation of the transcription factor Stat5a and the tyrosine kinase Jak2 were markedly decreased compared to M1 cells, suggesting a role of activated Stat5a in the induction of differentiation. These results demonstrate a direct correlation between alterations in the Jak/Stat signal pathway and the inability to differentiate after cytokine treatment of myeloid leukaemia cells.

Topics: Animals; Cell Differentiation; Cell Division; Cell Nucleus; DNA-Binding Proteins; Growth Inhibitors; Interleukin-6; Janus Kinase 2; Leukemia Inhibitory Factor; Leukemia Inhibitory Factor Receptor alpha Subunit; Leukemia, Myeloid; Lipopolysaccharides; Lymphokines; Mice; Milk Proteins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cytokine; Receptors, Interleukin-6; Receptors, OSM-LIF; RNA, Messenger; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Tretinoin; Tumor Cells, Cultured

Human C/EBP epsilon is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBP epsilon, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBP epsilon mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBP epsilon was the only C/EBP family member that was easily detected by RT-PCR. No C/EBP epsilon mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBP epsilon. Northern blot and RT-PCR analyses showed that C/EBP epsilon mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBP epsilon protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBP epsilon protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38-), purified from humans had very weak expression of C/EBP epsilon mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBP epsilon mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBP epsilon, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBP epsilon is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased

Topics: Acute Disease; Alitretinoin; Blotting, Western; Calcitriol; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Line; DNA-Binding Proteins; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; HL-60 Cells; Humans; Leukemia, Myeloid; Monocytes; Neoplasm Proteins; Nuclear Proteins; Oligonucleotides, Antisense; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tretinoin

The relationship between differentiation of human myeloid cells and apoptosis remains unclear. Recent studies have shown that terminal differentiation need not necessarily lead to the apoptotic demise of myeloid cells, while other studies have shown that induction of differentiation is associated with increased resistance to apoptosis-inducing agents, such as chemotherapy and gamma-irradiation. Such results are pertinent to the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome, where differentiating agents and hemopoietic growth factors are being combined with chemotherapy to enhance response and limit toxicity. To elucidate the factors governing apoptosis in human AML blasts, we have studied the cytotoxic effect of idarubicin on HL60, U937 and KG1 cells, after incubation with all-trans retinoic acid (ATRA), 1, 25(OH)2 D3, and granulocyte-macrophage colony-stimulating factor (GM-CSF ). We show that prior incubation of human myeloid leukemic cells with ATRA or 1,25(OH)2 D3 induced resistance to idarubicin-induced apoptosis, which was modulated by coincubation with GM-CSF. The altered chemosensitivity of cells depended on the degree of G0/G1 cell-cycle arrest induced by incubation with ATRA, 1, 25(OH)2 D3, and GM-CSF and was independent of differentiation status or Bcl-2 oncoprotein expression. These findings suggest that cell-cycle arrest in human leukemic cells can be induced by exogenous agents and may promote drug resistance. Determining the mechanisms by which cell-cycle arrest is induced may permit understanding of the processes by which the cells escape cytotoxic drug-mediated apoptosis.

Topics: Acute Disease; Antibiotics, Antineoplastic; Apoptosis; Calcitriol; Cell Cycle; Cell Differentiation; Drug Resistance, Neoplasm; Granulocyte-Macrophage Colony-Stimulating Factor; HL-60 Cells; Humans; Idarubicin; Leukemia, Myeloid; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

Differanisole A, 3,5-dichloro-2-hydroxy-4-methoxy-6-n-propylbenzoic acid, inhibited growth of human myeloid leukemia cells. The compound induced G1 arrest and granulocytic differentiation of HL-60 cells, although the differentiation-inducing effect was modest. Differanisole A and 9-cis retinoic acid (9cisRA) synergistically inhibited the growth and induced functional and morphologic differentiation of HL-60 and NB4 cells, whereas the combined treatment with differanisole A and all-trans retinoic acid or 1alpha,25-dihydroxyvitamin D3 was less effective. Similar results were obtained in primary culture of leukemia cells from a patient with acute promyelocytic leukemia. The synergistic effect on growth inhibition and induction of differentiation required simultaneous treatment with differanisole A and 9cisRA. Differanisole A and an RXR-specific ligand (Ro47-5944) cooperatively inhibited the cell growth, while the combined effect of differanisole A and an RAR-specific ligand Am80 was just additive. Differanisole A in combination with 9cisRA may have implications for therapy of acute promyelocytic leukemia patients.

Topics: Alitretinoin; Antibiotics, Antineoplastic; Calcitriol; Cell Differentiation; Cell Division; Chlorobenzoates; Flow Cytometry; Granulocytes; HL-60 Cells; Humans; Leukemia, Myeloid; Ligands; Receptors, Retinoic Acid; Retinoids; Tetrazolium Salts; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

We examined the effect of vesnarinone, an oral cardiotonic, on the growth and differentiation of human myeloid leukemia cells. Vesnarinone alone markedly induced erythroid differentiation of HEL cells. All-trans-retinoic acid also induced erythroid differentiation of the cells, and the differentiation was greatly enhanced by combined treatment with vesnarinone and retinoic acid. HEL cells are highly resistant to some anticancer drugs, including vincristine, but treatment with vesnarinone greatly increased the sensitivity of HEL cells to vincristine. Enhancement of vincristine sensitivity by vesnarinone was not as significant for other leukemia cells. Expression of P-glycoprotein in HEL cells was effectively inhibited by vesnarinone, suggesting that the restoration of vincristine sensitivity is associated with decrease of P-glycoprotein expression in HEL cells. The plasma level of vesnarinone required to induce differentiation of leukemia cells is 30 micrograms/mL, which could be achieved with oral administration. These results suggest that vesnarinone should be useful in differentiation therapy for some types of myelogenous leukemia.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcium; Cardiotonic Agents; Cell Differentiation; Drug Interactions; Drug Resistance; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Muramidase; Pyrazines; Quinolines; Stimulation, Chemical; Tretinoin; Tumor Cells, Cultured; Vincristine

mcl-1 was identified as an "early-induction" gene that increases in expression during the differentiation of ML-1 human myeloblastic leukemia cells. The mcl-1 gene product proved to be a member of the bcl-2 gene family and, like bcl-2, to have the capacity to promote cell viability. The pattern of expression of mcl-1 has now been characterized, the aim being to determine whether increased expression is consistently associated with differentiation-induction and whether expression is also associated with other changes in proliferative state or cell viability. Expression of the mcl-1 mRNA was found to increase rapidly in ML-1 cells exposed to inducers of monocyte/macrophage differentiation (phorbol esters or lymphocyte conditioned medium), but not cells exposed to an inducer of granulocyte differentiation (retinoic acid). Expression also increased rapidly in response to certain cytotoxic agents (colchicine and vinblastine), but did not increase during serum stimulation or growth-arrest in reduced serum. Increased expression of mcl-1 occurred during the initiation of cell differentiation or death and was not inhibited by cycloheximide, in agreement with the designation of mcl-1 as an early-induction gene. Increased transcription contributed to the increase in expression, and turnover of the mcl-1 mRNA was rapid. These findings suggest that mcl-1 may serve as a modulator of cell viability that can undergo rapid upregulation as well as downregulation, with upregulation harbingering the initiation of cell differentiation or death.

Topics: Base Sequence; Blood; Cell Death; Cell Differentiation; Cell Division; Colchicine; Culture Media, Conditioned; Cycloheximide; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; Lymphocytes; Macrophages; Molecular Sequence Data; Monocytes; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Phorbol Esters; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Vinblastine

Investigating 208 patients with acute haematological malignancies, we found that stem cell factor receptor (SCFR) was expressed on high numbers of blast cells from the vast majority of patients (93%) with refractory anaemia with excess of blasts in transformation. SCFR was also detected in 62% of AMLs, in which it was directly associated to the expression of CD7, interleukin 6 receptor and CD34, and inversely to that of CD11b and CD14. SCFR-positive cases were preferentially represented in AML-M1 (70%) and in AML-M2 (83%) subsets, whereas only 45% of the remaining samples (M3-M4-M5) exhibited SCFR positively. Interestingly, 50% of cases with acute promyelocytic leukaemia expressed SCFR and this molecule was heterogenously regulated by in vitro treatment with all-trans retinoic acid.

Topics: Acute Disease; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Lymphocyte Subsets; Proto-Oncogene Proteins c-kit; Tretinoin

Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All-trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage.

Topics: Acute Disease; Antineoplastic Agents; Base Sequence; Binding Sites; DNA Mutational Analysis; DNA, Neoplasm; Drug Resistance, Neoplasm; Exons; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Molecular Sequence Data; Myelodysplastic Syndromes; Neoplasm Proteins; Neoplastic Stem Cells; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin; Tumor Cells, Cultured

Angelmicin B is a new microbial substance which inhibits src tyrosine kinase activity and oncogenic signal transduction. We investigated the effect of angelmicin B on the proliferation and differentiation of the HL-60 human myeloid leukemia cell line. Angelmicin B caused the dose-dependent inhibition of cell proliferation and induction of differentiation along the myelomonocytic pathway, as determined by morphological changes, nitroblue tetrazolium (NBT) reduction, and non-specific esterase and lysozyme activities at concentrations ranging from 0.1 to 0.5 microgram/ml. Also, it induced significantly the differentiation of mouse myeloid leukemia M1 cells. A similar concentration of angelmicin B inhibited the growth of the myeloid leukemia cell lines K562, HEL, KU812, ML-1, U937 and THP-1, but did not induce differentiation of these cells significantly. The differentiation of HL-60 cells was enhanced by combined treatment with angelmicin B and 1 alpha, 25-dihydroxyvitamin D3 (VD3), retinoic acid or tumor necrosis factor-alpha (TNF alpha). Angelmicin analogs (A1, A2, B, C and D) had almost equivalent effects on the differentiation of HL-60 cells, although angelmicins C and D inhibited src tyrosine kinase activity less than the other analogs. The effective concentrations of angelmicin B in src kinase inactivation was about 100-fold higher than those required for the growth inhibition and differentiation induction. These findings indicate that the differentiation-inducing activity of angelmicins is not associated with their src kinase-inhibiting activity, and may be associated with the modulation of other signal pathway(s).

Topics: Animals; Anthraquinones; Antibiotics, Antineoplastic; Cell Differentiation; Cell Division; Cholecalciferol; HL-60 Cells; Humans; Leukemia, Myeloid; Mice; Monocytes; Nitroblue Tetrazolium; Oncogenes; Protein-Tyrosine Kinases; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Reactive metabolites of benzene (BZ) play important roles in BZ-induced hematotoxicity. Although reactive metabolites of BZ covalently bind to DNA, the significance of DNA adduct formation in the mechanism of BZ toxicity is not clear. These studies investigated the covalent binding of the BZ metabolites hydroquinone(HQ) and 1,2,4-benzenetriol(BT) using the DNA [32P]postlabeling method and explored the potential relationship between DNA adduct formation and cell differentiation in human promyelocytic leukemia (HL-60) cells, a model system for studying hematopoiesis. Maturation of HL-60 cells to granulocytes, as assessed by light and electron microscopy, was significantly inhibited in cells that were pretreated with HQ or BT prior to inducing differentiation with retinoic acid (RA). The capacity of RA-induced cells to phagocytose sheep red blood cells (RBC) and to reduce nitroblue tetrazolium (NBT), two functional parameters characteristic of mature, differentiated neutrophils, was also inhibited in cells pretreated with HQ or BT. These BZ metabolite treatments induced DNA adduct formation in HQ- but not in BT-treated cells. These results indicate that whereas HQ and BT each block granulocytic differentiation in HL-60 cells, DNA adducts were observed only following HQ treatment. Thus DNA adduct formation may be important in HQ but not in BT toxicity.

Topics: Benzene Derivatives; Cell Death; Cell Differentiation; Cell Division; DNA; DNA Adducts; Granulocytes; HL-60 Cells; Humans; Hydroquinones; Leukemia, Myeloid; Mutagens; Phosphorus Radioisotopes; Tretinoin

The bcl-2 protein suppresses apoptosis and the bax protein opposes the cytoprotective effect of bcl-2. A decrease in bcl-2 levels has been implicated in the induction of apoptosis during the terminal differentiation of HL60 myeloid leukaemia cells. We show here that bax protein also declined with a time course similar to the downregulation of bcl-2 following treatment of HL60 with phorbol myristate acetate (PMA), dimethyl sulphoxide (DMSO) or retinoic acid (RA). Decreased bcl-2 protein expression in induced cells was associated with down-regulation of its mRNA. By contrast, the decrease in bax occurred by a post-transcriptional mechanism. Co-ordinate downregulation of bcl-2 and bax proteins may fine-tune the induction of apoptosis during cellular differentiation.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Differentiation; Down-Regulation; Gene Expression Regulation, Developmental; Granulocytes; HL-60 Cells; Humans; Leukemia, Myeloid; Macrophages; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Splicing; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Administration, Oral; Antineoplastic Combined Chemotherapy Protocols; Drug Administration Schedule; Humans; Injections, Intramuscular; Interferon alpha-2; Interferon-alpha; Leukemia, Myeloid; Male; Middle Aged; Recombinant Proteins; Sarcoma; Skull Base Neoplasms; Tretinoin

All trans retinoic acid and vitamin D3 derivatives are well known for their antileukemic activity, while the precise mechanism of this effect remains to be clarified. Using human leukemic U937 and THP-1 promonocytic cell lines, we analyzed the effect of all-trans retinoic acid (RA) and/or 1,25-dihydroxyvitamin D3 (VD) on the generation of nitric oxide (NO), a potent antitumoral mediator. U937 cell differentiation with VD or with both RA and VD (RA/VD) correlated with gene transcription and functional expression of inducible nitric oxide synthase (iNOS). Nitrites and L-citrulline were also detected in U937 cell supernatants as soon as 24 hours following cell incubation with VD or RA/VD, but not in cells treated with RA alone. Inhibition of iNOS activity by NG-monomethyl-L-arginine (LNMMA) significantly decreased in vitro U937 cell differentiation with VD and RA/VD as shown by the expression of cell differentiation markers (CD14 and CD68) and by the capacity of these cells to undergo a luminol-dependent chemiluminescence in response to opsonized zymosan. Similar results were obtained using the THP-1 cell line strengthening the role of NO in the VD- and RA/VD-induced growth arrest and terminal differentiation of promonocytic leukemia cells.

Topics: Antineoplastic Agents; Calcitriol; Cell Differentiation; Drug Synergism; Humans; Leukemia, Myeloid; Nitric Oxide; Nitric Oxide Synthase; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (ATRA) has proven useful in acute promyelocytic leukemia (APL). In order to reduce the side effects and to improve the efficacy of this compound, conventional chemotherapy, and anthracyclines in particular, are frequently added either during remission induction or in consolidation therapy. In this study we aimed at investigating the rationale of the combination of ATRA plus idarubicin in two human leukemic cell lines, HL-60 and K562, that display a different sensitivity to ATRA treatment. The effects of ATRA were compared with those of two clinically active retinoids, 13-cis-retinoic acid (13-cis-RA) and 9-cis-retinoic acid (9-cis-RA). Both in HL-60 and in K562 cells, the majority of the combinations of ATRA and idarubicin were synergistic, while the combinations with 9-cis-RA and 13-cis-RA were more effective in HL-60 and K562 cells, respectively. A 72 h pre-incubation with retinoids was able to further increase the cytotoxicity of ATRA plus idarubicin in the two cell lines. Intracellular idarubicin accumulation was enhanced by retinoids, as demonstrated by a cytofluorimetric method. Our results could contribute to provide a rationale for ATRA plus idarubicin combinations not only in APL but also in acute leukemia of other cytotypes.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Drug Interactions; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Idarubicin; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (ATRA) increases the sensitivity of AML blast cells to cytosine arabinoside (Ara-C) or daunorubicin (DNR) when ATRA is given after drug. We have proposed that down-regulation of bcl-2 is part of the mechanism by which ATRA regulates drug sensitivity. To test this hypothesis cDNA encoding bcl-2 was transfected into cells of the continuous lines OCI/AML-2 and OCI/AML-5. Four transfectant lines were isolated; three contained transfected bcl-2 in the sense orientation (AML5-BCL2sa, AML5-BCL2sb and 2-bcl2) and one with anti-sense bcl-2(AML5-bcl2as). The presence of the transfected gene was demonstrated by Northern blot; translation of the sense transfected genes into protein was demonstrated by Western blotting. Lines with sense-oriented transfected bcl-2 were significantly less sensitive to Ara-C or H2O2 than the parental lines; the cells with anti-sense transfected genes were more sensitive than their parent but the difference did not reach statistical significance. The effect of ATRA on bcl-2 expression was compared in sense-transfected cells and their parents; by Northern blotting it was shown that the endogenous but not the transfected genes were down-regulated after ATRA exposure. The capacity of cells with transfected genes to respond to ATRA was tested by obtaining Ara-C survival curves for ATRA-treated cells. Compared to controls not exposed to ATRA, the transfected cells showed little or statistically insignificant changes in Ara-C sensitivity after ATRA treatment. We conclude that data from the transfectants provides evidence that expression of bcl-2 is a determinant of sensitivity to Ara-C and H2O2; and that the effect of ATRA on sensitivity requires the presence of bcl-2 genes in association with regulatory elements.

Topics: Acute Disease; Antineoplastic Agents; Cell Survival; Cytarabine; Drug Resistance, Neoplasm; Humans; Hydrogen Peroxide; Leukemia, Myeloid; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transfection; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

The mechanisms by which hematopoietic progenitor cells become lineage-committed remain poorly understood. A cloned subline of the AML14 cell line (AML14.3D10) that spontaneously differentiates to eosinophilic myelocytes in the absence of cytokine stimulation was obtained by limiting dilution. This subline exhibits augmented expression of interleukin-5 (IL-5) receptor alpha subunit mRNA and synthesizes all major eosinophil granule proteins. Exposure of this cell line to all-trans retinoic acid (ATRA) causes loss of eosinophilic granules and fast green staining within 48 hours, without cell death. In addition, mRNA for the IL-5 receptor alpha subunit becomes undetectable by 48 hours and the cells lose responsiveness to IL-5. Major basic protein, measured as a marker of eosinophilic granule content, decreases from more than 16 pg/cell to undetectable levels by 5 days after ATRA. Concomitant with the loss of major basic protein and fast green staining, surface expression of CD16 becomes detectable and is maximum by 10 days after ATRA. mRNA for the granulocyte colony-stimulating factor (G-CSF) receptor becomes detectable by day 5, and the cells become responsive to G-CSF. At this time, the cells appear morphologically as mature neutrophils and can reduce nitroblue tetrazolium. With continued culture, the neutrophilic cells die and the culture becomes repopulated with eosinophilic myelocytes. These findings show that it is possible to change the differentiation program of hematopoietic cells even after they show evidence of advanced lineage commitment. The AML14.3D10 subclone of AML14 will be a valuable model for study of the transcriptional regulation of the eosinophil and neutrophil differentiation programs and lineage-specific gene expression.

