trichostatin-a and Leukemia--Promyelocytic--Acute

Topics: Aminoglycosides; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Benzoates; Clinical Trials as Topic; Combined Modality Therapy; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gemtuzumab; Hematopoietic Stem Cell Transplantation; Humans; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Oxides; Practice Guidelines as Topic; Recurrence; Remission Induction; Tetrahydronaphthalenes; Tretinoin

Curcumin (Cur), a promising anticancer drug, kills tumor cells through either diminishing or promoting reactive oxygen species (ROS) generation. In this study, it was investigated whether trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor and a new anticancer drug, could improve the anticancer activity of low concentrations of Cur in human leukemia cells (HL-60). HL-60 cells were treated with Cur, TSA or their combinations; cell proliferation arrest, lactate dehydrogenase (LDH) release and cell viability were measured as indicators of cell damage. Reactive oxygen species (ROS) accumulation and the acetylation of histones were also measured. The cytotoxicity of Cur and TSA increased in a time and dose-dependent manner. Low Cur (no more than 20 microM) diminished the ROS generation in HL-60 cells, while high Cur (50 and 100 microM) promoted that. In contrast, TSA showed no influence on ROS generation. When their effects on histone acetylation were determined, low Cur showed no effect, while TSA significantly increased that. As expected, combinations of low Cur and TSA could not only diminish ROS generation, but also increase histone acetylation, and hence showed a more significant cytotoxicity in HL-60 cells. Since the extra ROS generation may also harm normal cells, instead of using high Cur, combining low Cur with TSA is obviously a better strategy to improve the anticancer activity of Cur.

Topics: Acetylation; Acetylcysteine; Antineoplastic Agents, Phytogenic; Antioxidants; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; Drug Synergism; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Indicators and Reagents; L-Lactate Dehydrogenase; Leukemia, Promyelocytic, Acute; Protein Synthesis Inhibitors; Reactive Oxygen Species

To investigate the potential role and the mechanism of PLZF-RARalpha/RARalpha-PLZF double fusion gene in the pathogenesis of acute promyelocytic leukemia (APL) in vivo at systematic biological level, PLZF-RARalpha/RARalpha-PLZF double transgenic mouse model was established by intercross; the integration and expression of fusion genes were analyzed by PCR and RT-PCR; the disease phenotype was detected by morphological and pathological examination of peripheral blood and bone marrow cells, as well as flow cytometry assays; the effects of ATRA with or without tricostatin A on bone marrow blast cells from PLZF-RARalpha/RARalpha-PLZF double TM were observed. The results showed that leukemia occurred in 5 PLZF-RARalpha/RARalpha-PLZF double TM 7, 7, 9, 11 and 11 months respectively, out of them two (40%) with classic APL features, the others (60%) with chronic myeloid leukemia through an observation period of 18 months. The leukemia occurrence of PLZF-RARalpha/RARalpha-PLZF TM was about 10%, which was similar to PLZF-RARalpha TM as that reported before. The latency was over 6 months, not earlier than PLZF-RARalpha TM only. No morphologic changes of PLZF-RARalpha/RARalpha-PLZF double TM blast cells to ATRA were observed, but increased cytoplasmic-nuclear ratio and nuclear condensation in bone marrow blast cells were found in combination of ATRA with tricostatin A. It is concluded that PLZF-RARalpha/RARalpha-PLZF double fusion gene transgenic mice have heterogeneity of pathogenesis. HDAC inhibitors such as trichostatin A, in combination with ATRA, induce differentiation of the blast/promyelocytic cells from PLZF-RARa/RARa-PLZF double TM, but not ATRA alone.

