tretinoin and Blast-Crisis

Topics: Blast Crisis; Humans; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Tretinoin

Hemorrhagic death is the leading cause of treatment failure in acute promyelocytic leukemia (APL). Our ability to identify patients at greatest risk of hemorrhage, and to actively prevent hemorrhage, remains limited. Nevertheless, some data is available to guide contemporary clinical practice and future investigation. Circulating disease burden, best represented by the peripheral WBC / blast count, is the most consistent predictor of major and fatal bleeding risk. In contrast, laboratory markers of disseminated intravascular coagulation (DIC) appear to be poor predictors. A number of interventions have been posited to reduce bleeding risk. Prompt initiation of all-trans retinoic acid (ATRA), avoidance of invasive procedures, transfusion support, and cytoreduction all have theoretical merit. Though they lack strong evidence to support their benefit with respect to bleeding, they are associated with limited risks, and are therefore advisable. Low-dose therapeutic heparin and antifibrinolytics have not shown the ability to positively modify bleeding risk, and heparin has been associated with harm. Thrombomodulin has shown promise in limited retrospective studies however further prospective data are needed. rFVIIa may have a role in cases of life-threatening bleeding however evidence is largely anecdotal. Additional studies evaluating the above interventions, and investigating other potential interventions are needed.

Topics: Blast Crisis; Factor VIII; Hemorrhage; Heparin; Humans; Leukemia, Promyelocytic, Acute; Thrombomodulin; Tretinoin

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

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

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

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

An understanding of the prognostic factors associated with the various forms of induction mortality in patients with acute promyelocytic leukemia (APL) has remained remarkably limited. This study reports the incidence, time of occurrence, and prognostic factors of the major categories of induction failure in a series of 732 patients of all ages (range, 2-83 years) with newly diagnosed APL who received all-trans retinoic acid (ATRA) plus idarubicin as induction therapy in 2 consecutive studies of the Programa de Estudio y Tratamiento de las Hemopatias Malignas (PETHEMA) Group. Complete remission was attained in 666 patients (91%). All the 66 induction failures were due to induction death. Hemorrhage was the most common cause of induction death (5%), followed by infection (2.3%) and differentiation syndrome (1.4%). Multivariate analysis identified specific and distinct pretreatment characteristics to correlate with an increased risk of death caused by hemorrhage (abnormal creatinine level, increased peripheral blast counts, and presence of coagulopathy), infection (age>60 years, male sex, and fever at presentation), and differentiation syndrome (Eastern Cooperative Oncology Group [ECOG] score>1 and low albumin levels), respectively. These data furnish clinically relevant information that might be useful for designing more appropriately risk-adapted treatment protocols aimed at reducing the considerable problem of induction mortality in APL.

Topics: Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Child; Child, Preschool; Creatinine; Disease-Free Survival; Female; Hemorrhage; Humans; Idarubicin; Infections; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Remission Induction; Risk Factors; Sex Factors; Survival Rate; Syndrome; Treatment Failure; Tretinoin

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

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

An in vitro synergism between different inducers of AML cell differentiation has been previously observed. Therefore, we treated 53 myelodysplastic (MDS) patients with a low dose combination of cis-retinoic acid (cRA, 20-40 mg/day) and 1,25 alpha (OH)2 cholecalciferol [(OH)2D3, 1-1.5 micrograms/day] +/- intermittent 6-thioguanine (30 mg/m2/day). The latter was reserved for patients with bone marrow (BM) blast excess (> or = 5%). The treatment was well tolerated, without major toxicity. Among 25 patients with BM blasts less than 5%, we observed one complete, eight partial and four minor responses (response rate 52%) with a median response duration of 8 months (2 +/- 24). Median survival, which did not correlate with response, is projected at 76 months. Thirty-one patients with BM blast excess (> or = 5%), including three of the previous group who progressed to refractory anemia with excess of blasts (RAEB), were treated with the three-drug protocol. One complete, 12 partial and six minor responses were obtained (response rate 61%) with a median response duration of 6 months (2-29+). A significant difference in survival (P < 0.005) was observed between the 19 responders (median 25 months) and the 12 non-responders (median 9 months). A reduction in the transfusion need was observed in 41% of the transfusion-dependent patients with blast excess and in 53% of those without blast excess. Therefore, combined differentiating therapy seems more effective than previously reported single agent treatments and should be considered for a larger randomized study to assess its actual impact on survival of MDS patients.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Blood Transfusion; Bone Marrow Transplantation; Cholecalciferol; Female; Humans; Male; Middle Aged; Myelodysplastic Syndromes; Remission Induction; Survival Analysis; Thioguanine; Tretinoin

