trichostatin-a and Leukemia

The antiparasitic clioquinol (CQ) represents a class of novel anticancer drugs by interfering with proteasome activity. In the present study, we found that CQ induced blood cancer cell apoptosis by inhibiting histone deacetylases (HDACs). CQ accumulated the acetylation levels of several key proteins including histone H3 (H3), p53, HSP90, and α-tubulin. In the mechanistic study, CQ was found to down-regulate HDAC1, -3, -4, and -5 in both myeloma and leukemia cells. Computer modeling analysis revealed that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp-267. Moreover, co-treatment with CQ and zinc/copper chloride led to decreased Ac-H3. Furthermore, CQ inhibited the activity of Class I and IIa HDACs in the cell-free assays, demonstrating that CQ interfered with HDAC activity. By inhibiting HDAC activity, CQ induced expression of p21, p27, and p53, cell cycle arrest at G1 phase, and cell apoptosis. This study suggested that the HDAC enzymes are targets of CQ, which provided a novel insight into the molecular mechanism of CQ in the treatment of hematological malignancies.

Topics: Antipruritics; Apoptosis; Chlorides; Clioquinol; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Leukemic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; K562 Cells; Leukemia; Male; Mouthwashes; Multiple Myeloma; Neoplasm Proteins; U937 Cells; Zinc Compounds

Although the protein-coding genes are subject to histone hyperacetylation- mediated regulation, it is unclear whether microRNAs are similarly regulated in the T cell leukemia Jurkat.. To determine whether treatment with the histone modifier Trichostatin A could concurrently alter the expression profiles of microRNAs and protein-coding genes.. Changes in histone hyperacetylation and viability in response to drug treatment were analyzed, respectively, using western blotting and flow cytometry. Paired global expression profiling of microRNAs and coding genes was performed and highly regulated genes have been validated by qRT-PCR. The interrelationships between the drug-induced miR-494 upregulation, the expression of putative target genes, and T cell receptor-mediated apoptosis were evaluated using qRT-PCR, flow cytometry, and western blotting following lipid-mediated transfection with specific anti-microRNA inhibitors.. Treatment of Jurkat cells with Trichostatin A resulted in histone hyperacetylation and apoptosis. Global expression profiling indicated prominent upregulation of miR-494 in contrast to differential regulation of many protein-coding and non-coding genes validated by qRT-PCR. Although transfection with synthetic anti-miR-494 inhibitors failed to block drug-induced apoptosis or miR-494 upregulation, it induced the transcriptional repression of the PVRIG gene. Surprisingly, miR-494 inhibition in conjunction with low doses of Trichostatin A enhanced the weak T cell receptor- mediated apoptosis, indicating a subtle pro-survival role of miR-494. Interestingly, this prosurvival effect was overwhelmed by mitogen-mediated T cell activation and higher drug doses, which mediated caspase-dependent apoptosis.. Our results unravel a pro-survival function of miR-494 and its putative interaction with the PVRIG gene and the apoptotic machinery in Jurkat cells.

Topics: Apoptosis; Cell Proliferation; Gene Expression; Histones; Humans; Hydroxamic Acids; Jurkat Cells; Leukemia; MicroRNAs

Some drugs can be used to treat multiple diseases, suggesting potential patterns in drug treatment. Determination of drug treatment patterns can improve our understanding of the mechanisms of drug action, enabling drug repurposing. A drug can be associated with a multilayer tissue-specific protein-protein interaction (TSPPI) network for the diseases it is used to treat. Proteins usually interact with other proteins to achieve functions that cause diseases. Hence, studying drug treatment patterns is similar to studying common module structures in multilayer TSPPI networks. Therefore, we propose a network-based model to study the treatment patterns of drugs. The method was designated SDTP (studying drug treatment pattern) and was based on drug effects and a multilayer network model. To demonstrate the application of the SDTP method, we focused on analysis of trichostatin A (TSA) in leukemia, breast cancer, and prostate cancer. We constructed a TSPPI multilayer network and obtained candidate drug-target modules from the network. Gene ontology analysis provided insights into the significance of the drug-target modules and co-expression networks. Finally, two modules were obtained as potential treatment patterns for TSA. Through analysis of the significance, composition, and functions of the selected drug-target modules, we validated the feasibility and rationality of our proposed SDTP method for identifying drug treatment patterns. In summary, our novel approach used a multilayer network model to overcome the shortcomings of single-layer networks and combined the network with information on drug activity. Based on the discovered drug treatment patterns, we can predict the potential diseases that the drug can treat. That is, if a disease-related protein module has a similar structure, then the drug is likely to be a potential drug for the treatment of the disease.