Topics: Animals; Antigens, CD34; Bone Marrow Cells; Cell Differentiation; Chorionic Gonadotropin; Disease Models, Animal; Female; Flow Cytometry; Humans; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Oncogene Proteins, Fusion; Pedigree; Receptors, Chemokine; 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

This study identified missense mutations in the ligand binding domain of the oncoprotein PML-RARalpha in 5 of 8 patients with acute promyelocytic leukemia (APL) with 2 or more relapses and 2 or more previous courses of all-trans retinoic acid (RA)-containing therapy. Four mutations were novel (Lys207Asn, Gly289Arg, Arg294Trp, and Pro407Ser), whereas one had been previously identified (Arg272Gln; normal RARalpha1 codon assignment). Five patients were treated with repeat RA plus phenylbutyrate (PB), a histone deacetylase inhibitor, and one patient experienced a prolonged clinical remission. Of the 5 RA + PB-treated patients, 4 had PML-RARalpha mutations. The Gly289Arg mutation in the clinical responder produced the most defective PML-RARalpha function in the presence of RA with or without sodium butyrate (NaB) or trichostatin A. Relapse APL cells from this patient failed to differentiate in response to RA but partially differentiated in response to NaB alone, which was augmented by RA. In contrast, NaB alone had no differentiation effect on APL cells from another mutant case (Pro407Ser) but enhanced differentiation induced by RA. These results indicate that PML-RARalpha mutations occurred with high frequency after multiple RA treatment relapses, indicate that the functional potential of PML-RARalpha was not correlated with clinical response to RA + PB treatment, and suggest that the response to RA + PB therapy in one patient was related to the ability of PB to circumvent the blocked RA-regulated gene response pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Binding Sites; Butyrates; Cell Differentiation; DNA Mutational Analysis; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Mutation, Missense; Neoplasm Proteins; Oncogene Proteins, Fusion; Phenylbutyrates; Prognosis; Protein Binding; Recurrence; Transcriptional Activation; Treatment Outcome; Tretinoin

Histone acetylation and deacetylation are closely linked to transcriptional activation and repression, respectively. In acute promyelocytic leukemia (APL), histone deacetylase inhibitors (HDACIs) have a synergistic effect with all-trans retinoic acid (ATRA) in vitro to induce differentiation. Here we report in vitro and in vivo effects of a HDACI, FK228 (formerly FR901228 or depsipeptide), on the human APL cell line NB4. FK228 had a strong and irreversible cytotoxicity compared with another HDACI, trichostatin A. In vivo administration of ATRA or FK228 alone partly inhibited the growth of established tumors of NB4 subcutaneously transplanted in NOD / Shi-scid / scid mice, and the combination was synergistically effective. Histopathological examination revealed that the combination induced apoptosis and differentiation as well as histone acetylation. Intravenous injection of NB4 in NOD / Shi-scid / scid mice followed by combination treatment significantly prevented leukemia death, whereas single administration did not. These findings suggest that FK228 is a promising agent to enhance ATRA-sensitivity in the treatment of APL.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Cell Differentiation; Depsipeptides; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Kinetics; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Peptides, Cyclic; Tretinoin; Tumor Cells, Cultured

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Northern; Blotting, Western; Cell Cycle; Cell Differentiation; Cell Division; DNA, Complementary; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Situ Nick-End Labeling; Leukemia, Promyelocytic, Acute; Mice; Mice, Transgenic; Microscopy, Fluorescence; Models, Chemical; Oligonucleotide Array Sequence Analysis; Phenylbutyrates; Protein Binding; Receptors, Retinoic Acid; Remission Induction; Retinoic Acid Receptor alpha; Time Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation; Vorinostat

Histone deacetylase (HDAC) appears to play an important role in the pathogenesis of acute promyelocytic leukaemia (APL) as it is recruited by both PML-RARalpha and PLZF/RAR alpha in leukaemic cells with t(15;17) and t(11;17) respectively. Recent studies have demonstrated that HDAC inhibitors can be therapeutically used in various neoplastic disorders including APL. Cell differentiation was considered the major mechanism of the anti-leukaemic effects of HDAC inhibitors in APL. However, most of these studies either evaluated the effect of HDAC inhibitors in combination with all-trans retinoic acid (ATRA) or focused on the less common form of APL with t(11;17). To investigate the cellular effects of HDAC inhibitors, including sodium butyrate, trichostatin A, and suberoylanilide hydroxamic acid (SAHA), we used two APL cell lines, NB4 and the ATRA-resistant derivative NB4.306. Moreover, primary cells from five patients with cytogenetic evidence for t(15;17) were also studied. Our results demonstrated that HDAC inhibitors induce distinct caspase-dependent apoptosis in APL, which showed both concentration-and time-dependence. In addition, changes in the apoptosis-regulatory proteins, daxx, bcl-2 and bax were analysed. HDAC inhibitors induced downregulation of daxx, but no significant changes were detected in bcl-2 or bax. In conclusion, apoptosis induced by HDAC inhibitors in APL could provide an effective strategy for treatment of patients with t(15;17).