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

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

A 37-year-old patient was transferred to Haematology from the ENT Emergency Department where he had been admitted due to tonsillitis. He displayed anaemia and leukopenia and had agranulocytosis in the study. A day later the patient had blast crisis, and was diagnosed with myeloid acute leukaemia. Due to blast crisis the patient experienced sudden back pain, with oliguria and renal function deterioration followed by anaemia, in the context of haemolysis consistent with thrombotic microangiopathy, and as such, we were consulted. We began treatment with plasmapheresis and on the following day we performed haemodialysis (we carried out a total of 12 sessions of plasmapheresis until haemolysis disappeared). Five days later there was respiratory failure, and the patient was consequently transferred to the Intensive Care Unit, where he continued treatment with plasmapheresis and haemodialysis. The patient remained anuric thereafter, requiring haemodialysis, with no sign of renal recovery. Once platelet levels normalised with haematology chemotherapy, a percutaneous renal biopsy was performed, which confirmed the diagnosis of cortical necrosis. Finally, the patient underwent renal replacement therapy by regular haemodialysis.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Hemolytic-Uremic Syndrome; Humans; Idarubicin; Ischemia; Kidney; Kidney Cortex Necrosis; Leukemia, Promyelocytic, Acute; Male; Plasma; Plasmapheresis; Renal Dialysis; Respiratory Insufficiency; Tonsillitis; Tretinoin

The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17) that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20-26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

Topics: Antibodies, Monoclonal; Binding Sites; Blast Crisis; Cell Line, Tumor; Cell Membrane; Humans; Leukemia, Promyelocytic, Acute; Plasminogen; Protein Binding; Receptors, Urokinase Plasminogen Activator; Tretinoin

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

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

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

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

All-trans retinoic acid (RA) treatment of patients with acute promyelocytic leukemia (APL) induces complete remission in more than 90% of the cases. Although RA therapy is well tolerated, about 25% of APL patients develop a potentially fatal condition called retinoic acid syndrome (RAS). Molecular mechanisms underlying the development of RAS pathogenesis, especially those that result in the damage of endothelial cells remain elusive. In the present study, we found that RA treatment induces the expression of interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta) in peripheral blast cells from APL patients. IFN-gamma and IL-1beta also exerted synergistic effect in driving human umbilical cord endothelial cells (HUVECs) and human lung microvascular endothelial cells (HLMVECs) into apoptosis. RA also upregulated the expression of CD38, an ectoenzyme responsible for the generation of the calcium messenger cyclic ADP-ribose. Importantly, RA-induced CD38 expression promoted strong attachment of leukemia cells to endothelial cells, and incubation of endothelial cells with either high concentration (100 ng/ml) of IFN-gamma alone or low concentration of IL-1beta and IFN-gamma (10 ng/ml, each) induced strong apoptotic responses as revealed by caspase-8 activation and DNA fragmentation. Our results suggest that these RA-induced events could contribute to the development of RAS pathogenesis in patients with APL.

Topics: ADP-ribosyl Cyclase 1; Antineoplastic Agents; Apoptosis; Blast Crisis; Blotting, Western; Cell Adhesion; Cell Differentiation; Cells, Cultured; Endothelium, Vascular; Humans; Interferon-gamma; Interleukin-1beta; Leukemia, Promyelocytic, Acute; Lung; Nitric Oxide; Receptors, Retinoic Acid; Respiration Disorders; Syndrome; Tretinoin; Umbilical Veins