Topics: Breast Neoplasms; Drug Repositioning; Female; Gene Ontology; Gene Regulatory Networks; Humans; Hydroxamic Acids; Leukemia; Male; Models, Biological; Prostatic Neoplasms; Protein Interaction Maps; Protein Synthesis Inhibitors; Transcriptome

Umbilical cord blood is a valuable source of hematopoietic stem cells. While cytokine stimulation can induce ex vivo hematopoietic cell proliferation, attempts have been made to use epigenetic-modifying agents to facilitate stem cell expansion through the modulation of cellular epigenetic status. However, the potential global effect of these modifying agents on epigenome raises concerns about the functional normality of the expanded cells. We studied the ex vivo expansion of cord blood hematopoietic stem and progenitor cells (HSPCs) by histone deacetylase (HDAC) inhibitors, trichostatin A and valproic acid. Treatment with HDAC inhibitors resulted in mild expansion of the total hematopoietic cell number when compared with cytokine stimulated sample. Nevertheless, we observed 20-30-fold expansion of the CD34

Topics: Cell Culture Techniques; Cells, Cultured; Fetal Blood; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Valproic Acid

SUV39H1 is a histone 3 lysine 9 (H3K9)-specific methyltransferase that is important for heterochromatin formation and the regulation of gene expression. Chaetocin specifically inhibits SUV39H1, resulted in H3K9 methylation reduction as well as reactivation of silenced genes in cancer cells. Histone deacetylase (HDAC) inhibitors inhibit deacetylases and accumulate high levels of acetylation lead to cell cycle arrest and apoptosis. In this study, we demonstrated that treatment with chaetocin enhanced apoptosis in human leukemia HL60, KG1, Kasumi, K562, and THP1 cells. In addition, chaetocin induced the expression of cyclin-dependent kinase inhibitor 2B (p15), E-cadherin (CDH1) and frizzled family receptor 9 (FZD9) through depletion of SUV39H1 and reduced H3K9 methylation in their promoters. Co-treatment with chaetocin and HDAC inhibitor trichostatin A (TSA) dramatically increased apoptosis and produced greater activation of genes. Furthermore, this combined treatment significantly increased loss of SUV39H1 and reduced histone H3K9 trimethylation responses accompanied by increased acetylation. Importantly, co-treatment with chaetocin and TSA produced potent antileukemic effects in leukemia cells derived from patients. These in vitro findings suggest that combination therapy with SUV39H1 and HDAC inhibitors may be of potential value in the treatment of leukemia.

Topics: Acetylation; Adolescent; Adult; Aged; Apoptosis; Cadherins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p15; DNA Methylation; Enzyme Inhibitors; Frizzled Receptors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; HL-60 Cells; Humans; Hydroxamic Acids; K562 Cells; Leukemia; Leukemia, Myeloid, Acute; Male; Middle Aged; Piperazines; Promoter Regions, Genetic; Young Adult

We have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized.. This study examined the methylation status at histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI), Trichostatin A (TSA), and 5-aza-2'-deoxycytidine (5-Aza) on the cells.. We found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases.. This study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

Topics: Acetylation; Acute Disease; Adolescent; Adult; Aged; Antimetabolites, Antineoplastic; Azacitidine; Case-Control Studies; Child; Decitabine; DNA Methylation; Female; Histone Deacetylase Inhibitors; Histone-Lysine N-Methyltransferase; Histones; Humans; Hydroxamic Acids; Isothiocyanates; Leukemia; Male; Middle Aged; Protein Processing, Post-Translational; Young Adult

Histone deacetylase inhibitors (HDACi) have been extensively studied as potential candidates for treatment of various malignancies, including leukemia, since they not only induce growth inhibition, cell cycle arrest and apoptosis of cancer cells, but can also increase the sensitivity of cancer cells to chemotherapeutic drugs. The aim of this study was to investigate the effect of two HDACi, trichostatin A (TSA) and valproic acid (VPA), on etoposide-induced apoptosis in human leukemia cell lines.. Viability, apoptosis rate, caspase activity, mitochondrial membrane potential and expression of BCL2 mRNA were assessed in HL60 and U937 cell lines treated with 250 nM TSA or 1.25 mM VPA alone or followed by 5 μM etoposide.. Preincubation of HL60 cells with TSA or VPA significantly potentiated etoposide-induced cytotoxicity and apoptosis, which was associated with activation of caspases and loss of mitochondrial membrane potential. Similar effects were not observed in U937 cells. Expression of BCL2 mRNA was strongly down-regulated after treatment of cells with HDACi alone but did not show additive effect with etoposide.. Combination of HDACi with etoposide can have a synergistic effect on increased apoptosis in leukemia cells but this effect depends on the cancer cell type and other factors such as the concentration of drugs and the administration schedule.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase 3; Caspase 7; Drug Synergism; Etoposide; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; U937 Cells; Valproic Acid