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Antineoplastic Agents; Apoptosis; Butyrates; Carrier Proteins; Caspases; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Co-Repressor Proteins; Down-Regulation; Enzyme Inhibitors; Female; Flow Cytometry; Fluorescent Antibody Technique; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Leukemia, Promyelocytic, Acute; Middle Aged; Molecular Chaperones; Neoplasm Proteins; Nuclear Proteins; Translocation, Genetic; Tumor Cells, Cultured; Vorinostat

Acute promyelocytic leukaemia (APL) with t(11;17)/PLZF-RARalpha responds poorly to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3), in contrast to APL with t(15;17)/PML-RARalpha. Molecular studies have shown that histone deacetylase (HDAC) recruited by PLZF-RARalpha is associated with the ATRA resistance. Here, we analysed in vitro the differentiation of APL cells with t(11;17) using ATRA, As203, granulocyte colony-stimulating factor (G-CSF), HDAC inhibitor trichostatin A (TSA), or combinations of these. Although 1 microM ATRA, which stimulated the differentiation of APL cells with t(15;17), was insufficient to induce differentiation, 3 microM ATRA induced terminal differentiation into granulocytes. As203 alone or in combination with ATRA induced neither differentiation nor apoptosis. However, the combination of TSA and 1 microM ATRA had a potent differentiating effect, although TSA alone had little effect. The combination of 1 microM ATRA and G-CSF did not induce differentiation. These results indicate that APL cells with t(11;17) need a higher concentration of ATRA than those with t(15;17) to differentiate and suggest that HDAC inhibitor is a promising differentiation enhancer in APL with t(11;17).

Topics: Adult; Aged; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cell Differentiation; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Drug Therapy, Combination; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Male; Oxides; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

Non-liganded retinoic acid receptors (RARs) repress transcription of target genes by recruiting the histone deacetylase complex through a class of silencing mediators termed SMRT or N-CoR. Mutant forms of RARalpha, created by chromosomal translocations with either the PML (for promyelocytic leukaemia) or the PLZF (for promyelocytic leukaemia zinc finger) locus, are oncogenic and result in human acute promyelocytic leukaemia (APL). PML-RARalpha APL patients achieve complete remission following treatments with pharmacological doses of retinoic acids (RA); in contrast, PLZF-RARalpha patients respond very poorly, if at all. Here we report that the association of these two chimaeric receptors with the histone deacetylase (HDAC) complex helps to determine both the development of APL and the ability of patients to respond to retinoids. Consistent with these observations, inhibitors of histone deacetylase dramatically potentiate retinoid-induced differentiation of RA-sensitive, and restore retinoid responses of RA-resistant, APL cell lines. Our findings suggest that oncogenic RARs mediate leukaemogenesis through aberrant chromatin acetylation, and that pharmacological manipulation of nuclear receptor co-factors may be a useful approach in the treatment of human disease.

Topics: Antineoplastic Agents; Cell Line; Cloning, Molecular; DNA-Binding Proteins; Drug Resistance, Neoplasm; Enzyme Inhibitors; Escherichia coli; Hematopoiesis; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Nuclear Proteins; Nuclear Receptor Co-Repressor 2; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Repressor Proteins; Retinoic Acid Receptor alpha; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sin3 Histone Deacetylase and Corepressor Complex; Transcription Factors; Tretinoin; Tumor Suppressor Proteins

The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

Topics: Binding Sites; Cell Differentiation; Cell Line; Cloning, Molecular; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Leukemia, Promyelocytic, Acute; Mutagenesis; Neoplasm Proteins; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Oncogene Proteins, Fusion; Protein Binding; Receptors, Retinoic Acid; Repressor Proteins; Retinoic Acid Receptor alpha; Transcription Factors; Tretinoin; Tumor Suppressor Proteins