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

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

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

The acute promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion product recruits histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activities on retinoic acid (RA)-target promoters causing their silencing and differentiation block. RA treatment induces epigenetic modifications at its target loci and restores myeloid differentiation of APL blasts. Using RA-sensitive and RA-resistant APL cell lines and primary blasts, we addressed the functional relevance of the aberrant methylation status at the RA-target promoter RARbeta2 and the mechanism by which methylation is reversed by RA. RA decreased DNMT expression and activity, which correlated with demethylation at specific sites on RARbeta2 promoter/exon-1, and the ability of APL blasts to differentiate in vitro and in vivo. None of these events occurred in an RA-resistant APL cell line containing a PML-RARalpha defective for ligand binding. The specific contribution of the HDAC and DNMT pathways to the response of APL cells to RA was also tested by inhibiting these enzymatic activities with TSA and/or 5-azacytidine. In RA-responsive and RA-resistant APL blasts, TSA and 5-azacytidine induced specific changes on the chromatin state at RA-target sites, increased the RA effect on promoter activity, endogenous RA-target gene expression and differentiation. These results extend the rationale for chromatin-targeted treatment in APL and RA-resistant leukemias.

Topics: Blast Crisis; Bone Marrow Cells; Cell Culture Techniques; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Modification Methylases; DNA Primers; DNA, Neoplasm; Exons; Histone Deacetylases; Humans; Karyotyping; Leukemia, Promyelocytic, Acute; Polymerase Chain Reaction; Promoter Regions, Genetic; Receptors, Retinoic Acid; Tretinoin

Acute leukemia with the t(11;17) expressing the PLZF-RARalpha gene fusion is a rare variant of acute promyelocytic leukemia (APL) that has been associated with poor clinical response to all-trans retinoic acid (ATRA) treatment. However, some recent reports have put into question the absolute refractoriness of this leukemia to ATRA. We describe here a patient with PLZF/RARalpha APL who was treated at relapse with ATRA and low-dose hydroxyurea. Complete hematologic remission was obtained through differentiation of leukemic blasts, as proven by morphologic, immunophenophenotypic, and genetic studies carried out in sequential bone marrow samples. Moreover, in vitro studies indicated that blast differentiation was potentiated by the addition of the histone deacetylase inhibitor tricostatin A, but not of hydroxyurea, to ATRA. Our findings indicate that the maturation block may be overcome and terminal differentiation obtained in this leukemia subset and support the view that sensitivity/refractoriness of this form to ATRA should be revisited.

Topics: Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Cell Differentiation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins, Fusion; Remission Induction; Tretinoin

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

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

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

Although retinoic acid receptor alpha (RARalpha) agonists induce the maturation of t(15;17) acute promyelocytic leukemia (APL) cells, drug treatment also selects leukemic blasts expressing PML-RARalpha fusion proteins with mutated ligand-binding domains that no longer respond to all-trans retinoic acid (ATRA). Here we report a novel RARalpha-independent signaling pathway that induces maturation of both ATRA-sensitive and ATRA-resistant APL NB4 cells, and does not invoke the ligand-induced alteration of PML-RARalpha signaling, stability or compartmentalization. This response involves a cross-talk between RXR agonists and protein kinase A signaling. Our results indicate the existence of a separate RXR-dependent maturation pathway that can be activated in the absence of known ligands for RXR heterodimerization partners.

Topics: Amino Acid Sequence; Base Sequence; Benzoates; Blast Crisis; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Cyclic AMP-Dependent Protein Kinases; Dimerization; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptor Cross-Talk; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Signal Transduction; Transcription Factors; Translocation, Genetic; Tretinoin

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

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

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

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

A 44-year-old woman was diagnosed as having acute promyelocytic leukemia (APL) in April 1988. On her first admission, chromosomal translocation (15; 17), +8, and +12 was detected. When she was readmitted to our hospital with the second relapse in May 1990, t(3; 13) and +8 was detected, instead of t(15;17). Complete remission was re-achieved with VP-16, MIT, and BHAC, but the third relapse occurred in September 1990. After obtaining informed consent, she was given etretinate 40 mg per day orally for 17 days, without any effect on leukemia. She was then given all-trans retinoic acid (ATRA) 60 mg per day orally for 29 days. Although a mild granulocytic recovery was observed, no sufficient hematological recovery was obtained (minor response). Besides common side effects of ATRA, such as dry skin and hypertriglycedemia, she had a myeloblastoma in the oral cavity, but it is unknown whether the symptom was a complication of ATRA therapy or not.

Topics: Administration, Oral; Adult; Blast Crisis; Female; Humans; Leukemia, Promyelocytic, Acute; Mouth Neoplasms; Tretinoin

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

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

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

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

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

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

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

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

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

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

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

The 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