Topics: Base Sequence; Biomarkers; Blood Proteins; Cell Differentiation; Cell Lineage; Clone Cells; Cytoplasmic Granules; Eosinophil Granule Proteins; Eosinophils; Granulocyte Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Molecular Sequence Data; Neutrophils; Receptors, IgG; Receptors, Interleukin; Receptors, Interleukin-5; Ribonucleases; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, three proteins, AML1a, AML1b and AML1c, are produced by alternative splicings. We previously showed that AML1 is potentially involved in myeloid cell differentiation. Here we analyzed the expression of AML1 in myeloid cell lines. It was revealed that AML1b and AML1c are the major AML1 proteins in these cell lines and that prior to morphological and functional differentiation, their expressions increase in U937 cells when treated with all-trans retinoic acid. These data suggest that the expression of AML1 is associated with myeloid cell differentiation.

Topics: Alternative Splicing; Binding Sites; Bone Marrow Cells; Cell Differentiation; Core Binding Factor Alpha 2 Subunit; DNA-Binding Proteins; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Monocytes; Neoplasm Proteins; Protein Binding; Proto-Oncogene Proteins; RNA, Messenger; Transcription Factor AP-2; Transcription Factors; Tretinoin; Tumor Cells, Cultured

1,25(OH)2D3 and two stereoisomers of retinoic acid, all trans and 9-cis retinoic acid, are regulators of cell proliferation and differentiation. The aim of this study was to evaluate the effects of a combination of 1,25(OH)2D3 and retinoic acid (all trans or 9-cis) on proliferation and cell differentiation of the human promyelocytic leukemia cell line HL60, and to test the reversibility of the induced differentiation. Cell proliferation was inhibited as expected by 1,25(OH)2D3 and all trans retinoic acid alone (IC50 of cell survival was 4 x 10(-7) M, 9 x 10(-6) M and 9 x 10(-7) M for 1,25(OH)2D3, all trans and 9-cis retinoic acid, respectively). Combination of 1,25(OH)2D3 and either form of retinoic acid resulted in a partially additive decrease in cell proliferation. 1,25(OH)2D3 induced a monocytic differentiation (100% CD14+ cells with 10(-7) M 1,25(OH)2D3), while retinoic acid led to a predominantly granulocytic differentiation (36 and 42% CD67+ cells with 10(-6) M all trans and 9-cis retinoic acid, respectively). Additive effects on differentiation were observed upon combination of subtherapeutical doses of the drugs, achieving a mainly monocytic population, demonstrating the dominant role of 1,25(OH)2D3 in determining the direction of differentiation. The effects on proliferation and differentiation of the solitary drugs were reversible, while the proliferation arrest and differentiation induced by the combination persisted and even progressed after withdrawal of the drugs. We conclude that 1,25(OH)2D3 and retinoic acid (all trans or 9-cis) exert additive effects on inhibition of proliferation and induction of cell differentiation of HL60 cells, leading to a persistent differentiation, even after drug withdrawal.

Topics: Antigens, CD; Calcitriol; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; HLA-DR Antigens; Humans; In Vitro Techniques; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

Treatment of HL-60 with phorbol myristate acetate (PMA) for 30 min, or all-trans retinoic acid (RA) for 60 min, results in hyperphosphorylation (3-5x) of topoisomerase II (p170, topo II) in vivo. RA and PMA activate a coprecipitating kinase, respectively inducing 1.6 and 2.7-fold increases in phosphorylation of topo II in immunoprecipitates. The activity of the co-precipitating kinase is inhibited by heparin and unlabelled GTP suggesting that casein kinase II (CKII) is, at least in part, responsible for the topo II hyperphosphorylation in response to differentiation signals. Although following dephosphorylation of the enzyme with alkaline phosphatase there was virtual abrogation of activity, the differentiation associated hyperphosphorylation had little impact on the decatenation activity of topo II in nuclear extracts. There were, however detectable changes in topo II function in vivo which affected the formation of the etoposide stabilised cleavable complex, but only after PMA treatment. PMA resulted in a rapid reduction in etoposide induced cleavage, 30 min treatment with PMA reducing cleavage by 20%. However, treatment with RA for 1 or 2 h when hyperphosphorylation was maximal did not affect cleavage. Immunoband depletion assays suggested that differentiation associated changes in chromatin structure rather than alterations in the enzyme per se are responsible for the reduction in cleavable complex formation following PMA treatment. Etoposide cytotoxicity was significantly reduced following just 30 min PMA treatment, but not reduced and even possibly enhanced by retinoic acid treatment. These findings are relevant not only to the dissection of the role of topo II in differentiation but also to its exploitation as a therapeutic target.

Topics: Antigens, Neoplasm; Casein Kinase II; Cell Differentiation; Cell Nucleus; Cell Survival; DNA Topoisomerases, Type II; DNA-Binding Proteins; Enzyme Activation; Etoposide; Humans; In Vitro Techniques; Isoenzymes; Leukemia, Myeloid; Peptide Mapping; Phorbol Esters; Phosphorylation; Protein Serine-Threonine Kinases; Time Factors; Tretinoin; Tumor Cells, Cultured

The human promyelocytic cell line HL-60 can be differentiated with retinoic acid (RA) along the granulocytic pathway. Numerous studies have identified many synergistic combinations of RA with cytostatics, cytokines and other inducers. A combination of RA with the crude thymus extract Thymex-L increased differentiation of HL-60 cells as confirmed by two functional assays and morphology, whereas the extract itself did not show any effect. The functional markers phagocytosis-associated chemiluminescence and nitroblue tetrazolium reduction were more enhanced (up to 4-fold with 1000 micrograms/ml Thymex-L) than morphology. The effect was found over a wide RA concentration range (10(-11) - 10(-6) M) and was dependent on extract concentration. The half-maximal induction of both functional markers was reached at 400 micrograms/ml. To achieve the same effect with the combination in comparison with RA alone, an RA dose reduction of about 100-fold was estimated. The effect was also seen when the cells were pretreated with the thymus extract for two days. The enhancement of RA action by Thymex-L was not correlated with an increase of extracellular or intracellular RA concentration. The active compound in Thymex-L is heat stable and bigger than 5 kDa as confirmed by gelfiltration. The defined thymus peptides thymosin alpha 1, prothymosin alpha 1 and thymopentin were unable to synergistically enhance HL-60 differentiation. These data suggest that the treatment with a thymus extract can increase the sensitivity of HL-60 cells for RA. This may have clinical implications.

Topics: Cell Differentiation; Drug Synergism; Humans; Leukemia, Myeloid; Thymus Extracts; Tretinoin; Tumor Cells, Cultured

The role of p21 RAS in the proliferation and differentiation of myeloid cells has been studied by analysing the changes in the level of expression of p21 RAS proteins by flow cytometry upon differentiation down the granulocytic and monocytic pathways. Differentiation resulted in upregulated p21 RAS expression despite a marked decline in the number of dividing cells. On the other hand, growth inhibition, without differentiation, resulted in a decline in expression. Cell cycle analysis showed that the increase in p21 RAS occurred throughout the cell cycle. These results suggest that p21 RAS has a role in the process of myeloid differentiation.

Topics: Cell Cycle; Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Genes, ras; Hematopoiesis; Humans; Leukemia, Myeloid; Proto-Oncogene Proteins p21(ras); Tetradecanoylphorbol Acetate; Tretinoin

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.

Topics: 3T3 Cells; Animals; Binding Sites; Cell Differentiation; Dimethyl Sulfoxide; Dimethylformamide; DNA, Recombinant; Gene Expression Regulation, Neoplastic; Globins; Homeodomain Proteins; Humans; Leukemia, Myeloid; Macrophages; Membrane Glycoproteins; Mice; NADPH Oxidase 2; NADPH Oxidases; Neutrophils; Nuclear Proteins; Promoter Regions, Genetic; Protein Binding; Repressor Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Transformation, Genetic; Tretinoin; Tumor Cells, Cultured

Among the Retinoic Acid (RA) derivatives, retinamides, and in particular N-(4-hydroxyphenyl) retinamide (4-HPR), are currently being investigated in selected cases of cancer chemoprevention. The cellular target range, however, seems to be limited, as cells of hemopoietic origin are virtually incapable of terminal differentiation upon addition of the compound. We have reconsidered the effect of 4-HPR on HL-60 cells by taking advantage of a mutant clone, generated in our laboratory, unresponsive to RA but highly responsive to dimethylsulfoxide (DMSO). We show here that this clone, upon addition of 4-HPR, although unable of undergoing full differentiation, shows considerable reduction of clonal growth. Moreover, the combination of 4-HPR and RA resulted in a much greater effect than the administration of 4-HPR alone. We suggest that 4-HPR and RA, at least in terms of mediating growth inhibition, may follow different metabolic pathways.

Topics: Animals; Cell Differentiation; Cell Division; Clone Cells; Dimethyl Sulfoxide; Drug Resistance; Drug Screening Assays, Antitumor; Fenretinide; Flow Cytometry; Leukemia, Myeloid; Mice; Phenotype; Tretinoin; Tumor Cells, Cultured

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

We studied 18 patients with acute promyelocytic leukaemia and 13 with relapsed APL. We found a significantly elevated EGF in acute leukaemia, especially in APL, being 418.59 +/- 19.2 micrograms in the 24-h urine that was much higher than that of the normal controls. When eight APL patients achieved complete remission by RA treatment, the EGF value decreased to 149.9 +/- 27.3 micrograms in the 24-h urine near to normal. In 13 patients with relapsed APL, EGF rose to 446.9 +/- 82.6 micrograms in the 24-h urine. Most interestingly, this elevated EGF could be detected before the relapse by 5 +/- 0.84 months in seven out of eight APL with relapse. We suggest that the unaccountably elevated EGF during remission period may be an indicator of the occurrence of relapse.

Topics: Acute Disease; Adult; Aged; Base Sequence; Biomarkers, Tumor; Cell Differentiation; Epidermal Growth Factor; Female; Humans; Immunologic Factors; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm, Residual; Oncogene Proteins, Fusion; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Remission Induction; Tretinoin

ATRA is extremely effective for inducing clinical remission in APML. The presence of PML/RAR-alpha fusion gene produced as a result of the unique chromosomal translocation in APML is a marker of the sensitivity to ATRA therapy. Further research is needed to elucidate the mechanisms by which the development of the fusion protein in APML leads to the arrest of myeloid differentiation. ATRA leads to rapid resolution of the coagulopathy associated with APML. There is a major clinical benefit since coagulopathy often causes early fatal hemorrhage. The effectiveness of ATRA in APML can serve as a paradigm for the use of differentiation therapy in human malignancies.

Topics: Acute Disease; Blood Coagulation; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Myelodysplastic Syndromes; Tretinoin

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in many eukaryotic cell lines. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. The regulatory subunits of cyclic AMP-dependent protein kinase are retinoylated in the human myeloid leukemia cell line HL60 (Takahashi, N., Liapi, C., Anderson, W. B., and Breitman, T. R. (1991) Arch. Biochem. Biophys. 290, 293-302), and cytokeratins are retinoylated in normal human keratinocytes (Takahashi, N., Jetten, A. M., and Breitman, T. R. (1991) Biochem. Biophys. Res. Commun. 180, 393-400). We show, in this study, that the intermediate filament protein vimentin is retinoylated in HL60 cells during a 24-h exposure to 100 nM [3H]RA. We found that a retinoylated protein of M(r) 55,000 coeluted on anion exchange chromatography and comigrated on either one- or two-dimensional polyacrylamide gel electrophoresis with a protein that also was stained on immunoblots by an anti-vimentin antibody. About 50% of the [3H]RA was released from this M(r) 55,000 retinoylated protein after hydrolysis with either NH2OH (1 M, pH 10) or CH3OH, 0.1 M KOH. These results indicated that a large fraction of the RA was bound to vimentin by an ester bond. Both the M(r) 55,000 retinoylated protein and immunoreactive vimentin were associated with cell nuclei isolated by two procedures. They were detached during exposure to a nonionic detergent buffer, suggesting that they are bound to the nuclear envelope. These results indicate that retinoylation is a new modification of vimentin that may be an early event in RA-induced differentiation of HL60 cells.

Topics: Cell Differentiation; Chromatography, Ion Exchange; Electrophoresis, Gel, Two-Dimensional; Humans; Leukemia, Myeloid; Proteins; Rosaniline Dyes; Staining and Labeling; Tretinoin; Tumor Cells, Cultured; Vimentin

Several human myeloid leukemia cell lines growing in vitro can be induced to differentiate to more mature monocyte/macrophage-like cells by treatment with protein kinase C-activating phorbol esters, such as PMA. In addition to PMA, cells of the THP-1 myeloid leukemia cell line acquire macrophage-like characteristics after treatment with all-trans retinoic acid (RA). To analyze the signal transduction mechanisms induced by RA, we first compared the effects of PMA and RA on the expression of genes which are known to be regulated during monocytic differentiation. Both RA and PMA effectively down-regulated c-myc expression, while c-myb expression decreased only after PMA treatment. Expression of the beta 2-integrin genes, CD11a and CD11b, was clearly increased after both of these treatments. Their effects on the src-family tyrosine kinase genes were different: hck expression was similarly induced by these agents but lyn expression was stronger and more rapid after RA treatment. RA also enhanced lyn mRNA production rapidly in HL-60, indicating that the activation of lyn gene expression is common in monocytic and granulocytic maturation of myeloid leukemia cells. To examine whether the AP-1 enhancer activity is involved in RA-induced monocytic differentiation, THP-1 cells were transiently transfected with a chloramphenicol acetyl transferase (CAT)-reporter gene containing 5 copies of the AP-1 binding sites. In contrast to PMA, RA did not induce any CAT activity in these cells, thus suggesting that the RA-induced changes in the expression of those genes described above were not dependent on the AP-1 enhancer activity.

Topics: CD18 Antigens; Cell Differentiation; Evaluation Studies as Topic; Gene Expression; Genes, myc; Humans; Integrins; Leukemia, Myeloid; Models, Biological; Monocytes; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myb; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin; Tyrosine

The intermediate filament proteins which include vimentin, desmin, and the keratins are one of three major classes of cytoskeletal proteins in eukaryotic cells. In this study we found that most of the vimentin of undifferentiated HL60 and cells induced to differentiate either along the monocytoid pathway by 12-O-tetradecanoylphorbol-13-acetate (TPA) or along the granulocytic pathway by retinoic acid was soluble in a buffer containing 1% Triton X-100/0.6 mol/l KCl in which the intermediate filament proteins usually are not soluble. HL60 vimentin separated on polyacrylamide gel electrophoresis into two proteins of Mr 55,000 and 54,000 that we detected by immunoblotting. The Mr 55,000 species was the major form in undifferentiated HL60 cells and cells induced by retinoic acid. The distribution of both forms of vimentin changed during induction of differentiation by TPA and after 24 h the Mr 54,000 species was predominant. After an additional 24 h exposure to TPA the relative levels of the two forms of vimentin approached equivalence and a high level of vimentin degradation products was seen. These results suggest that TPA may increase vimentin degradation along a pathway that has a Mr 54,000 intermediate. In addition, the high levels of soluble vimentin in HL60 cells suggests that these cells may be a good model for studying components involved in vimentin assembly.

Topics: Antibodies, Monoclonal; Cell Differentiation; Desmin; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Keratins; Leukemia, Myeloid; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin; Tumor Cells, Cultured; Vimentin

The human myeloid leukemia cell lines HL-60, U-937, and THP-1 were used to analyze the alterations of transforming growth factor beta (TGF-beta) during hematopoietic cell growth and differentiation. Differentiation of these cell lines was induced by the phorbol ester phorbol 12-myristate 13-acetate, tumor necrosis factor alpha or by retinoic acid. Northern hybridization analyses indicated that the basal levels of TGF-beta 1, latent TGF-beta binding protein, and type II TGF-beta receptor (T beta IIR) mRNAs were low in untreated cells. Major increases of these mRNAs were observed in cells treated with phorbol 12-myristate 13-acetate, with maximal induction after 12-72 h of stimulation. Retinoic acid and tumor necrosis factor alpha elevated significantly only the expression of T beta IIR mRNA. TGF-beta 1 induced its receptor mRNA in HL-60 and U937-1SF cells but not in THP-1 cells. These changes in gene expression were related to the differentiation of myeloid leukemia cells. Affinity labeling with 125I-TGF-beta 1 indicated that type I TGF-beta receptor was coregulated with T beta IIR. Types I and II receptors were coprecipitated by T beta IIR-specific antibodies. Differentiation of myeloid cells induced secretion of latent TGF-beta 1 protein, as shown by immunoblotting, but significant changes in the levels of active TGF-beta were not observed. These results indicate that the genes involved in TGF-beta signal transduction are coordinately up-regulated during myeloid differentiation.

Topics: Amino Acid Sequence; Animals; Antibodies; Base Sequence; Cell Differentiation; Cell Line; Cloning, Molecular; DNA Primers; Fibrosarcoma; Gene Expression; Humans; Kinetics; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Lung; Mink; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

In the present study we investigated the membrane expression of selectin ligands (CD15/Le(x), CDw65/VIM2, CD15s/sLe(x), beta 2 integrins (CD11a/LFA-1, CD11b/Mac-1) and CD45 phosphatase isoforms (CD45RA, CD45O) on leukaemic cells from 28 patients with acute myeloid malignancies cultured with and without all-trans retinoic acid (ATRA). Within each adhesion system. ATRA was able to differentially regulate distinct molecules. Furthermore, it was able to exert effects specific for acute promyelocytic leukaemia (APL) blast cells, as well as to induce a series of non-cytotype-restricted phenotypic changes. An impressive feature of ATRA induction was a simultaneous increase in the expression of CD15, CDw65 and CD11b on leukaemic promyelocytes. The sialylated antigen CD15s, however, showed results independent from the other two carbohydrates (CD15 and CDw65), suggesting a differential enzymatic regulation within the selectin ligands system. In spite of the well-recognized expression of CD11a throughout all stages of normal myeloid differentiation, APL blast cells were found to virtually lack LFA-1 expression. Moreover, ATRA was unable to promote an up-regulation of this antigen in APL, while inducing a frequent down-modulation in non-APL cases constitutively expressing this antigen. In APL cases ATRA generated an asynchronous phenotype (CD15+, CDw65+, CD11b+, CD11a-), undetectable on normally maturing myeloid cells, but consistent with the concept that incomplete differentiation, in terms of surface molecule expression, can be sufficient to obtain therapeutic results.

Topics: Acute Disease; Antigens, CD; CD11 Antigens; Cell Adhesion Molecules; Cell Differentiation; Humans; Leukemia, Myeloid; Lewis X Antigen; Neoplasm Proteins; Tretinoin

Myelodysplastic syndrome (MDS)-derived leukemia cell line P39/Tsugane could be induced to apoptosis by a variety of agents including metabolic inhibitors, a calcium ionophore and differentiation-inducing agents. As evaluated by characteristic morphological changes and oligonucleosomal lengths DNA ladder, the levels of apoptosis in P39 cells induced by actinomycin D, or A23187, were far greater than in other myeloid lines examined in this study. When 22-oxa-1 alpha, 25(OH)2D3 (D3), dimethyl sulfoxide (DMSO) and all-trans retinoic acid (RA) were used as differentiation-inducers, varying degrees of apoptosis were seen. D3 induced monocytoid differentiation, but not apoptosis above the control level. On the other hand, RA induced profound apoptosis concomitant with the progressive expression of differentiation markers. Studies on morphology, functions and phenotypes of P39 cells exposed to differentiation inducers suggest that the incidence of apoptosis was not affected by the process of differentiation, but cells in the process of varying degrees of differentiation may die via apoptosis. Moreover, RA-treated P39 cells are unique in the simultaneous occurrence of profound apoptosis and differentiation. We propose that RA-treated P39 differentiation model is ideally suited for the study of MDS.

Topics: Apoptosis; Calcimycin; Cell Differentiation; Dactinomycin; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid; Leukemia, Myelomonocytic, Chronic; Tretinoin; Tumor Cells, Cultured

Camptothecin (CPT) and 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP16) at concentrations of 0-10 nM and 0-400 nM, respectively, have weak synergistic effects with retinoic acid (RA) on the induction of differentiation and growth inhibition of HL-60 cells. However, pretreatment of HL-60 cells with the above respective concentrations of CPT or VP16 for 1 h, had no influence on the differentiation of HL-60 cells induced by the following treatment with RA: when HL-60 cells were pretreated with CPT or VP16 at concentrations of 80 nM or 16 microM, respectively, the following treatment with RA had a synergistic effect on the induction of differentiation and growth inhibition. Morphology and biochemical markers were used to show that the differentiation-inducing effect of RA on HL-60 cells was potentiated by pretreatment with higher concentrations of 80 nM CPT or 16 microM VP16. The present results suggest that combination of CPT or VP16 at low concentration with RA or pretreatment with higher concentrations of camptothecin or VP16, followed by RA treatment may be useful for increasing the differentiation effect of RA and decreasing the side effects of RA.

Topics: Camptothecin; Carboxylesterase; Carboxylic Ester Hydrolases; Cell Cycle; Cell Differentiation; DNA, Neoplasm; Drug Synergism; Etoposide; Growth Inhibitors; Humans; In Vitro Techniques; Leukemia, Myeloid; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Tretinoin; Tumor Cells, Cultured

Treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), is associated with induction of monocytic differentiation. Since PKC can act immediately upstream to the cytoplasmic Raf-1 serine/threonine protein kinase, we studied activation of Raf-1 during induction of the differentiated monocytic phenotype. The results demonstrate that Raf-1 is activated during TPA-induced monocytic differentiation of HL-60 cells. In contrast, there was little effect of TPA on this kinase in an HL-60 variant, designated HL-525, which is resistant to TPA-induced differentiation. Treatment of both HL-60 and HL-525 cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with Raf-1 activation and induction of the monocytic phenotype. Since Raf-1 can activate the mitogen-activated protein (MAP) kinases, we also studied the relationship between MAP kinase activation and monocytic differentiation. Treatment of HL-60, but not HL-525, cells with TPA was associated with increased MAP kinase activity as determined by phosphorylation of myelin basic protein and the c-Jun Y peptide. Okadaic acid-induced differentiation of both HL-60 and HL-525 cells was similarly accompanied by increases in MAP kinase activity. These findings indicated that activation of Raf-1/MAP kinase signaling is associated with induction of a differentiated monocytic phenotype and that okadaic acid bypasses a defect in this cascade in TPA-treated HL-525 cells. While recent studies have shown that HL-525 cells are deficient in PKC beta, the present results demonstrate that PKC beta expression is up-regulated in the HL-525 variant by treatment with retinoic acid. The results also demonstrate that retinoic acid-treated HL-525 cells respond to TPA with activation of Raf-1 and MAP kinase, as well as induction of monocytic differentiation. Taken together, the results indicate that activation of Raf-1/MAP kinase signaling is associated with monocytic differentiation and that stimulation of serine/threonine protein phosphorylation by TPA or okadaic acid is sufficient for reversal of the leukemic HL-60 phenotype.

Topics: Amino Acid Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Enzyme Activation; Ethers, Cyclic; Gene Expression; Genes, jun; Humans; In Vitro Techniques; Leukemia, Myeloid; Molecular Sequence Data; Monocytes; Okadaic Acid; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The ability of cytokines (IFN alpha, IFN gamma, TNF alpha, IL-1 alpha, IL-6), all-trans retinoic acid, 1,25(OH)2-vitamin D3 and the tumor promoting phorbol ester TPA to regulate cell surface expression of protectin (CD59 antigen) on human hematopoietic and non-hematopoietic neoplastic cell lines was examined with the aid of immunocytofluorometric measurements. The tumor promoting phorbol ester TPA induced a marked up-regulation of protectin in all examined cell lines with the exception of promyelocytic leukemia HL-60, where TPA significantly decreased protectin cell surface expression. All-trans retinoic acid weakly down-regulated cell surface protectin on K-562, while 1,25(OH)2-vitamin D3 produced such effect on HL-60 cells. None of the examined cytokines induced a significant protectin down-regulation in the examined cell lines.

Topics: Antigens, CD; Breast Neoplasms; Calcitriol; Carcinoma; CD59 Antigens; Cell Membrane; Cytokines; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Female; Flow Cytometry; Fluorescent Antibody Technique; Glioma; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Membrane Glycoproteins; Ovarian Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Up-Regulation

The efficiency of retinoic acid (RA)-induced differentiation was dependent on the position of HL-60 cells in the cell cycle. Our results demonstrated that cells at the G1/S border were more efficiently induced to differentiate by short exposure to RA than cells at other phases of the cell cycle. Synchronization of cells in G1/S phase by aphidicolin (APH) or mimosine (MIMO) increased the sensitivity of cells to RA short exposure treatment. Pretreatment with sphinganine (SP), a protein kinase C (PKC) inhibitor, potentiated RA-induced cell differentiation. By cell cycle analysis, SP was found to block the cell progression through the G1/S phase. Consequently, cells accumulated in the G1/S phase of the cell cycle. The present data therefore suggest a possible mechanism of action of SP to enhance RA-induced differentiation.

Topics: Aphidicolin; Cell Cycle; Cell Differentiation; DNA; Granulocytes; Humans; In Vitro Techniques; Leukemia, Myeloid; Mimosine; RNA; Sphingosine; Tretinoin; Tumor Cells, Cultured

Regulatory molecules that affect the growth culture of blast cells from acute myeloblastic leukemia (AML) may also alter drug sensitivity, a phenomenon that may be called regulated drug sensitivity. Previous studies have shown: (i) blast cells exposed to retinoic acid before cytosine arabinoside (Ara-C) usually show increased sensitivity, but after some retinoic acid exposure times, sensitivity may be decreased; (ii) factor-sensitive or responsive blasts cultured with granulocyte colony-stimulating factor (G-CSF) are regularly more Ara-C-sensitive than when cultured with granulocyte-macrophage CSF (GM-CSF). This paper is concerned with the effects of schedule on drug sensitivity as regulated by either retinoic acid or the myelopoietic growth factors, G-CSF and GM-CSF. We measured the effects of retinoic acid on the sensitivity of blasts cells from the two continuous AML lines to Ara-C or arabinofuranosyl 5-azacytosine (Ara-AC). Cells from seven patients with AML were tested for Ara-C sensitivity in conjunction with retinoic acid. The cells were treated with retinoic acid before or after administration of the drug. Both increases and decreases in Ara-C sensitivity were seen for both schedules. Consistent increases in Ara-C sensitivity were obtained when retinoic acid was included in the methylcellulose cultures used to determine clonogenic cell recovery at each drug dose. In studies of growth factors, a single factor-dependent cell line (OCI/AML-5) was used to compare the effects of G-CSF and GM-CSF on Ara-C sensitivity. An experimental design was used that permitted factors to present in culture for 24 h before Ara-C, during the next 24 h period with the drug, for a subsequent day in suspension without drug, and during the 5-7 days required for colony formation in methylcellulose cultures. G-CSF and GM-CSF were most effective in increasing or decreasing Ara-C, respectively, when the factor under test was included in the methylcellulose cultures. Thus, like retinoic acid, growth factors influenced drug sensitivity when they were present after the drug had been removed. These data, therefore, are compatible with the hypothesis that repair mechanism may contribute to regulated drug sensitivity.

Topics: Acute Disease; Aged; Cell Survival; Cytarabine; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; In Vitro Techniques; Leukemia, Myeloid; Male; Methylcellulose; Middle Aged; Tretinoin; Tumor Cells, Cultured

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

Annexin VIII is a calcium- and phospholipid-binding protein with anticoagulant activity. Annexin VIII mRNA was found to be specifically expressed in acute promyelocytic leukemia (APL) cells; it was not found in other types of acute myeloid leukemia (AML) nor in lymphoid malignancies. Using Northern blot analysis we investigated annexin VIII expression in 142 continuous human leukemia and lymphoma cell lines at the mRNA level. While the only APL cell line, NB-4, was indeed positive, other cell lines also displayed annexin VIII mRNA: 4/22 myeloid cell lines, 8/23 monocytic cell lines, 2/8 megakaryoblastic cell lines, 5/26 lymphoma-derived cell lines, 2/10 myeloma cell lines and 1/44 lymphoid leukemia cell lines. The strongest expression was seen in NB-4 and in the Hodgkin's disease derived cell line HDLM-2. Treatment of NB-4 cells with all-trans retinoic acid (ATRA) or the phorbol ester TPA induced terminal differentiation and down-regulated annexin VIII mRNA expression rapidly within a few hours; vitamin D3 was ineffective in this regard; the protein kinase C activator Bryostatin 1 up-regulated the expression. A panel of initially negative cell lines could not be induced by any of these biomodulators to transcribe annexin VIII. The half-life (T1/2) of annexin VIII mRNA was about 3-4 h using actinomycin D as transcription inhibitor. Treatment with ATRA or TPA prior to exposure to actinomycin shortened the T1/2 to 2 h while Bryostatin 1 extended it to 6h. As 21/141 non-APL cell lines were positive, annexin VIII cannot be used as a marker gene for APL cells; however, it might be associated with myelomonocytic or erythro-megakaryoblastic precursor cells. Annexin VIII gene expression might play a unique role in the proliferation and/or differentiation of leukemic cells and could be associated with the particular abnormal hemostasis of some leukemias.

Topics: Annexins; Blotting, Northern; Bryostatins; Cell Differentiation; Cholecalciferol; Dactinomycin; Gene Expression Regulation, Neoplastic; Half-Life; Humans; Lactones; Leukemia; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Lymphoma; Macrolides; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Earlier reports seemed to indicate that the cytoplasmic intermediate filament protein vimentin and the nuclear intermediate-type filament proteins A and C lamins are expressed in a coordinate manner in human myeloid cells. We have comparatively studied the expression of the vimentin and the A/C lamin genes at the RNA and protein levels in human U-937 promonocytic and HL-60 promyelocytic cells treated with differentiation inducers. 12-O-Tetradecanoyl phorbol-13-acetate and cytosine arabinoside produced a coordinate and stable stimulation of both vimentin and A/C lamin expression in U-937 cells. A stable increase in vimentin expression was also produced by sodium butyrate and by dibutyryl cyclic AMP in U-937 cells and by dimethyl sulfoxide in HL-60 cells. In contrast, these agents produced only a transient increase in A/C lamin expression (maximum mRNA levels at 6-24 h), which later returned to expression levels similar to or even lower than those in untreated cells. Retinoic acid greatly inhibited vimentin expression in HL-60 cells, but it had little effect on A/C lamin expression. Taken together, the present results suggest that there are important differences in the mechanisms which regulate the expression of the vimentin and nuclear lamin genes, as well as in their implication in the differentiation of human myeloid cells.

Topics: Bucladesine; Butyrates; Butyric Acid; Cell Differentiation; Cytarabine; Gene Expression; Humans; In Vitro Techniques; Lamins; Leukemia, Myeloid; Nuclear Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vimentin

We investigated the interactive regulation of the plasminogen activators (PAs) and their inhibitors (PAIs) by all-trans-retinoic acid (RA) in the presence and absence of the phorbol ester, phorbol myristate acetate (PMA), in four developmentally distinct human myeloid leukemic cell lines. Treatment of HL-60, K562, THP-1, and U937 cells with PMA resulted in an induction of urokinase-type PA (u-PA), the u-PA receptor (u-PAR), and PAI types 1 and 2 (PAI-1 and PAI-2). The addition of RA alone failed to alter gene expression or antigen production of PAI-1, PAI-2, or u-PAR. However, RA potentiated PMA-mediated induction of PAI-2 mRNA in HL-60 and U937 cells and PAI-2 antigen in all four cell lines. The effect of PMA on u-PA mRNA was also potentiated by RA in HL-60 and U937 cells. A similar, but transient, effect was seen on u-PA antigen levels. Run-on transcription analysis confirmed that these effects were due at least in part to changes in gene template activity. Furthermore, RA did not potentiate the effects of PMA on either u-PAR or PAI-1. In fact, in U937 cells, RA inhibited PMA-induced PAI-1 antigen secretion by approximately 60%. It would seem that interactive regulation of these genes allows for greater diversity of control, which may, in turn, be required for localized control of plasminogen-dependent extracellular proteolysis generated by monocytes/macrophage during cell migration and tissue remodeling.

Topics: Drug Synergism; Enzyme Induction; Humans; Leukemia, Myeloid; Plasminogen Activator Inhibitor 2; Plasminogen Activators; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Templates, Genetic; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Prothymosin alpha (ProT alpha) is a nuclear protein related to cell proliferation. Its gene is highly activated during postnatal development at stages containing many proliferating but also differentiating cells. In this report, a study on ProT alpha gene expression during differentiation of human myeloid leukemic (HL-60) cells was undertaken to analyze the possible association of ProT alpha to cell differentiation. When HL-60 cells were induced to differentiate to granulocytes (using retinoic acid) or monocyte/macrophages (using 12-O-tetradecanoylphorbol-13-acetate), a marked down-regulation in the levels of ProT alpha transcript was found. When cell division of immature HL-60 cells was interrupted by either treatment with hydroxyurea or serum starvation, ProT alpha gene expression was not significantly altered. These findings suggest that loss of ProT alpha mRNA in induced HL-60 cells is a differentiation-related event. Examination of the stability of ProT alpha mRNA showed that the stabilization of the ProT alpha transcript is differentially regulated in the two HL-60 lineages. Nuclear run-on experiments revealed that during HL-60 differentiation, the transcriptional activity of the ProT alpha gene does not experience significant variations.

Topics: Cell Differentiation; Down-Regulation; Humans; Hydroxyurea; Leukemia, Myeloid; Protein Precursors; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymosin; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Retinoic acid is known to induce differentiation of human myeloid leukemia cells in vitro. Recently, all-trans retinoic acid has been used to induce remissions in patients with acute promyelocytic leukemia, probably through differentiation of the leukemia cells. Myeloblastin (mbn) is a protease that has been identified in the human leukemia cell line HL-60. Downregulation of this protease can inhibit proliferation and induce differentiation of HL-60-derived leukemia cells. Here we have investigated the regulation of mbn messenger RNA (mRNA) expression in two human leukemia cell lines, HL-60 and NB4, treated with all-trans retinoic acid. Under this treatment, downregulation of mbn mRNA was observed in both cell lines, but was considerably delayed in NB4 cells that carry the t(15;17) translocation characteristic of acute promyelocytic leukemia. We have found that multiple mechanisms were involved in the control of mbn mRNA expression. These mechanisms were different in HL-60 and NB4 cells. Our results show that in HL-60 cells, all-trans retinoic acid rapidly decreased transcription of mbn. In contrast, in the t(15;17)-positive NB4 cells treated with all-trans retinoic acid, upregulation of mbn mRNA expression was followed by a late downregulation, both achieved via posttranscriptional mechanisms.

Topics: Cell Cycle; Cell Differentiation; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Cycloheximide; Dactinomycin; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Myeloblastin; RNA, Messenger; RNA, Neoplasm; Serine Endopeptidases; Time Factors; Transcription, Genetic; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

Low concentrations of geranylgeranylacetone (GGA), known as an antiulcer agent (Teprenone), induces differentiation of various human myeloid leukemia cell lines. The cell lines examined in the present study were myeloblastic ML1, histiocytic U937, promyelocytic HL60, and multipotential K562. All of these cell lines were induced to differentiate by 20 microM GGA, as measured by NBT staining. Neither polyprenylacetones, with more or fewer isoprene units than the geranylgeranyl group, nor polyprenylalcohols had no differentiation-inducing activity. GGA used in combination with RA or TNF-alpha increased ML1 cell differentiation. The present results suggest that GGA may be a useful agent in differentiation therapy of leukemia.

Topics: Cell Differentiation; Diterpenes; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Leukemia, Myeloid; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

All-trans-retinoic acid (ATRA) induces complete remissions in the majority of patients with acute promyelocytic leukemia. Despite continuous p.o. ATRA administration, many patients relapse after a short remission duration. In these patients, ATRA plasma concentrations were found to be very low, which was related to induction of retinoic acid-binding protein and increased drug catabolism by cytochrome P-450-mediated reactions. An i.v. ATRA formulation, which can be achieved by encapsulating ATRA into liposomes, may have the potential to overcome these unwanted effects. We investigated the ability of liposomal ATRA (L-ATRA) to induce differentiation of human myeloid leukemia cell lines (HL-60, KG-1, and THP-1). Cellular differentiation, as assessed by morphological criteria and by the expression of a mature myeloid cell surface antigen (CD11b on HL-60 and KG-1 cells), was induced by culture in the presence of L-ATRA. The ability of L-ATRA-treated HL-60 cells to reduce nitroblue tetrazolium demonstrated that they were functionally differentiated cells. Cell cycle analysis revealed significant growth inhibition of the cells after L-ATRA treatment. Following culture with L-ATRA, cells from five patients with acute promyelocytic leukemia were found to be morphologically and immunophenotypically mature myeloid cells. L-ATRA was as effective as free ATRA in inducing differentiation of the cell lines and of the cells from patients with acute promyelocytic leukemia. We conclude that L-ATRA effectively induces differentiation and may be a useful parenteral ATRA formulation for overcoming the pharmacological mechanisms that lead to "retinoid resistance."

Topics: Cell Cycle; Cell Differentiation; Granulocytes; Humans; In Vitro Techniques; Leukemia, Myeloid; Liposomes; 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

Methylation and DNase I-hypersensitive sites of the myeloperoxidase gene in human myeloid leukemia HL-60 cells were studied by Southern blot hybridization using the myeloperoxidase gene probes. Digestion of DNA with a methylation-sensitive restriction endonuclease indicated that a CpG in the CCGG sequence located 3.53 kbp upstream of the myeloperoxidase gene was unmethylated in HL-60 cells expressing the gene, whereas it was methylated in K562 cells and human placenta not expressing the gene. The site in HL-60 cells remained unmethylated after retinoic acid- or 12-O-tetradecanoyl-phorbol-13-acetate-induced differentiation that arrests myeloperoxidase synthesis. Digestion of isolated nuclei with various amounts of DNase I indicated that four DNase I-hypersensitive sites were in an upstream region of the myeloperoxidase gene in HL-60 cells and three sites were within the gene. In retinoic acid-induced cells, the bands of the hypersensitive site near the 5' side of the gene and that in the first intron became weak, while that of the site in the fifth intron became strong. The bands of these hypersensitive sites were weak in K562 cells. The implications of these changes in tissue-specific expression and developmental down-regulation of the myeloperoxidase gene are discussed.

Topics: Base Sequence; Cell Differentiation; Chromatin; Deoxyribonuclease I; Gene Expression Regulation, Neoplastic; Genes; Humans; Leukemia, Myeloid; Methylation; Molecular Sequence Data; Peroxidase; Regulatory Sequences, Nucleic Acid; Tretinoin; Tumor Cells, Cultured

Twenty-seven novel nucleobases and nucleosides were synthesized by structural modification of uracil, and their effects on growth and differentiation of human myeloid leukemia HL-60 cells were examined. Some of the compounds inhibited the growth of HL-60 effectively. The nitroblue tetrazolium (NBT)-reducing activities of cells treated with the concentrations of these compounds for 50% inhibition of growth were compared. TI-66 (2,4-dibenzyl-6-fluoro-7,7,8,8-tetramethyl-cis-2,4-diazabicyclo-[4.2.0] octane-3,5-dione) was the most effective inducer of NBT-reducing activity and morphological differentiation of HL-60 cells into cells of the myelomonocytic lineage. TI-66 was also effective for induction of differentiation of another human myelogenous leukemia cell line, ML-1 cells, but not for differentiation of human erythroid leukemia K562 or HEL cells, or monocytic U937 cells. The effect of TI-66 in inducing differentiation of HL-60 cells was additive or more than additive in combination with retinoic acid or vitamin D3. Adenine or hypoxanthine alone induced NBT-reducing activity of the cells, and at suboptimal concentrations these compounds enhanced the effect of TI-66, but the enhanced NBT-reducing activities did not exceed the maximal activity induced by TI-66 alone. Simultaneous treatment of HL-60 cells with hypoxanthine reduced the growth inhibition by TI-66 alone. TI-66 was about 150 times more potent on a molar basis than adenine in inducing differentiation of HL-60 cells. These results suggest that nucleobase analogs such as TI-66 should be useful for differentiation therapy of some types of myelogenous leukemia.

Topics: Cell Differentiation; Cell Division; Cholecalciferol; Humans; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured; Uracil

Human IMP dehydrogenase (IMPDH; EC 1.1.1.205) was recently found to consist of two molecular species (types I and II) with high expression of type II isozyme in leukemic cells. Here we report that the low level of type II mRNA in normal lymphocytes was up-regulated by phytohemagglutinin stimulation (3.2-fold) and Epstein-Barr viral transformation (5.7-fold). The type II mRNA expression in quiescent HL-60 cells was also elevated 2.8-fold by serum stimulation. Conversely the enhanced level of type II IMPDH mRNA in HL-60 cells was down-regulated to less than 5% along with differentiation induced by retinoic acid (1 microM), phorbol-12-myristate-13-acetate (33 nM), or dimethyl sulfoxide (1.25%) independent of end-stage phenotype. By contrast, type I IMPDH mRNA was expressed constitutively in the various states of proliferation and differentiation. The type II IMPDH stringently linked with cell proliferation should be a crucial target for antileukemic and immunosuppressive chemotherapy.

Topics: Blotting, Northern; Cell Division; Dimethyl Sulfoxide; Gene Expression; Guanosine; Humans; IMP Dehydrogenase; Leukemia, Myeloid; Lymphocyte Activation; Lymphocytes; Ribavirin; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The role of CD11/CD18 leukocyte adhesion molecules and their ligands in mediating non-major histocompatibility complex (MHC) restricted lymphocyte cytotoxicity is controversial. In order to examine the role of target cell intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of lymphocyte function-associated antigen (LFA-1) (CD11a/CD18), we exposed the human leukemia cell line, HL-60, to a variety of agents implicated in modulating ICAM-1 expression and/or sensitivity to lymphocyte cytolysis. Exposure of HL-60 cells to retinoic acid (RA), interferon (IFN)-alpha, IFN-beta, and IFN-gamma induced protection from lymphokine-activated killer (LAK) cytolysis. Only RA and IFN-gamma induced ICAM-1 expression. Tumor necrosis factor and vitamin D3, which also induced ICAM-1 expression, increased HL-60 sensitivity to LAK lysis. Granulocyte-macrophage colony-stimulating factor also increased sensitivity to LAK lysis; ICAM-1 was not induced. The state of cellular differentiation and expression of class I and II MHC antigens also did not correlate with sensitivity to LAK cytolysis. Exposure of untreated HL-60 cells and HL-60 cells expressing ICAM-1 to monoclonal antibody (mAb) versus ICAM-1 did not modulate LAK sensitivity. Exposure of LAK cells to mAb versus LFA-1 partially inhibited cytolysis; mAb versus CD18 inhibited cytolysis more completely. HL-60 cells were resistant to natural killer lysis; exposure to the various experimental agents did not alter sensitivity. We conclude that leukemic cell sensitivity to LAK cytolysis can be modulated by a variety of agents. Although our results suggest a role for leukocyte CD11/CD18 adhesion molecules in LAK cytolysis, the poor correlation between ICAM-1 expression and sensitivity to LAK lysis suggest that interactions other than LFA-1/ICAM-1 conjugation may be more central to the processes involved.

Topics: Antibodies, Monoclonal; Cell Adhesion Molecules; Cell Death; Cell Differentiation; Cell Division; Cell Membrane; Cholecalciferol; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Intercellular Adhesion Molecule-1; Interferon-alpha; Interferon-beta; Interferon-gamma; Killer Cells, Lymphokine-Activated; Leukemia, Myeloid; Lymphocytes; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We analyzed the maintenance of acute myeloid leukemia clonogenic cells (AML CFU-L) in liquid culture in the presence of five potential differentiation inducers: trans-retinoic acid, 1,25-dihydroxy vitamin D3, interferon gamma, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), used singly and with two combined. The culture medium contained either fetal bovine or human serum from normal donors. CFU-L recovery after 7 days was compared to that observed in control cultures. Of AML cases and HL60 cells, 11/15 displayed greater than 50% CFU-L reduction in response to one or more inducers. In 8/9 responsive cases that underwent the nitroblue tetrazolium (NBT) reduction test, an increase in the percentage of functionally mature (NBT-positive) cells was detected. The combination of retinoic acid with interferon gamma was most effective in reducing CFU-L recovering (8 responsive/15 AML cases), G-CSF and M-CSF displayed either inhibitory or stimulatory activity in different AML cases. The type of serum employed generally did not affect the response to inducers of differentiation. No significant inhibition of the recovery of granulocyte-macrophage colony-forming units was determined by the five inducers in experiments with three normal bone marrow samples. Our experiments indicate that biological differentiation inducers can reduce AML CFU-L self-renewal and increase the proportion of differentiated cells at concentrations that do not affect normal myelopoiesis and could be achieved during treatments in vivo.

Topics: Acute Disease; Animals; Calcitriol; Cell Transformation, Neoplastic; Drug Synergism; Granulocyte Colony-Stimulating Factor; Humans; Interferon-gamma; Leukemia, Myeloid; Macrophage Colony-Stimulating Factor; Recombinant Proteins; Tretinoin; Tumor Stem Cell Assay

Retinoic acid (RA)-treated HL-60 cells subjected to density arrest (DA) and double thymidine block (TB) synchronization demonstrated image feature changes associated with cellular proliferation and differentiation. RA-treated TB cells demonstrated an increased level of morphologic differentiation (assessed by differential counts and quantitation of nuclear shape) and more rapid functional differentiation (assessed by superoxide production and expression of complement receptors) than RA-treated DA cells. By comparison to DA cells, TB cells had less variation in virtually all image features values. A Kruskal-Wallis test of image features ranked total optical density (TOD) of Feulgen-stained cells, nuclear area and shape factor as the top three features regardless of synchronization method. Statistically significant changes in image feature values of RA-treated cells were first noted on day 1. The computer-assisted ability to discriminate RA-treated cells on a given day after induction from paired controls by means of an unsupervised learning algorithm increased over a seven-day period for both DA and TB cells. However, in the dichotomous (RA-treated versus untreated) classification scheme employed, which did not account for continuous levels of morphologic differentiation, there was no advantage in the use of the TB over DA procedure.

Topics: Cell Count; Cell Cycle; Cell Differentiation; Cell Division; DNA, Neoplasm; Granulocytes; Hematopoiesis; Humans; Image Processing, Computer-Assisted; Leukemia, Experimental; Leukemia, Myeloid; Staining and Labeling; Thymidine; Time Factors; Tretinoin; Tumor Cells, Cultured

Induction of differentiation, inhibition of cell growth, and localization of nucleophosmin in HL-60 cells under the treatment of retinoic acid (RA) were studied. Bright nucleolar fluorescence was observed in control promyelocytic growing cells. The addition of RA in the culture system resulted in time- and dose-dependent induction of differentiation, cell growth inhibition, and nucleophosmin translocation from nucleoli to nucleoplasm. Unlike the control cells, many fewer nucleophosmin-associated preribosomal ribonucleoprotein particles (pre-rRNPs) could be obtained from nucleoli of RA-treated cells. Addition of sphinganine, an inhibitor of protein kinase C, facilitated the RA-induced differentiation, nucleophosmin translocation, and cell growth inhibition. Cells treated with sphinganine were more responsive to RA. Differentiation, translocation of nucleophosmin, and inhibition of cell growth occurred with lesser doses of RA or in shorter incubation times in the presence of sphinganine. Significant numbers of HL-60 cells could be rescued from the effects of RA upon the removal of RA after 2-h drug exposure. Pretreatment but not posttreatment of HL-60 cells with sphinganine, however, modulated the reversibility of the effects induced by short-exposure RA treatment. These results indicated that RA therapy can be improved by the pretreatment or the concurrent use of a modulator of protein kinase C activity. Nucleophosmin translocation as observed by immunofluorescence may be a simple and rapid method for assessing inhibition of cellular growth in response to differentiation inducers such as RA in cancer chemotherapy.

Topics: Cell Compartmentation; Cell Differentiation; Cell Division; Drug Administration Schedule; Drug Synergism; Growth Inhibitors; In Vitro Techniques; Leukemia, Myeloid; Nuclear Proteins; Nucleophosmin; Ribonucleoproteins; Sphingosine; Tretinoin; Tumor Cells, Cultured

We have examined the role of retinoic acid (RA), the biologically active metabolite of vitamin A, in expression of the IL-1 beta gene in the human myeloid leukemia cell line THP-1 and in human monocytes. Both protein kinase C-activating phorbol esters, e.g., PMA, and LPS induce IL-1 beta expression in these cells. Physiologic RA concentrations alone were not able to induce any IL-1 beta production, but they strongly enhanced the PMA-induced IL-1 beta protein production and mRNA accumulation in both human monocytes and in THP-1 cells. Nuclear run-off analysis revealed that the enhancing effect was at the transcriptional level. RA also slightly potentiated LPS-induced IL-1 beta expression in THP-1 cells but not in human monocytes. These data suggest that RA can be a strong up-regulator of IL-1 production, but its strength varies depending on the nature of the activating signal.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Blotting, Northern; Bucladesine; Gene Expression; Humans; Interleukin-1; Isoquinolines; Leukemia, Myeloid; Lipopolysaccharides; Monocytes; Piperazines; Protein Kinase Inhibitors; RNA, Messenger; Sulfonamides; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.

Topics: Azides; Carrier Proteins; Cell Line; Chromatography, Ion Exchange; Cyclic AMP; Cyclic AMP Receptor Protein; Electrophoresis, Gel, Two-Dimensional; Humans; Leukemia, Myeloid; Molecular Weight; Neoplasm Proteins; Precipitin Tests; Protein Kinases; Sodium Chloride; Tretinoin

Retinoic acid (RA) induces HL-60 and THP-1 leukemic cell lines to differentiate into granulocyte-like and monocyte-like cells. Limited data are available concerning the effects of RA on components of the cyclic AMP pathway in human myeloid leukemic cells. We showed previously a decrease in adenylate cyclase activity in the presence of histamine, prostaglandin E1 and forskolin in RA-treated HL-60 cells as compared to untreated cells. We examined the elements of the signal transduction pathway utilized by RA in the human myeloid cell line HL-60 and the human monocytic cell line THP-1. We therefore studied the effect of RA on the activity of the stimulatory G-protein (Gs). We demonstrate that addition of RA to two human myeloid leukemia cell lines, HL-60 and THP-1, does not induce a reduction of the 2 subunit of Gs (Gs alpha) RNA or Gs alpha protein in the plasma membrane but leads to a rapid decrease in the cholera toxin (CTX)-catalysed ADP-ribosylation of Gs alpha. In addition, this effect seems to be specific to RA, since there was no modification in Gs alpha ADP-ribosylation in the membranes of cells treated with dimethyl sulfoxide (DMSO), another inducer of differentiation in HL-60 cells.

Topics: Adenosine Diphosphate; Cell Differentiation; Cell Membrane; Cholera Toxin; Cyclic AMP; GTP-Binding Proteins; Humans; Leukemia, Experimental; Leukemia, Myeloid; Macromolecular Substances; NAD; Protein Processing, Post-Translational; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The subcellular distribution of the elastase in human myeloid leukemia HL-60 cells was studied in comparison with that in normal leukocytes. On differential centrifugation, most of the elastase activity of HL-60 cell lysates was recovered in the 105,000 x g supernatant, while that of human peripheral blood leukocyte lysates was recovered in the 500 x g precipitate (azurophil granule-rich fraction). Moreover, on Percoll density gradient centrifugation, the elastase activity in HL-60 cell extracts was recovered in the lightest fraction with none in the azurophil granule-rich fractions, whereas most of the activity in leukocyte extracts was recovered in the azurophil granule-rich fractions. This subcellular localization of elastase did not change when HL-60 cells differentiated into monocytes and granulocytes by induction with 12-O-tetradecanoyl phorbol-13-acetate and retinoic acid, respectively. Furthermore, on Sephadex G-75 gel filtration, the elastase activity in HL-60 cell extracts was eluted earlier than that in leukocyte extracts. The size estimation indicated that the elastase of HL-60 cells was 36-30 kDa, corresponding to the size of an elastase precursor reported. The relevance of a large form of the elastase in HL-60 cells to its subcellular localization is discussed.

Topics: Cell Differentiation; Cell Fractionation; Centrifugation, Density Gradient; Chromatography, Gel; Cytosol; Humans; Leukemia, Myeloid; Leukocytes; Pancreatic Elastase; Peroxidase; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

AML cells were cultured free of serum with G-CSF in combination with all-trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Acute Disease; Bone Marrow; Cell Differentiation; Cells, Cultured; Dinoprostone; Flow Cytometry; Fluorescent Antibody Technique; Granulocyte Colony-Stimulating Factor; Granulocytes; Humans; Kinetics; Leukemia, Myeloid; Staining and Labeling; Tretinoin

The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furth

Topics: Antibiotics, Antineoplastic; Autoradiography; Cell Line; Culture Techniques; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Epitopes; Glycosylation; Humans; Leukemia, Myeloid; Membrane Glycoproteins; Phenotype; Tetradecanoylphorbol Acetate; Tretinoin; Tunicamycin

1. The effect of recombinant human interleukin-6 on the antiproliferation and differentiation inducing actions of all-trans retinoic acid in HL-60 human myeloid leukaemia cells was studied in short-term liquid suspension culture. 2. Interleukin-6 alone showed no significant effect on HL-60 human myeloid leukaemia cells. 3. The addition of interleukin-6 to all-trans retinoic acid-treated cultures of HL-60 human myeloid leukaemia cells significantly enhanced the desired antiproliferation effect of all-trans retinoic acid. 4. The combination of interleukin-6 with all-trans retinoic acid reduced the doses of all-trans retinoic acid required to induce the same differentiation of HL-60 cells as single agent by between 1.7- and 4.8-fold; that is, the efficacy of all-trans retinoic acid in inducing the differentiation of human myeloid leukaemia HL-60 cells was increased up to 4.8 times by its combination with interleukin-6. 5. The combination of all-trans retinoic acid and interleukin-6 could provide an effective alternative therapy for elderly myeloid leukaemia patients.

Topics: Cell Differentiation; Cell Division; Humans; Interleukin-6; Leukemia, Myeloid; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Differentiation inducing agents in double and triple combinations can induce differentiation in primary culture of more than 80% of blast cells from some AML patients. In the present study, the interactions between these differentiating agents have been analysed using Berenbaum's general algebraic solution and three new, potentially clinically useful synergistic combinations: have been identified all trans retinoic acid (RA) + hexamethylene bisacetamide (HMBA), cytosine arabinoside (Ara-C) + HMBA and RA + Ara-C + HMBA. A measure of the effectiveness of these combinations was that the doses of Ara-C and HMBA required to induce 50% differentiation were decreased about 10-fold and 5-fold, respectively, in combination with 1 microM RA. The new synergistic combinations are important not only to limit toxicity but also because multiple drug combinations may better overcome the inherent molecular heterogeneity of the differentiation defect in AML patients. They warrant clinical trial in AML patients who are either unsuitable for or are unresponsive to conventional cytotoxic chemotherapy.

Topics: Acetamides; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cytarabine; Drug Synergism; Humans; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

Topics: Cell Division; Drug Synergism; Humans; In Vitro Techniques; Interleukins; Leukemia, Myeloid; Tretinoin

Retinoic acid, vitamin D3, and dexamethasone are known inducers of myeloid leukemic cell differentiation. Recent evidence indicates that these drugs mediate their biological effects through binding to a nuclear receptor which belongs to the steroid/thyroid hormone receptor superfamily. This paper shows that the ligands of the other receptors of this family, estrogens, progesterone, androgens and thyroid hormone, do not induce leukemic cell differentiation. However, thyroid hormone potentiates, by one order of magnitude, the dose-response effect of retinoic acid in HL-60 cells.

Topics: Cell Differentiation; Cholecalciferol; Dose-Response Relationship, Drug; Drug Synergism; Granulocytes; Humans; Leukemia, Experimental; Leukemia, Myeloid; Oxygen; Stimulation, Chemical; Thyroid Hormones; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

A unique oncofetal protein (OFP) previously identified in rat fetal tissue and rat and human solid tumors, is now shown to be present in rat and human leukemia cells by use of a monoclonal antibody-based assay. Using a highly specific anti-rat OFP monoclonal antibody OFP has been unquivocally immunolocalized to the cytoplasm of the rat leukemia cells. The factor is rapidly released to the circulation as 50 and 55 kD species which share the immunological determinants. When leukemia cells are transplanted to normal rats, OFP increases in the circulation in a biphasic manner which may be due to immune clearance since circulating anti-OFP antibodies have been demonstrated. Induction of differentiation in the human HL-60 leukemia cell line by 13-cis-retinoic acid caused a down regulation of OFP synthesis, both intra- and extra-cellular levels dropping to essentially zero. Induction of differentiation with dibutyryl cyclic AMP caused a cessation of secretion of OFP, with a marked increase in its intracellular concentration, a condition resembling the retention in fetal cells. Leukemia cells add to a growing list of tumors previously shown to produce OFP, suggesting that OFP is intimately involved in some facet of tumorigenesis.

Topics: Animals; Antigens, Neoplasm; Cell Differentiation; Down-Regulation; Fluorescent Antibody Technique; Gene Expression Regulation, Leukemic; Humans; Leukemia, Experimental; Leukemia, Myeloid; Molecular Weight; Neoplasm Transplantation; Rats; Rats, Inbred F344; Tretinoin; Tumor Cells, Cultured

The human myeloid leukemia cell line HL60 is an in vitro model to study myeloid differentiation. HL60 cells differentiate along different cell type lineages in response to a variety of compounds. The direction of differentiation is usually inducer-specific. However, the response of HL60 cells to sodium n-butyrate (NaB) is pleiotropic. NaB induces HL60 along the monocytic, neutrophilic, eosinophilic, and basophilic pathways. In this study we saw that physiologic concentrations of all-trans-retinoic acid (RA) switched the direction of NaB-induced differentiation from monocytic to granulocytic. We showed previously (Breitman & He, Cancer Res 1990: 50: 6268-6273) that combinations of RA and NaB synergistically induce HL60 to cells that reduce nitroblue tetrazolium. The present study shows that this synergy was even greater if the parameter measured was mature granulocytes. Our results raise the possibility that the endogenous RA in the serum used to grow cells in culture may affect the direction of differentiation of HL60 cells induced by NaB. Furthermore, our results may provide additional rationale for the use of combinations of RA and NaB in the treatment of some leukemias.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Drug Synergism; Granulocytes; Humans; Leukemia, Myeloid; Monocytes; Tretinoin; Tumor Cells, Cultured

Human myeloblastic leukemia cell line ML-1 was induced to differentiate by 1 mumol/l all-trans-retinoic acid (RA) or by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). After 4-6 days of the induction several differentiation-associated characteristics were observed: (1) Ability to stimulate respiration burst in the ML-1 induced cells as detected by nitroblue tetrazolium (NBT) test or by chemiluminescence (CL). (The percentage of the NBT-positive cells was up to 99% in the RA-induced cells and up to 85% in the 1,25(OH)2D3-induced cells.) (2) Substantially higher phagocytosis of colloid iron, latex or Staphylococcus particles was found in the induced cells. (3) The 1,25(OH)2D3-induced ML-1 cells expressed the monocytic enzyme NaF-inhibitable alpha-naphthyl butyrate esterase and the surface monocytic antigen CD-14. (4) A majority of the induced cells lost the morphological features of blast cells; while the 1,25(OH)2D3-treated cells acquired certain features of monocyte-macrophage differentiation, the RA-treated cells displayed several granulocytic characteristics. (5) Cytofluorometric DNA assay after treatment of the cells with colcemid showed that the decline observed in the growth rate of the induced cells was connected with their arrest in G1/G0 phase of the cell cycle. The obtained results indicate granulocytic differentiation of the RA-induced ML-1 cells and monocyto/macrophage differentiation of the 1,25-(OH)2-D3 induced cells.

Topics: Acid Phosphatase; Alkaline Phosphatase; Antigens, Differentiation, Myelomonocytic; Calcitriol; Carboxylic Ester Hydrolases; Cell Differentiation; Cell Division; Demecolcine; Humans; Iron; Leukemia, Myeloid; Lipopolysaccharide Receptors; Naphthols; Oxygen Consumption; Peroxidase; Tretinoin

The human promyelocytic THP-1 cell line has been found to support the growth of Leishmania parasites. THP-1 cells, differentiated with retinoic acid, cease replication while remaining in suspension. 72 +/- 8% of THP-1 cells became infected after inoculation with promastigotes of several Old and New World Leishmania species. The resulting amastigotes (19 +/- 5 per infected cell) were easy to harvest, capable of reinfecting cultures of normal human cells and, in the case of L. major and L. infantum, caused specific lesions in BALB/c mice. This culture system should facilitate biochemical and immunological studies on amastigotes and be of use in screening anti-parasite drugs.

Topics: Animals; Humans; Leishmania; Leishmania donovani; Leishmania tropica; Leukemia, Myeloid; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred BALB C; Monocytes; Tretinoin; Tumor Cells, Cultured

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.

Topics: Cell Cycle; Cell Differentiation; Granulocytes; Leukemia, Myeloid; Oligonucleotides, Antisense; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fes; Proto-Oncogene Proteins c-myb; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Vitamin D

We examined the effect of transforming growth factor-beta (TGF-beta) alone and in combinations with other factors on the growth and differentiation of the human promyelocytic cell line HL60 and the human monoblastic cell line U937. Treatment with TGF-beta alone did not significantly affect growth or differentiation of HL60 cells, while it significantly inhibited proliferation and induced monocytic differentiation of a small percentage of U937 cells. Combinations of TGF-beta and tumor necrosis factor-alpha (TNF-alpha) acted in synergy to inhibit cell proliferation and to induce monocytic differentiation of both HL60 and U937 cells. In contrast, no synergy was observed when HL60 cells were treated with TGF-beta in various combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and retinoic acid. Examination of TNF-alpha receptor expression on HL60 and U937 cells showed that these cell lines expressed comparable levels of high-affinity TNF-alpha binding sites. Treatment of HL60 and U937 cells with TGF-beta did not induce significant changes in TNF-alpha receptor expression in either cell line. In contrast, HL60 cells expressed much lower levels of TGF-beta receptors than did U937 cells. Treatment of both HL60 and U937 cells with TNF-alpha induced a dose-dependent increase in expression of TGF-beta receptors, suggesting that the synergy between TNF-alpha and TGF-beta may result, at least in part, from upregulation of TGF-beta receptor expression by TNF-alpha.

Topics: Cell Differentiation; Cell Division; Drug Synergism; Humans; In Vitro Techniques; Interferon Type I; Interferon-gamma; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Monocytes; Receptors, Cell Surface; Transforming Growth Factors; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Monocytotropic human immunodeficiency virus type 1 (HIV-1) isolates from patients with acquired immunodeficiency syndrome (AIDS) infect mononuclear phagocytes as well as activated T cells, but do not usually infect immature human myeloid cell lines in vitro. The HL-60 promyelocytic/myeloblastic cell line and the promonocytic line, U937, were susceptible to productive infection by monocytotropic HIV-1 isolates (HIV-1JR-FL and HTLV-IIIBa-L) after treatment with retinoic acid, dimethyl sulfoxide, dibutyryl cAMP, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Virus production was only detected when these compounds were added before virus infection. Virus replication did not correlate with CD4 receptor expression because undifferentiated HL-60 cells express CD4 and the level of CD4 expression did not increase after differentiation in the presence of retinoic acid, 1,25(OH)2D3, or TPA. A mature monocytic cell line (THP-1) was capable of infection without pretreatment, and treatment with differentiating agents enhanced virus production. A chronically infected cell line (J-HL-60) was isolated after HIV-1JR-FL infection of HL-60 cells treated with retinoic acid. Virus production in this cell line was enhanced more than 10-fold after differentiation in the presence of 1,25(OH)2D3 or TPA. The majority of virus production by 1,25(OH)2D3-treated J-HL-60 cells was associated with the mature, adherent population. Molecular analysis of a cloned line of J-HL-60 showed integration of a single DNA provirus. These results suggest that cellular factors associated with precursor cell differentiation along the myelomonocytic pathway are required for optimal replication of monocytotropic HIV-1 strains in vitro.

Topics: Bucladesine; Calcitriol; CD4 Antigens; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; DNA, Viral; Gene Expression; HIV Infections; HIV-1; Humans; Leukemia, Myeloid; Tetradecanoylphorbol Acetate; Thymidine; Tretinoin; Tritium; Virus Replication

The effects of TPA (12-0-tetradecanoylphorbol-13-acetate) and RA (retinoic acid) were investigated on the cell lines HL60 (acute promyelocytic leukemia) and K562 (erythroleukemia) and on cells from patients with several kinds of leukemia. There were 14 cases of acute lymphocytic leukemia (ALL), 2 cases of chronic lymphocytic leukemia (CLL), 23 cases of acute myeloid leukemia (M1-M7), 5 cases of chronic myelocytic leukemia in blast crisis (CML-BC) and 2 mixed leukemias. In almost all of the cases examined, after TPA exposure cells from patients with proven myeloid leukemia became adherent to the substrate, while lymphoid leukemia cells remained in suspension, allowing the differentiation of lymphoid from myeloid blasts. The only exception was in one case of CLL, which had cells that became adherent with long filamental projections. In addition, increased phagocytosis following TPA exposure permitted characterization of M7 as this was the only myeloid leukemia negative for phagocytosis. Further discrimination between the subtypes of myeloid leukemia could be based on the increased lysozyme production seen after TPA in M4 and M5. Esterase positivity allowed the discrimination of M1 cells, which were negative before and after TPA treatment. In agreement with the results of other authors, TPA and RA led to independent ways of differentiation, granulocytic-like lineage and monocytic-like cells being favored by RA and TPA, respectively. The capacity of the same cell to differentiate into more than one lineage, depending on whether RA or TPA was used, was only seen in the present study with M3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Transformation, Neoplastic; Diagnosis, Differential; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tetradecanoylphorbol Acetate; Tretinoin

Differentiation induction therapy provides an alternative for treatment of acute myeloid leukaemia (AML) patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + actinomycin D (Act-D) + dimethylformamide (DMF) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in 24-well tissue-culture plates containing RPMI 1640 culture medium with 20% fetal calf serum, 10% autologous serum and 10% 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 5 nM actinomycin D and/or 100 mM dimethylformamide alone and in combination with each other for 6 days at 37 degrees C in a humidified incubator and an atmosphere containing 5% CO2. The triple combination of 10(-6) M retinoic acid + 5 nM actinomycin D + 100 mM dimethylformamide induced 90% of the blasts from 22 of the 24 AML patients to differentiate. The combination of N-methylformamide (a compound similar to dimethylformamide) with cyclophosphamide significantly increased the in vivo activity with no concomitant increase in its reversible hepatotoxicity. Since several polar compounds related to dimethyl-formamide, e.g. hexamethylene bisacetamide and N-methylformamide, are currently undergoing phase II clinical trials, it may be feasible to combine one of these with retinoic acid and/or actinomycin D in the treatment of AML patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Dactinomycin; Dimethylformamide; Drug Interactions; Drug Screening Assays, Antitumor; Humans; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

Butylated hydroxytoluene (BHT), which has both antioxidant and membrane active properties, has been reported to affect cellular differentiation. We studied its effect on the bipotent lineage differentiation of the important HL-60 human myeloblastic leukemia cell line using reduction of nitroblue tetrazolium, cell cycle analysis, population growth rate, monoclonal antibodies, and morphology. BHT markedly accelerated retinoic acid-induced myelocytic differentiation and dihydroxyvitamin D3-induced monocytic differentiation in a concentration and time-dependent manner. Butylated hydroxyanisole (BHA) had a comparable effect. Preincubation with the compounds was not necessary to evoke the acceleration Other antioxidants and inhibitors of eicosanoid synthesis were inactive. We conclude that the important food preservatives BHT and BHA accelerate the kinetics of terminal differentiation of human leukemia and that this effect is likely due at least in part to their membrane active properties.

Topics: Antigens, Differentiation, Myelomonocytic; Antioxidants; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Calcitriol; Cell Cycle; Cell Differentiation; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Leukemia, Myeloid; Nitroblue Tetrazolium; Tretinoin; Tumor Cells, Cultured

Increased calcium influx associated with differentiation of four human myeloid leukemic cell lines: HL-60, KG-1, U-937 and K-562, to either monocytic or granulocytic direction was demonstrated. Calcium influx was measured employing two methods; measurement of radioactive calcium influx rate at 4 degrees C and employing the fluorescent probe, fura-2 acetoxymethyl ester. The increase in Ca2+ influx was demonstrated with three chemically unrelated differentiation inducers: retinoic acid, 1 alpha, 25 dihydroxy vitamin D3 and dimethyl sulfoxide. Inhibitors of calcium uptake such as verapamil diltiazem and cromolyn, partially reduced differentiation, suggesting that differentiation of myeloid leukemic cell lines is dependent on the availability of extracellular calcium.

Topics: Calcitriol; Calcium; Calcium Channel Blockers; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Extracellular Space; Humans; Leukemia, Experimental; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (RA), sodium n-butyrate (NaB), hexamethylene bisacetamide (HMBA), and dimethyl sulfoxide (DMSO) induce differentiation of the human acute myeloid leukemia cell line HL60. In the clinic, RA, NaB, or HMBA induce complete or partial remissions. However, the achievement and maintenance of effective plasma concentrations and toxicity have been problems. These difficulties led us to study the interaction of RA with these inducers. We found that combinations of RA with either NaB, HMBA, or DMSO synergistically induced terminal differentiation of HL60. A measure of the effectiveness of these combinations was that the doses of NaB, HMBA, and DMSO required alone to induce half-maximal differentiation were decreased about 4-fold in combination with normal plasma concentrations of about 30 nM RA. RA or NaB alone did not enhance the growth of HL60 cells. In contrast, HMBA or DMSO alone increased growth of HL60 cells even at concentrations that did not induce differentiation. The addition of RA reduced the promotion of growth and increased the extent of terminal differentiation seen with HMBA and DMSO alone. These data suggest that treatment of some malignancies with combinations of RA with HMBA or NaB may maintain differentiation-inducing effects and decrease the problems associated with the achievement and maintenance of effective plasma concentrations as single agents.

Topics: Acetamides; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Humans; Leukemia, Myeloid; Tretinoin; Tumor Cells, Cultured

Human myeloid leukaemia (U-937 and HL-60) cells when incubated at low cell densities with human recombinant gamma-interferon underwent functional maturation without any loss of proliferative potential relative to uninduced cells. In addition, the proportion of cells in S,G2/M and levels of c-myc oncogene (mRNA and protein) were maintained at the same level as those of untreated control cells. However, cells grown under similar conditions but with retinoic acid matured to the same extent but became growth inhibited with concomitant reductions in the proportion of cells in S,G2/M and levels of c-myc mRNA and protein. These studies indicate firstly that c-myc levels are regulated independently from differentiation in myeloid (non lymphoid) cells, secondly that gamma-interferon can induce differentiation without growth arrest under conditions of low cell density and thirdly emphasise the close association of c-myc expression with proliferative capacity.

Topics: Cell Cycle; Cell Differentiation; Cell Division; Cell Line; Flow Cytometry; Gene Expression Regulation, Leukemic; Humans; Interferon-gamma; Kinetics; Leukemia, Myeloid; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We studied the chemotactic peptide receptor/cytoskeletal interactions in HL-60 cells induced to differentiate with different agents and attempted to correlate these observations with the acquisition of different functional responses. Dibutyryl cyclic AMP-treated cells showed rapid superoxide anion production in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and slow, sustained response to phorbol myristate acetate (PMA). Retinoic acid-induced cells showed a slow, sustained response to both FMLP and PMA. Interferon-gamma-treated cells produced no superoxide anion on stimulation with FMLP, whereas tumor necrosis factor (TNF)-treated cells showed a slight response. Chemotactic peptide receptor association was the same in the HL-60 cells treated with different agents, despite marked differences in the superoxide anion generation and actin polymerization responses to FMLP and PMA in these cells. In mature neutrophils chemotactic peptide receptor association with the cytoskeleton was not affected by either pertussis or cholera toxin. However, both toxins inhibited FMLP-induced actin polymerization and superoxide anion generation. This suggested involvement of a G-protein similar to Gt, rather than Gi or Gs. Neither toxin had any effect on PMA-induced superoxide anion generation. These observations indicate that receptor association with the cytoskeleton may not have a significant role in affecting signal recognition and response. Among the several possible roles suggested, clearance of the occupied receptors may be the most important role of the cytoskeletal association. HL-60 cells induced to differentiate with different agents (because of their varied functional responses) might prove very useful in dissecting the molecular mechanisms regulating stimulus-induced activation of neutrophils.

Topics: Bucladesine; Cell Differentiation; Cholera Toxin; Cytoskeleton; Interferon-gamma; Leukemia, Myeloid; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Formyl Peptide; Receptors, Immunologic; Superoxides; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Virulence Factors, Bordetella

The human cell line U 937 spontaneously expresses monocytic maturation and can be induced into macrophage-like cells when treated with retinoic acid, sodium butyrate, or 2,3-O-tetra decanoylphorbol-13-acetate. We have selected a subclone, designated UM 384, that expresses granulocytic characteristics and can be induced to mature to granulocytes after exposure to retinoic acid, actinomycin D, and dimethylsulfoxide, and to monocyte-like cells when treated with sodium butyrate and phytohemagglutinin-stimulated leukocyte-conditioned medium. These cells retain the same constitutive markers as the parent line including histocompatibility leukocyte antigens and karyotype but share numerous chromosomal abnormalities, mainly t(X;8) (p21;q12).

Topics: Antigens, Differentiation; Butyrates; Butyric Acid; Cell Differentiation; Clone Cells; Dactinomycin; Granulocytes; Histocytochemistry; Humans; In Vitro Techniques; Leukemia, Myeloid; Microscopy, Electron; Monocytes; Tretinoin; Tumor Cells, Cultured

We studied the changes of protein phosphorylation in human leukemic cells by granuloid inducers, using two-dimensional electrophoresis. The phosphorylation of 22 kD, pI 6.0 and 5.8 proteins (pp22) in HL-60 cells or myeloid leukemic cells from patients, was enhanced by treatment with granuloid inducers such as retinoic acid, dimethyl sulfoxide or G-CSF, in common with prostaglandin E2 and theophylline, or dibutyryl c-AMP, which increased intracellular c-AMP. In contrast, pp22 phosphorylation was not induced by the monocytes/macrophages inducer in HL-60 cells, or by the granuloid inducers in lymphoid cells. This phosphorylation occurred within 30 min and continued for more than 48 h. These pp22 proteins were present in the cytosol and phosphorylated on the serine residues. We now present a possibility that granuloid differentiation in myeloid cells is closely linked with these pp22 phosphorylation.

Topics: Cell Differentiation; Cell Line; Colony-Stimulating Factors; Dimethyl Sulfoxide; Granulocyte Colony-Stimulating Factor; Granulocytes; Humans; Leukemia, Myeloid; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Subcellular Fractions; Tretinoin

Adherence reactions involving human leukocytes are mediated by a family of glycoprotein surface antigens composed of three different alpha subunits designated alpha L, alpha M, and alpha X, each of which is associated with a single beta subunit in an alpha 1 beta 1 heterodimer structure. We cloned the cDNA for the common beta subunit and investigated beta subunit mRNA expression in HL-60 promyelocytic leukemia cells and human granulocytic cells. Leukocyte adherence receptor beta subunit mRNA transcripts were present in low levels in HL-60 myeloblasts and promyelocytes and increased 10-fold or greater with chemically induced differentiation to more mature granulocytes (using retinoic acid and dimethylformamide) or monocyte/macrophages (using phorbol myristate acetate). Levels of beta subunit mRNA expression were also increased both in normal human peripheral blood granulocytes and in granulocytes from patients with chronic myelogenous leukemia. Nuclear run-off assays indicated that the increased steady state level of the beta subunit mRNA in retinoic acid-differentiated HL-60 cells was secondary to enhanced beta subunit gene transcription. We conclude that mRNA levels for the beta subunit of the receptor on human leukocytes that mediates cellular adherence are increased in more mature granulocytic cells compared to immature myeloid precursors and that this enhanced mRNA expression is transcriptionally regulated.

Topics: CD18 Antigens; Cell Adhesion; Cell Differentiation; Cloning, Molecular; Dimethylformamide; DNA; Gene Expression Regulation; Granulocytes; Humans; Leukemia, Myeloid; Macromolecular Substances; Membrane Glycoproteins; Nucleic Acid Hybridization; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

When the human myeloid leukemia cell line (HL60) is induced to differentiate with retinoic acid (RA), there is a concentration-dependent increase in transglutaminase (TGase) activity which peaks on day 5. While dibutyryl 3',5'-cyclic adenosine monophosphate (db-cAMP) alone produced only a slight increase in TGase activity in HL60 cells, the concomitant addition of db-cAMP (100 microM) with RA (10(-12)-10(-4) M) potentiates RA induction of TGase activity. Maximal increases in TGase activity (2- to 10-fold) were observed with 10(-4)-10(-7) M RA and when db-cAMP was present from 24 to 48 h after the addition of RA. The cyclic nucleotide enhancement was dose-dependent from 10 to 100 microM of cAMP. Less marked increases were observed with 8-bromo-cAMP and with the phosphodiesterase inhibitor theophylline. Although the simultaneous addition of PGE1 or PGE2 (10(-8)-10(-6) M) produced no enhancement of RA-induced TGase activity, adding PGE1 or PGE2 24 or 48 h following RA treatments produced an enhancement of TGase activity. The phosphodiesterase inhibitor potentiated the increases produced by db-cAMP and the prostaglandins. Dibutyryl cAMP enhanced the ability of RA to induce the cells to reduce nitroblue tetrazolium (NBT), a functional measure of differentiation, at lower concentrations of RA and with shorter treatment durations. cAMP potentiates RA-induced TGase activity in HL60 cells and the combination appears to be associated with enhanced RA-induced differentiation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Alprostadil; Bucladesine; Butyrates; Butyric Acid; Cell Division; Cyclic AMP; Dinoprostone; Enzyme Induction; Humans; Leukemia, Myeloid; Theophylline; Transglutaminases; Tretinoin; Tumor Cells, Cultured

The effects of all-trans retinoic acid (RA) were tested on the growth in vitro of myeloid progenitors from peripheral blood or bone marrow, in 25 patients with chronic myeloid leukemia (CML), ten of whom were either in accelerated or blastic phase, and in nine patients with myeloproliferative disease (MPD). The responses were compared with 12 normal bone marrow controls obtained from patients with lymphoma. Clonal growth in CML blastic and accelerated phase was inhibited to the greatest degree (mean 49 +/- 9% (SEM) of control at 0.3 microM RA). The responses in CML chronic phase and MPD were more heterogeneous, but significant inhibition was seen at higher concentrations of RA (50 +/- 12% CML chronic phase, 58 +/- 26% MPD at 3.0 microM RA). At 0.3 microM and 1.0 microM RA there were significant differences between the CML chronic phase and the CML blastic phase patients (p less than 0.02 and p less than 0.05 respectively). At these concentrations there was no significant inhibition on normal bone marrow myeloid progenitors. Inhibition was independent of the proportions of progenitors in S phase, as assessed by tritiated thymidine suicide. Preincubation of cells from selected patients with RA for 48 hours before culture in agar resulted in a significant degree of inhibition (48 +/- 8% of control). Inhibition was prevented by delaying the addition of RA from 24 to 48 hours from the beginning of the culture, indicating that RA exerts an early direct effect on myeloid progenitors.

Topics: Blast Crisis; Cell Division; Drug Resistance; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Leukemia, Myeloid; Myeloproliferative Disorders; Neoplastic Stem Cells; Tretinoin

The expression of the X-CGD gene, which encodes the heavy-chain subunit of the phagocyte cytochrome b, was studied during induced myeloid differentiation of HL-60 cells. Incubation of the cells with a combined regimen of retinoic acid and dimethyl formamide resulted in granulocytic morphological differentiation and acquisition of nitroblue tetrazolium reduction, a measure of superoxide generation. During the 5-day course of induced differentiation, the levels of X-CGD mRNA transcripts rose 13-fold, with a 2-fold increase detectable within 3 h of exposure to retinoic acid. Relative transcription rates for the X-CGD gene, determined by nuclear runoff, increased two- to eightfold after 24 to 72 h of induced differentiation. However, the greater change in X-CGD mRNA levels than that in transcription rates implies the involvement of posttranscriptional regulation as well. Fractionation by centrifugal elutriation into phases of the cell cycle showed expression of X-CGD transcripts predominantly in G1 cells before induction and in all phases of the cell cycle 24 h after induction. Thus the rapid increase in X-CGD expression in induced cells reflects the acquisition of functional competence and not the concomitant cessation of proliferation or shift in cell cycle distribution.

Topics: Cell Differentiation; Cytochrome b Group; Gene Expression Regulation; Granulocytes; Humans; Leukemia, Myeloid; Phagocytes; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2-pH-unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na+/H+ exchange system and a Na+-dependent HCO3-/Cl- exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H+ exchange system with no change in the activity of the bicarbonate-dependent system. The properties of interaction of the Na+/H+ exchanger of U937 cells with Na+, Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.

Topics: Amiloride; Bicarbonates; Cell Differentiation; Cells, Cultured; Energy Transfer; Flow Cytometry; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid; Lithium; Sodium; Spectrometry, Fluorescence; Tretinoin

Activity of the Ca2+/phospholipid-dependent protein kinase C has been shown to increase during differentiation of the human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide and retinoic acid (Zylber-Katz, E., and Glazer, R. I. (1985) Cancer Res. 45, 5159-5164). Antipeptide antibodies were prepared that specifically recognize the alpha, beta, and gamma isozymes of protein kinase C in rat brain cytosol and HL-60 cell extracts. The three isozymes do not share a common tissue distribution pattern. The gamma enzyme is abundant in brain but a relatively minor component in HL-60 cells; the opposite is true for the alpha enzyme. All three isozymes increase at least 2-fold in abundance in HL-60 cells exposed to 1.2% dimethyl sulfoxide for 48 h. The increase in abundance of the alpha and beta isoforms reaches 7- and 5-fold, respectively, by 96 h without further increase in the abundance of the gamma isozyme. Similarly, all three isozymes increase at least 1.5-fold in abundance after 48 h and 3-fold after 96 h with 1 microM retinoic acid. No further increase in the abundance of any of the isozymes is seen between 96 and 144 h of incubation with retinoic acid. The increase in protein kinase C activity is not limited to the cytosolic forms of the enzyme; a parallel increase in membrane-associated protein kinase C is also observed during differentiation. Approximately 10% of total protein kinase C activity is membrane-associated in both control and differentiating cells. These studies provide the first immunochemical evidence that all three protein kinase C isozymes increase during HL-60 cell differentiation, and they suggest that the increase in the isozyme levels may be coordinately regulated.

Topics: Amino Acid Sequence; Animals; Brain; Cattle; Cell Differentiation; Cytosol; Dimethyl Sulfoxide; Electrophoresis, Polyacrylamide Gel; Humans; Isoenzymes; Leukemia, Myeloid; Molecular Sequence Data; Protein Kinase C; Rabbits; Rats; Sequence Homology, Nucleic Acid; Tetradecanoylphorbol Acetate; Tissue Distribution; Tretinoin; Tumor Cells, Cultured

Human promyelocytic leukemia (HL-60) cells display a novel voltage-dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL-60 cells after exposure to phorbol-12-myristate-13-acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl-. From a holding potential of -80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1-2 X 10(-6) M). These properties appear to differ significantly from the Ca2+-activated K+ channel and the delayed rectifier. The increase of this voltage-activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.

Topics: Calcium; Cell Differentiation; Cell Line; Cesium; Chlorides; Electric Conductivity; Humans; Ion Channels; Leukemia, Myeloid; Macrophages; Potassium; Tetradecanoylphorbol Acetate; Tretinoin

In previous studies we have shown that the synthetic retinoid (E)-4[2-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1- propenyl]benzoic acid (TTNPB) stimulates the growth of myeloid progenitors from normal and myelodysplastic patients. In the present study we compared TTNPB with 13-cis-retinoic acid (RA) in its potential to inhibit cell growth and to induce morphological differentiation and functional activity in two cell lines established in vitro from either acute promyelocytic (HL-60) or acute myelomonocytic (LK) patients. Both agents, 10(-6) M, were found to effectively inhibit cell growth and cause a significant decrease in number of immature granulocytes in both cell lines. However, while in HL-60 cells this decrease was associated with a concomitant increase in fully mature granulocytes (neutrophil-like cells) the maturation of LK cells was blocked at the metamyelocyte stage. Study of the functional activity of the induced cells revealed that the rate of superoxide (O-2) production, as assayed by superoxide dismutase-inhibitable ferricytochrome c reduction, was faster in RA treated HL-60 cells than in TTNPB treated cells (0.41 vs. 0.25 nmol. O2-/10(6) cells/60 min). Superoxide production by LK cells treated by either TTNPB or RA was negligible. The percentage of O2(-)-producing cells was determined cytochemically by their ability to reduce the dye nitroblue tetrazolium (NBT). The results showed that production of O2-by LK cells exposed to TTNPB or RA was negligible by this method as well. A higher percentage of HL-60 cells reduced NBT following incubation with RA than with TTNPB (93 +/- 4% vs 26 +/- 2%), but neither of the two retinoids affected the ability of LK cells to reduce NBT. TTNPB thus proved less effective than RA in inducing morphological and functional differentiation in HL-60 cells, whereas in LK cells both agents inhibited cell growth but induced only partial cell differentiation.

Topics: Benzoates; Cell Differentiation; Humans; Leukemia, Myeloid; Retinoids; Superoxides; Tretinoin; Tumor Cells, Cultured

The effect of glucocorticosteroids, retinoids, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the tumor promoter phorbol myristate acetate (TPA) on the expression of transglutaminase activity in vitro differentiating bone marrow-derived mouse and rat mononuclear phagocytes (BMDMP) and mouse and human myeloid leukemia cell lines was assessed. Dexamethasone was found to induce an increase of about 100% in transglutaminase activity in mouse and rat BMDMP. The effect was time- and dose-dependent, and specific for steroids with glucocorticoid activity. Retinoic acid (RA) suppressed transglutaminase activity in mouse BMDMP (approximately 50%) and enhanced it in rat BMDMP (100-200%). Other retinoids were less effective. 1,25(OH)2D3 had little effect on transglutaminase expression in mouse BMDMP and suppressed it in rat BMDMP (approximately 60%). TPA exerted a suppressive effect (approximately 50%) on transglutaminase activity of both rat and mouse BMDMP. In murine (P388D1 and J774.2) and human (ML3, HL-60, KG-1, HEL, U937) myeloid leukemia cell lines, dexamethasone enhanced transglutaminase activity to a varying degree (100-1,000%), RA suppressed it in P388D1 cells (approximately 70%) and enhanced it in the other cell lines (100-1,500%), 1,25(OH)2D3 induced a rather small augmentation of enzyme expression, whereas TPA suppressed enzyme expression (70-100%). The species-specific differences previously observed by us for the effect of RA, dexamethasone and 1,25(OH)2D3 on the formation of BMDMP from mouse and rat bone marrow progenitor cells are now shown to extend also to effects on expression of transglutaminase activity. From a mechanistic point of view it is of interest that dexamethasone uniformly enhanced transglutaminase activity, whereas TPA suppressed it. RA and 1,25(OH)2D3 induced either suppression or enhancement in the various cell types, with no correlation between the direction of the effect of the two agents. The data suggest that modulation of transglutaminase activity by the four agents occurs via disparate mechanisms.

Topics: Animals; Bone Marrow Cells; Calcitriol; Cell Differentiation; Cell Line; Dexamethasone; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Macrophages; Male; Mice; Mice, Inbred BALB C; Phagocytes; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Transglutaminases; Tretinoin

The effects of retinoic acid on the differentiation of human monocytic leukemia cell lines containing aneuploid (THP-1-Cs5) or diploid chromosomes (THP-1-R) were studied and compared. The induction of cell adhesion to a substratum, phagocytosis of sheep red blood cells (SRBC) or IgG-coated SRBC, pinocytosis of dextran sulfate, and NBT dye reduction by the cells were examined. The occurrence of these processes was much greater in RA-treated THP-1-Cs5 cells than in RA-treated THP-1-R cells. Of all these functional activities, the most remarkable differences between the two cell types were seen for cell adhesion and phagocytosis of SRBC. Morphological changes in RA-treated THP-1-Cs5 cells were observed by light and electron microscopy. RA-treated THP-1-Cs5 cells had a moderately-developed Golgi apparatus, and abundant lysosomes, mitochondria and lipid droplets in the cytoplasm. Among various retinoids examined, RA was the strongest inducer of the differentiation of the THP-1-Cs5 cells into mature cells. These findings suggest that THP-1-Cs5 cells which contain aneuploid chromosomes are more efficiently functionally differentiated by RA than are THP-1-R cells.

Topics: Aneuploidy; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Chromosomes, Human; Diploidy; Humans; Leukemia, Myeloid; Microscopy, Electron; Phagocytosis; Tretinoin

Cells of the human myeloid leukaemia cell line ML-1 were exposed to differentiation inducing doses of dimethylsulfoxide (DMSO) and retinoic acid (RA). DMSO (but not RA) caused an inhibition of cell growth which was reversible. Some granulocytic maturation associated changes were induced by both agents: nuclear segmentation in 10-20% of cells, B43.4 antigen positivity. In contrast, several other markers were not induced: nucleoli persisted in almost 100% cells including the segmented ones, the nuclear membrane regions did not stain with Victoria blue B (which stains these regions in normal neutrophils), the expression of other antigens of mature neutrophils was also not induced. Maturation asynchrony and non-physiological segmentation of round and oval nuclei were observed. Examination of nucleoli on a single cell level revealed a reversible decrease of pre-rRNA synthesis in 28-48% of the induced cells. These results indicate that terminal differentiation did not occur and confirm the dissociation in induction of various differentiation markers.

Topics: Antigens, Surface; Cell Differentiation; Cell Nucleolus; Cell Survival; Coloring Agents; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid; Organic Chemicals; RNA Precursors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced differentiation of a human monocytic leukemia cell line, THP-1. RA- or TPA-treated cells stopped proliferating, became adherent to plastic surfaces, and acquired the ability to phagocytose yeast cells, plain sheep RBCs, and IgG-coated sheep RBCs. The morphological and functional changes, induced by RA or TPA, were associated with a 20-50-fold increase in cellular transglutaminase activity. This increase in enzyme activity was found to be due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. The induction of tissue transglutaminase was a specific response of THP-1 cells to differentiation and was not observed with agents that did not induce their morphological or functional differentiation. Dibutyryl cyclic AMP potentiated the RA-induced expression of tissue transglutaminase. A 15-min exposure to TPA was sufficient to induce differentiation and expression of tissue transglutaminase in THP-1 cells. In contrast, RA required a continuous exposure (48 h) to induce similar changes in morphology or enzyme activity. These results support the view that differentiation of cells of the monocytic lineage is associated with an induction and accumulation of the protein cross-linking enzyme tissue transglutaminase.

Topics: Bucladesine; Cell Differentiation; Dimethyl Sulfoxide; Humans; Immunosorbent Techniques; Interferon-gamma; Leukemia, Myeloid; Macrophages; Phagocytosis; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transglutaminases; Tretinoin; Vitamin A

Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation.

Topics: Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; DNA Replication; Heme; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Receptors, Cell Surface; Receptors, Transferrin; Tetradecanoylphorbol Acetate; Transferrin; Tretinoin

WEHI-3B D+ monomyelocytic leukemia cells were induced to differentiate to mature granulocytes when treated with either 30 nM aclacinomycin A or 7 microM retinoic acid. Differentiation was assessed by the appearance of mature granulocytic phenotypes, as measured by the ability to reduce nitro blue tetrazolium, morphological changes, an increase in cell surface Fc receptors, as well as the loss of proliferative capacity. Maximum differentiation occurred 3 days after drug exposure. Analysis of DNA histograms of treated cells indicated that cells accumulated in the G1 phase of the cell cycle after 8 h of exposure to either inducer, with maximum accumulation occurring by 20 h; this arrest was observed prior to the phenotypic appearance of mature cells. The minimum interval of time necessary to commit cells to a differentiation pathway, which was less than one doubling time (9.2 h), closely paralleled the initial accumulation of cells in the G1 phase of the cell cycle. Since drug exposure for more than one cell division was required for maximum differentiation, the observed kinetics of maturation is consistent with a stochastic model. These studies support the idea that this cell line would be a particularly good model for extrapolation of findings with differentiating agents in culture to therapeutic monitoring in animals, since WEHI-3B D+ leukemia cells can be readily propagated in vivo in BALB/c mice.

Topics: Aclarubicin; Animals; Cell Cycle; Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Leukemia, Myeloid; Mice; Naphthacenes; Neutrophils; Receptors, Fc; Time Factors; Tretinoin

Four monocytoid cell lines, JOSK-I, -S, -M, and -K, were newly established successfully from peripheral blood of two cases of acute monocytic leukemia and one case each of acute myelomonocytic leukemia and chronic myelogenous leukemia in myelomonocytic blast crisis. In order to establish permanent cell lines, cultures of leukemic blasts were initiated in 96-well microtiter plates. Each cell line grew in a suspension culture with a doubling time of 24-32 h and has been serially maintained for over 20 mo. Each line had immature monocytic properties as judged from the results of cytological, immunochemical, and functional analyses. The cells showed a positive reaction for alpha-naphthyl butyrate esterase which was completely inhibited by sodium fluoride and exhibited immature monocytic features on electron microscopic observation. They also had surface markers specific for the monocyte-macrophage lineage. Chromosome analyses showed that each line had a variety of marker chromosomes; furthermore, these established lines exhibited high potentialities involving morphological and functional differentiation into more mature monocytic cells when induced by several chemical inducers. We also found that two of the established cell lines produced much interleukin 1 activity without any stimuli. These new lines might be valuable for studying the regulation of monocyte-macrophage differentiation and host defense mechanisms.

Topics: Aged; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Female; Humans; Interleukin-1; Karyotyping; Leukemia, Myeloid; Macrophages; Male; Middle Aged; Monocytes; Phagocytosis; Philadelphia Chromosome; Tetradecanoylphorbol Acetate; Tretinoin

The human monocytic leukemia cell line, THP-1, is induced to differentiate into more functionally mature monocyte (macrophage)-like cells by incubation with retinoic acid at concentrations of 10nM or higher. There is no apparent morphological change accompanying this functional maturation. These induced cells show increases in nitroblue tetrazolium reduction, immunoerythrophagocytosis, hexose monophosphate shunt activity, and 5'-nucleotidase and NAD+-glycohydrolase activities. Prostaglandin E2, dibutyryl cyclic adenosine 3':5'-monophosphate, or T-lymphocyte-derived differentiation-inducing activity, all inactive or less active alone, increase the extent of differentiation of THP-1 in combination with 10nM retinoic acid. THP-1 is also induced to differentiate by 0.1nM or higher concentrations of cholera toxin. Furthermore, 24,24-difluoro-1 alpha,25-dihydroxyvitamin D3 induces less differentiation of THP-1 compared to retinoic acid. Dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate show no induction of functional differentiation. THP-1 thus joins the list of leukemic myelomonocytic cell lines (e.g., the promyelocytic HL-60 and the monoblast-like U-937) that are blocked at a relatively late stage of maturation and which differentiate in response to retinoic acid.

Topics: 5'-Nucleotidase; Bucladesine; Cell Differentiation; Cell Line; Cholera Toxin; Culture Media; Dinoprostone; Drug Synergism; Glycoproteins; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Myeloid; Lymphokines; NAD+ Nucleosidase; Nucleotidases; Prostaglandins E; Tretinoin

Treatment of chronic phase chronic myelogenous leukemia with hydroxyurea or busulfan rarely induces cytogenetic (true) remissions. Intensive chemotherapy induces brief true remissions in approximately 50% of patients. We added 13-cis-retinoic acid to daunorubicin, cytosine arabinoside, and thioguanine to determine if it could increase the incidence and duration of remission induced by cytotoxic chemotherapy. Of the 17 evaluable patients, one patient (6%) achieved complete remission, and seven patients (41%) achieved partial remissions. The median duration of remission was 1.6 months. We conclude that 13-cis-retinoic acid does not increase the incidence and duration of remission in chronic phase chronic myelogenous leukemia.

Topics: Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Chromosomes, Human, 21-22 and Y; Cytarabine; Daunorubicin; Humans; Isotretinoin; Leukemia, Myeloid; Leukopenia; Liver; Middle Aged; Stomatitis; Thioguanine; Thrombocytopenia; Time Factors; Tretinoin

Two recently derived human myeloid leukemia cell lines, ML-1 and ML-2, were induced to differentiate by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or with retinoic acid for 5 to 12 days. They were then compared with similarly treated promyelocytic HL-60 cells. TPA-treated ML-1 and ML-2 cells became firmly surface adhesive with a fibroblastoid morphology, while TPA-treated HL-60 cells adhered as rounded macrophages. In contrast, retinoic acid induced only slight morphological changes in all three cell lines. The differentiation-related alterations of the surface membrane glycoproteins were followed by polyacrylamide gel electrophoresis after surface labeling by the periodate-NaB3H4 or galactose oxidase-NaB3H4 methods. The expression of surface membrane differentiation antigens was analyzed with a panel of monoclonal antibodies against myeloid, myelomonocytic, monocytic, and granulocytic determinants using FACS IV flow cytometry. The acquisition of surface adhesiveness by TPA-treated ML-1 and ML-2 cells coincided with the appearance of membrane surface proteins of varying molecular weights, ranging between 90,000 and 155,000, which were not labeled in untreated ML-1 and ML-2 cells. These findings and the results obtained by monoclonal antibody staining and FACS analysis indicate that treatment of the myeloid lines ML-1, ML-2, and HL-60 by TPA induced the expression of antigenic and membrane molecular features compatible with a monocytic-macrophage phenotype, while treatment by retinoic acid induced granulocytic differentiation. The ML-1 and ML-2 cells offer interesting models for studies on the membrane molecular events occurring when nonadherent, monocytic cells become surface adherent.

Topics: Antigens, Surface; Cell Differentiation; Cell Line; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Leukemia, Myeloid; Membrane Proteins; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Cell Differentiation; Cell Line; Cytarabine; Drug Synergism; Humans; Kinetics; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Tretinoin

The effect of increasing concentrations of retinoic acid (RA) on the in-vitro proliferation of normal and chronic myeloid leukemia (CML) granulo-monocyte precursors (CFU-GM) was studied. 10(-7)M RA added to semisolid cultures stimulated the growth of day 14 but not of day 7 normal CFU-GM, whereas in CML the growth of both populations was either unchanged or inhibited. Five-day and 10-day preincubation of normal bone marrow cells with RA augmented the number of day 14 CFU-GM (by up to 187% with 10(-6) M RA), whereas there was a marked decrease when CML cells were used. Total cellularity was not much affected, though a slight increase in liquid normal bone marrow cultures and a slight fall in CML cultures could be detected. These data point to a difference in the response to RA of normal and CML precursors. They may offer of preclinical basis for its employment to delay the blastic progression of CML.

Topics: Cell Differentiation; Cells, Cultured; Dose-Response Relationship, Drug; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Tretinoin

Eighteen patients with myelodysplastic syndrome received 13-cis-retinoic acid (1.0-mg/kg/day starting dose with 0.5-mg/kg increment escalations) in a phase I-II trial. Two partial responses involving the erythroid series were observed in four patients with primary refractory anemia with ring sideroblasts. One of two patients with chronic myelomonocytic leukemia also achieved a partial response. No other responses were found in the remaining patients, which included eight with refractory anemia with excess blasts. In six patients drug toxicity necessitated termination of the trial. Four patients had unexpected drug-induced thrombocytopenia; three of these had low platelet counts before treatment. Two of the six patients had other toxic effects. Further studies are warranted to evaluate the effectiveness of 13-cis-retinoic acid in patients with refractory anemia with ring sideroblasts and chronic myelomonocytic leukemia. At moderate doses significant toxic effects, including thrombocytopenia, are not uncommon.

Topics: Anemia, Refractory; Blood Cell Count; Drug Evaluation; Erythropoiesis; Hematopoietic Stem Cells; Hemoglobins; Humans; Isotretinoin; Leukemia, Myeloid; Myelodysplastic Syndromes; Thrombocytopenia; Tretinoin

Topics: Antineoplastic Agents; Aphidicolin; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Cytarabine; Diterpenes; DNA Replication; Formamides; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Mercaptopurine; Myeloproliferative Disorders; Reference Values; Tretinoin

Retinoic acid and other retinoids stimulate or inhibit a number of immune responses, but their mechanism of action on immune cells is not fully understood. However, retinoids have been shown to inhibit interferon production, so they could act by influencing the production of lymphokines. Hence we have studied the effect of retinoic acid on the production of interleukins (ILs) 1 and 3 in vitro. Models for the production of ILs were the murine macrophage cell line P388D1 and human peripheral blood mononuclear cells for IL 1 and the murine WEHI-3 cell line for IL 3. Retinoic acid stimulated IL 1 release by P388D1 cells in a dose-related fashion, starting at 10(-9) M and maximally at 10(-8)-10(-6) M. With peripheral blood mononuclear cells a maximal stimulation of IL 1 release was observed with 10(-7) M-retinoic acid. IL 3 release by WEHI-3 cells was also stimulated by retinoic acid in a dose-related fashion. The maximal response was obtained with 10(-8) M-retinoic acid. These results show that retinoic acid, in physiological concentrations, exerts selective effects on interleukin production in vitro, and this stimulation of IL 1 and IL 3 release may explain some of the immunostimulatory effects of retinoids in vivo. Moreover, since IL 1 is known to influence connective tissues and bone, an increase in IL 1 might also explain some of the changes observed in these tissues in vitamin A poisoning and with high-dose retinoid therapy.

Topics: Animals; Cell Line; Humans; Interleukin-1; Interleukin-3; Leukemia, Myeloid; Leukocytes; Lymphokines; Macrophages; Mice; Tretinoin

The human histiocytic lymphoma line U-937 consists of cells having characters of immature monocytes. We have demonstrated that these cells possess highly specific insulin receptors with binding properties similar to that found for mature human blood monocytes. 125I-insulin binding increased progressively with time to reach a maximum at 90 min at 22 degrees C and was proportional to the number of cells in the incubation medium. Insulin degradation as assessed by TCA precipitation was negligible. Scatchard analysis of the binding data was curvilinear and the total number of insulin binding sites was around 13,500. The average affinity profile gave an 'unoccupied site' affinity constant of 1.34 nM-1. When the U-937 cells were induced to differentiate into morphologically and functionally monocyte-like cells, after incubation with retinoic acid, the total number of binding sites decreased significantly with no change in the affinity of the hormone for its receptor.

Topics: Binding, Competitive; Cell Differentiation; Cell Line; Glucagon; Humans; Insulin; Leukemia, Myeloid; Receptor, Insulin; Somatostatin; Tretinoin

In order to better understand the mechanism by which changes in the fatty acid composition of cellular lipids occur in leukemia cell lines induced to differentiate, the activity of the first enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (EC 6.4.1.2) was measured in HL-60 promyelocytic leukemia cells before, during and after treatment with compounds that induce these cells to mature to neutrophillike cells. After 24 h of exposure to dimethylsulfoxide, retinoic acid, or butyric acid, no morphological or biochemical (nitroblue tetrazolium reduction) evidence of differentiation occurred, but acetyl-CoA carboxylase activity decreased 44, 44.5, and 49% respectively, compared to untreated cells. After 7 days of culture in the presence of these agents, 79, 83, and 72% of cells acquired the ability to reduce nitroblue tetrazolium (versus 15% of control cells) and enzyme activity decreased 92.7, 99.7, and 98%, compared to control cultures, with the three compounds respectively. Thus, some of the reported changes in fatty acid composition of leukemia cells with differentiation may arise, in part, from the depression of the de novo fatty acid biosynthetic pathway and the loss of acetyl-CoA carboxylase activity may be a useful marker for neutrophilic differentiation in HL-60 cells.

Topics: Acetyl-CoA Carboxylase; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Humans; Kinetics; Leukemia, Myeloid; Ligases; Neutrophils; Tretinoin

The alterations of stimulus-induced membrane potential changes, superoxide (O2-)-producing capacity and phagocytic activity during differentiation of human granulocytes were investigated in the human leukemia cell lines HL-60 and KG-1 differentiating in vitro and in human leukemic granulocytes obtained from chronic myelogenous leukemia patients. HL-60 cells incubated with dimethyl sulfoxide or with retinoic acid showed progressively increasing O2- production as well as membrane potential changes (depolarization) on contact with phorbol myristate acetate or the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, with a concomitant increase in the proportion of mature cells of the granulocytic type. Phagocytosis of latex particles, yeast, and oil droplets appeared 24 h after incubation with dimethyl sulfoxide and anteceded the increment of O2- production and membrane potential changes, both of which appeared concomitantly 3 d after incubation with dimethyl sulfoxide. Similar findings were observed when immature and mature granulocytes obtained from chronic myelogenous leukemia patients were stimulated by phorbol ester, the chemotactic peptide, or calcium ionophore A23187, and the amount of O2- production was parallel to the magnitude of membrane potential changes. HL-60 and KG-1 cells incubated for 1-6 d with phorbol myristate acetate showed neither O2- production nor membrane potential changes on contact with phorbol ester, chemotactic peptide, or A23187, although such cells resembled macrophages morphologically, and their phagocytic activity was significantly increased. O2- production and membrane potential changes in normal granulocytes induced by phorbol ester, chemotactic peptide and A23187 were inhibited by 2-deoxyglucose. These findings indicate that the O2--producing system and the system provoking membrane potential changes may develop concomitantly as human granulocytes mature and differentiate, and that the development of these systems and of phagocytic activity may be independently regulated.

Topics: Adult; Calcimycin; Cell Differentiation; Cell Transformation, Neoplastic; Deoxyglucose; Dimethyl Sulfoxide; Gramicidin; Granulocytes; Humans; Leukemia, Myeloid; Membrane Potentials; Phagocytosis; Superoxides; Tetradecanoylphorbol Acetate; Tretinoin

To study the effects of retinoic acid (RA) and indomethacin on myeloid differentiation, normal and chronic myeloid leukemic (CML) bone marrow cells lighter than 1.065 g/ml were incubated in liquid cultures and grown in agar. Retinoic acid (RA) (10(-8) M and 10(-6) M) increased the number of clusters (3-40 cells) formed in agar and also increased the neutrophil production in liquid cultures. Indomethacin (10(-6) M) did not change the cell growth in agar. In liquid culture, however, more macrophages and fewer neutrophils were produced in the presence of indomethacin. The results suggest that RA enhances neutrophil production by stimulating the proliferation and maturation of a cluster-forming cell within the myeloblast-promyelocyte compartment. The stimulatory effect of indomethacin on macrophage production is probably secondary to its inhibition of prostaglandin synthesis, which inhibits the proliferation of monocyte precursor cells. No major differences were noted between CML and normal cells in their responses to RA or indomethacin.

Topics: Cell Division; Cells, Cultured; Granulocytes; Humans; Indomethacin; Leukemia, Myeloid; Macrophages; Prostaglandins; Stem Cells; Tretinoin

Evidence is presented to indicate that the retiNOic acid (RA)-induced program of myeloid differentiation and growth arrest by HL-60 human promyelocytic leukemia cells was initiated by a signal originating at the cell membrane. Free RA and RA covalently immobilized on a solid substrate elicited similar kinetics of differentiation and G1/0-specific growth arrest. No evidence of cell-induced RA detachment from the solid substrate was found. The data explain why HL-60 cells which are deficient in cellular RA-binding protein (CRABP) nevertheless respond to RA.

Topics: Cell Differentiation; Cell Division; Cell Line; Cell Membrane; Humans; Leukemia, Myeloid; Tretinoin

Topics: Anilides; Cell Line; Humans; Leukemia, Myeloid; Phthalic Acids; Tretinoin

The effect of retinoic acid (all-trans) on the clonal growth of chronic myeloid leukemia marrow was studied. The results indicate that 3 X 10(-5) M, 3 X 10(-6) M, 3 X 10(-7) M and 3 X 10(-8) M of retinoic acid inhibits the colony stimulating activity of placental conditioned medium.

Topics: Bone Marrow; Cell Differentiation; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Tretinoin

Antigenic changes detected by myeloid-specific monoclonal antibodies on HL-60 cells induced to differentiate by various chemical mediators were investigated using flow cytometry. Antigen levels detected by monocyte-granulocyte-specific monoclonal antibodies AML-2-23, 61D3, and 63D3 increased dramatically after differentiation of HL-60 cells along the granulocytic pathway by the addition of dimethyl formamide (DMF), dimethylsulfoxide (DMSO), or cis-retinoic acid. The expression of these same antigens also increased in conjunction with monocytoid differentiation when HL-60 cells were treated with supernatants from leukocytes stimulated with phytohemagglutinin (PHA-LCM) or with mixed lymphocyte conditioned medium (MLC). In contrast, treatment of HL-60 cells with phorbol 12-myristate 13-acetate (PMA), which also induced differentiation along the monocyte pathway, had no effect on the expression of these monocyte-associated antigens. The expression of antigens on HL-60 cells recognized by the granulocyte-specified monoclonal antibodies PMN 6 and PMN 29 decreased after treatment of HL-60 cells with PMA, but remained constant after treatment with DMF, DMSO, cis-retinoic acid, PHA-LCM, or MLC. These results suggest that normal myeloid differentiation may be dependent on various signals and that morphological and cell surface marker maturity may, under some conditions, be separable. The utility of the HL-60 cell line as a model of myeloid differentiation and for evaluation of inductive signals is discussed.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Dimethylformamide; Epitopes; Granulocytes; Humans; Hybridomas; Leukemia, Myeloid; Mice; Tetradecanoylphorbol Acetate; Tretinoin

Three human myeloid leukemic cell lines (HL60, KG1, and ML3) and one histiocytic lymphoma line (U937) were induced to differentiate terminally to mature myelomonocytic cells with either 12-O-tetradecanoylphorbol-13-acetate (TPA) or lymphocyte-conditioned medium (LCM), which is known to contain differentiation-inducing factors. HL60 cells were also forced to differentiate along the myeloid series with retinoic acid (RA) or dimethyl sulfoxide (DMSO). The striking morphologic changes and the expression of differentiated markers on the induced cells (whether macrophage- or granulocyte-like) were always associated with an acquired or dramatically increased ability to stimulate proliferation and natural killer cell (NK) activity in human lymphocytes. Like HL60 cells after granulocytic differentiation, granulocytes freshly separated from the peripheral blood of healthy donors were also strong inducers of mixed lymphocyte reaction (MLR) responses. Analysis of the expression of HLA-DR antigens on the surface of undifferentiated and mature cells with two monoclonal antibodies directed against HLA-DR monomorphic determinants, indicated that 1) upon differentiation induced with RA, DMSO, and TPA the cells never acquired surface DR antigens, and 2) normal peripheral blood granulocytes lacked these antigens. In contrast, treatment with LCM always resulted in the expression of high levels of DR antigens on the differentiated macrophage-like cells. Taken together, these findings indicate that all mature myelomonocytic cells, either freshly separated from peripheral blood or obtained after forced in vitro differentiation of leukemic cells, express MLR stimulatory antigens that appear to be unrelated to DR determinants. The possibilities discussed are that such antigens are associated with other molecules encoded by the D region or with new surface structures unrelated to Ia but dependent on the stage of differentiation.

Topics: Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Granulocytes; Histiocytoma, Benign Fibrous; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Killer Cells, Natural; Leukemia, Myeloid; Lymphoma; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Calcitriol; Cell Differentiation; Cell Line; Colony-Forming Units Assay; Endotoxins; Humans; Isotretinoin; Kinetics; Leukemia, Experimental; Leukemia, Myeloid; Macrophages; Mice; Neutrophils; Tretinoin

13-cis-Retinoic acid (13-cRA) induces maturation and differentiation of neoplastic myeloid cell lines in vitro. We conducted a phase I clinical trial of 13-cRA in patients with myelodysplastic syndromes (MDS), using a single daily oral dose schedule. Seventeen patients with MDS and one each with acute nonlymphoblastic leukemia and chronic myelogenous leukemia in blast crisis were treated with 13-cRA at doses ranging from 20 to 125 mg/m2/day. Hepatotoxicity was dose-limiting and was manifested by hyperbilirubinemia and increased SGOT levels. This effect was seen only at the highest dose level of 125 mg/m2/day and was completely reversible upon cessation of the drug. Other toxic effects were mild, and included cheilosis, hyperkeratosis, stomatitis, and elevation of serum triglyceride levels. Fifteen patients with MDS were evaluable for therapeutic response. Five patients showed improvement in hematologic parameters. These responses included normalization of bone marrow blast count and increases in leukocyte count, platelet count, and/or hemoglobin concentration. Responses were generally not seen until at least 3 weeks of therapy were completed. We conclude that further study of 13-cRA in myelodysplastic syndromes is warranted and recommend that future studies utilize a starting dose of 100 mg/m2.

Topics: Administration, Oral; Adult; Aged; Blood Cell Count; Dose-Response Relationship, Drug; Drug Evaluation; Female; Humans; Isotretinoin; Leukemia, Myeloid; Liver; Male; Middle Aged; Myeloproliferative Disorders; Preleukemia; Tretinoin

Topics: Cell Differentiation; Cycloheximide; Dimethyl Sulfoxide; Gangliosides; Granulocytes; Humans; Leukemia, Myeloid; Membrane Potentials; Neuraminidase; Phagocytosis; Tretinoin

Necessary for growth and differentiation in many normal tissues and capable of inducing differentiation in human promyelocytic cell lines, retinoids were the subject of this study. Specifically, effects of 13-cis-retinoic acid and 13-trans-retinoic acid on the growth of normal human bone marrow cells in soft-agar system were studied. Both short-term incubation and continuous exposure to retinoic acid caused a decreased number of granulocyte colonies and an increased cluster-to-colony ratio. This effect was concentration-dependent. Examination of specimens stained with Wright-Giemsa or nitro blue tetrazolium stains showed a progressive increase in the percentage of immature granulocytic precursors with increasing concentrations of retinoic acid. No effect of retinoic acid was seen on a number of human tumor cell lines. Retinoic acid blocked both differentiation and proliferation and appeared to do so by specific, noncytotoxic mechanisms in normal human bone marrow cells.

Topics: Bone Marrow; Bone Marrow Cells; Burkitt Lymphoma; Cell Division; Cell Line; Clone Cells; Colony-Forming Units Assay; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Leukemia, Myeloid; Melanoma; Multiple Myeloma; Osteosarcoma; Tretinoin

Topics: Animals; Cell Differentiation; Cell Line; Humans; Interferons; Kinetics; Leukemia, Myeloid; Mercaptoethanol; Mice; Phytohemagglutinins; Tretinoin

Topics: Cell Differentiation; Cell Division; Cell Line; Dimethyl Sulfoxide; Granulocytes; Humans; Leukemia, Myeloid; Neuraminidase; Tretinoin

Mouse myeloid leukemia M1 cells were induced to differentiate in vitro into macrophages and granulocytes by various inducers, including dexamethasone. Prostaglandin F2 alpha inhibited the inductions by dexamethasone of phagocytic and lysozyme activities in M1 cells. Prostaglandin F2 alpha stimulated the production of differentiation-inhibiting activity (I-activity) in M1 cells. I-activity production by prostaglandin F2 alpha was decreased by simultaneous treatment with actinomycin D (5 ng/ml) but not with 5-fluoro-2'-deoxyuridine (10 ng/ml). The I-activity was inactivated by heating (70 degrees, 20 min) or by treatment with trypsin but not with mixed glycosidases or ribonuclease, suggesting that I-activity was due to a proteinous substance(s). B-Type prostaglandins also stimulated I-activity production, whereas A-, E- and D-type ones did not. Induction of prostaglandin E2. Retinoic acid stimulated the synthesis and release of prostaglandin F2 alpha and production of I-activity in M1 cells. Indomethacin completely inhibited induction of I-activity by retinoic acid. On the basis of these results, the relationship between I-activity production and prostaglandin F2 alpha production is discussed.

Topics: Animals; Cell Differentiation; Cells, Cultured; Dexamethasone; Dinoprost; Dinoprostone; Leukemia, Experimental; Leukemia, Myeloid; Mice; Mice, Inbred Strains; Muramidase; Phagocytosis; Prostaglandins E; Prostaglandins F; Tretinoin

Retinoic acid and retinol induced functional and morphological differentiation of human promyelocytic leukemia cells (HL-60) into mature granulocytes, but did not induce functional or morphological differentiation of mouse myeloid leukemia cells (M1). Cellular retinoic acid-binding protein, but not retinol-binding protein, was detected on HL-60 cells. Neither binding protein could be detected on M1 cells. These results suggest that retinoic acid-binding protein may be necessary for induction by retinoids of functional and morphological differentiation of myeloid leukemia cells.

Topics: Animals; Carrier Proteins; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Induction; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Muramidase; Neoplasm Proteins; Rats; Receptors, Retinoic Acid; Retinol-Binding Proteins; Tretinoin

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Blood Cells; Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Rabbits; Tetradecanoylphorbol Acetate; Tretinoin

The human leukemic cell lines HL60 and K562, were induced to differentiate terminally by chemical agents. The isoenzyme patterns of lactate dehydrogenase (LD) in the cells before and after differentiation were determined electrophoretically on agarose gels. In general, treatment of the leukemic cells with inducers of differentiation resulted in a quantitative shift of the isoenzyme pattern towards anodic or cathodic forms. This was correlated with the conversion of the chemically treated cells to morphologically more normal cells, as verified by light microscopy and/or synthesis of hemoglobin. The LD isoenzyme patterns of the chemically differentiated cells were: (a) characteristic for the particular cell type obtained rather than for the nature of the inducer used; and (b) not similar to those of normally differentiated cells of the corresponding lineage, indicating that incomplete differentiation had occurred.

Topics: Acetamides; Butyrates; Cell Differentiation; Cell Line; Cell Survival; Diamines; Dimethyl Sulfoxide; Erythropoiesis; Granulocytes; Hematopoiesis; Humans; Isoenzymes; L-Lactate Dehydrogenase; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Tetradecanoylphorbol Acetate; Tretinoin

Marrow culture studies revealed a spectrum of qualitative and quantitative defects in granulocyte-macrophage progenitors (GM-CFC) of patients with chronic myeloid leukemia in chronic phase and blastic crisis. Parallel culture studies and terminal transferase determinations revealed that a significant proportion of patients in blastic crisis possess two coexisting acute phase clones, one lymphoblastic and one myeloblastic. Measurement of response to and production of T cell growth factor showed that the leukemic blast cells from patients with TdT-positive blastic crisis produced the factor, but did not exhibit a proliferative response to exogenous factor. This phenotype was identical to that observed in TdT-positive acute lymphoblastic leukemia. Additional regulatory defects were identified in CML, since leukemic GM-CFC proliferation was resistant to inhibition by concentrations of prostaglandin E, which are markedly inhibitory for normal GM-CFC. The self-renewal or recloning capacity of GM-CFC was also identified as a unique feature of some patients with CML. The addition of retinoic acid to primary cultures of leukemic GM-CFC completely abolished this recloning capacity.

Topics: Bone Marrow; Cells, Cultured; Clone Cells; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Phenotype; Prostaglandins E; Tretinoin

Retinoic acid induced lysozyme activity in mouse myeloid leukemia M1 cells. It also stimulated the synthesis and release of prostaglandins such as prostaglandin F2alpha, E2, and D2 by the cells. The particulate fraction of retinoic acid-treated M1 cells converted arachidonate to prostaglandins, and this conversion was almost completely inhibited by indomethacin. Retinol, retinal and retinyl acetate, but not the pyridyl analog of retinoic acid, also induced lysozyme activity and stimulated synthesis and release of prostaglandins. Indomethacin inhibited the induction of lysozyme activity by retinoic acid. The induction of lysozyme activity and the stimulation of prostaglandin E2 production were dependent on the concentration of retinoic acid. Kinetic studies showed that stimulation of prostaglandin E2 production by retinoic acid was followed by induction of lysozyme activity. The tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol 12,13-didecanoate inhibited the induction of lysozyme activity by retinoic acid, but 4 alpha-phorbol didecanoate and phorbol did not. TPA and phorbol 12, 13-didecanoate, but not 4 alpha -phorbol didecanoate, also inhibited the stimulation of prostaglandin E2 production by retinoic acid. These results suggest that stimulation by retinoic acid of prostaglandin E2 production in M1 cells is a prerequisite for the induction of lysozyme activity. On the other hand, both retinoic acid and TPA inhibited the induction by dexamethasone of phagocytic activity, which is a typical functional marker of differentiation of M1 cells, without causing significant growth inhibition. Suboptimal concentrations of retinoic acid and TPA had synergistic inhibitory effects on the induction of phagocytic activity of M1 cells by dexamethasone.

Topics: Animals; Cell Line; Dinoprostone; Enzyme Induction; Indomethacin; Leukemia, Myeloid; Mice; Mice, Inbred Strains; Muramidase; Prostaglandins; Prostaglandins E; Tetradecanoylphorbol Acetate; Tretinoin

The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, proliferates continuously in suspension culture and consists predominantly (greater than 90%) of promyelocytes. These cells can be induced to differentiate to morphologically and functionally mature granulocytes by incubation with a wide variety of compounds, including butyrate and hypoxanthine and polar planar compounds such as dimethyl sulfoxide and hexamethylene bisacetamide. We have now found that retinoic acid (all-trans-retinoic acid) induces differentiation (as measured morphologically and by the ability to reduce nitroblue tetrazolium) of HL-60 at concentrations as low as 1 nM. Maximal differentiation (approximately 90%) occurs at 1 micro M, a concentration 1/500th to 1/160,000th the concentrations of butyrate (0.5 mM) and dimethyl sulfoxide (160 mM) that promote a similar increase in differentiation. Continuous exposure to retinoic acid is necessary for optimal differentiation, with the percentage of mature cells in the culture directly related to the length of time of exposure to retinoic acid. Retinoic acid and 13-cis-retinoic acid are equally effective in inducing differentiation of HL-60. Retinol (vitamin A), retinal, and retinyl acetate are approximately 1/1000th less potent. This study suggests that retinoids could provide a therapeutic tool in the treatment of acute myeloid leukemia, a disease that has been looked upon as primarily involving a block in myeloid differentiation, and indicates that retinoids, in addition to their well-characterized involvement in epithelial cell differentiation, may also be involved in the differentiation of certain hematopoietic cells.

Topics: Cell Differentiation; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Granulocytes; Hematopoiesis; Humans; Leukemia, Myeloid; Phagocytosis; Preleukemia; Structure-Activity Relationship; Time Factors; Tretinoin

Topics: Cell Differentiation; Cell Line; Dose-Response Relationship, Drug; Humans; Kinetics; Leukemia, Myeloid; Neutrophils; Phagocytosis; Structure-Activity Relationship; Tretinoin; Vitamin A

Retinoic acid, retinol, retinyl acetate, and retinal induced activities of lysosomal enzymes, such as lysozyme, acid protease, and acid phosphatase, in mouse myeloid leukemia cells (M1), while the pyridyl analog of retinoic acid had no effect. Retinoic acid was the most potent inducer of lysosomal enzyme activities. The induction of lysozyme activity by retinoic acid was inhibited by treatment with puromycin. The retinoids did not induce phagocytic and locomotive activities or morphological changes in M1 cells, and they inhibited the induction of these differentiation-associated properties by various inducers without inhibiting cell growth. Retinoic acid was the most potent inhibitor of induction of these differentiation-associated properties. The inhibitory effect of retinoic acid was found to be reversible. These results suggest that distinct mechanisms exist for control of induction of lysosomal enzyme activities and of other differentiation-associated properties of M1 cells, such as phagocytosis, morphological changes, and migration.

Topics: Animals; Cell Differentiation; Cell Line; Cell Movement; Dexamethasone; Diterpenes; Leukemia, Experimental; Leukemia, Myeloid; Lysosomes; Mice; Phagocytosis; Retinaldehyde; Retinyl Esters; Tretinoin; Vitamin A

The specific insulin binding activity of human promyelocitic HL 60 cell line during the myeloid and macrophagic differentiation induced by chemical compounds was investigated. Dimethyl sulfoxide (DMSO) and retinoic acid myeloid induced differentiation in HHL 60 cells was accompanied by a marked decrease of insulin receptors. In K 562 cell line, where DMS O has no effect on differentiation, the number of insulin receptors was only slightly affected. 12-0-tetradodecanoil phorbol 13-acetate (TPA) induced macrophagic differentiation of HL 60 cell line was also accompanied by a decrease of insulin binding activity. Our results support the hypothesis that during the process of terminal differentiation a decrease of insulin receptors occurs.

Topics: Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Granulocytes; Humans; Insulin; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Macrophages; Receptor, Insulin; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Alkaloids; Animals; Carcinogens; Cell Adhesion; Cell Line; Enzyme Induction; Female; Humans; Indoles; Irritants; Kinetics; Leukemia, Myeloid; Lyngbya Toxins; Mice; Ornithine Decarboxylase; Skin; Streptomyces; Structure-Activity Relationship; Tretinoin