Histone deacetylase (HDAC) inhibitors are a new class of epigenetic agents that were reported to enhance the cytotoxic effects of classical anticancer drugs through multiple mechanisms. However, which of the possible drug combinations would be the most effective and clinically useful are to be determined. We treated the HL60 and NB4 promyelocytic leukaemia cells with a combination of the ribonucleotide reductase (RR) inhibitor 3'-C-methyladenosine (3'-Me-Ado) and several hydroxamic acid-derived HDAC inhibitors, including two recently synthesized molecules, MC1864 and MC1879, and the reference compound trichostatin A (TSA). The results showed significant growth inhibitory and apoptotic synergistic effects with the combinations. Hence, we evaluated the effects of the combinations on cell cycle distribution and on the level of several proteins involved in the apoptotic process (p21, caspase-3, Bcl-2, Bax, AIF). Since HDAC inhibitors increased the G1-S transition block induced by 3'-Me-Ado, an effect on RR activity was hypothesized. Indeed, the HPLC evaluation of intracellular deoxyribonucleotide (dNTP) pools showed that both TSA and MC1864 induced a decrease in dNTPs, even if with a somewhat different pattern, suggesting that RR inhibition contributes to the observed synergism. Furthermore, while TSA was shown to activate the intrinsic apoptotic pathway, MC1864 induced a dose-dependent increase in ROS and AIF levels. Moreover, the treatment with the radical scavenger N-acetylcysteine determined a significant inhibition of MC1864- but not TSA-mediated synergistic effects. Hence, our findings are consistent with a possible role of HDAC inhibitor mediated-ROS induction in RR inhibition and in the potentiation of RR inhibitor-mediated apoptosis.

Topics: Adenosine; Antineoplastic Agents; Apoptosis; Cell Proliferation; Deoxyribonucleotides; G1 Phase; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Reactive Oxygen Species; Ribonucleotide Reductases

Here, we focus on epigenetic changes in leukaemia and MM (multiple myeloma) cells. We show how the histone signature, DNA methylation and levels of select tumour-suppressor proteins can be affected by inhibitors of HDACs (histone deacetylases) and Dnmts (DNA methyltransferases). Both inhibitors, TSA (trichostatin A) and 5-AZA (5-azacytidine), have the ability to change the histone signature in a tumour-specific manner. In MM cells, we observed changes in H3K4 methylation, while in leukaemia cells, H3K9 methylation was especially affected by select inhibitors. Compared with normal peripheral blood lymphocytes, tumour cell samples were characterized by increased H3K9 acetylation, increased H3K4me2, H3K9me2 and HP1α (heterochromatin protein 1α) levels and specific changes were also observed for DNA methylation. Additionally, we showed that the tumour suppressor pRb1 (retinoblastoma protein) is more sensitive to epigenetic-based anti-cancer stimuli than p53. We have found significant decrease in the levels of pRb1 and p53 in both myeloma and leukaemia cells after HDAC inhibition.

Topics: Antineoplastic Agents; Azacitidine; Chromobox Protein Homolog 5; DNA Methylation; DNA Modification Methylases; Epigenesis, Genetic; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leukemia; Multiple Myeloma; Retinoblastoma Protein; Tumor Suppressor Protein p53

Histone deacetylase inhibitors (HDACIs) inhibit deacetylases and the accumulation of high levels of acetylation results in chromatin remodeling events which may lead to cell cycle arrest and apoptosis. This work investigates the sensitivity of four leukemic cell lines to the HDACI, trichostatin A (TSA) as compared to normal lymphocytes with respect to acetylation and apoptotic levels. Specifically, this study analyzes the time kinetics of histone H4 and alpha-tubulin acetylation and associates these findings to the time course of TSA-induced PARP cleavage and DFF45 proteolysis. The results of this study show (1) that a non-responsive leukemic cell line to the apoptotic effects of TSA does not have increased acetylation levels in contrast to the responsive leukemic cell lines that show a hyperacetylated profile. This indicates that acetylation levels may be of special importance in accessing the potential sensitivities of leukemic cells to HDACIs, (2) TSA induced apoptosis in lymphocytes but at lower levels and (3) the lack of PARP cleavage and DFF45 proteolysis found in lymphocytes clearly differentiates the final stages apoptosis of human peripheral blood lymphocytes from those of the TSA-sensitive leukemic cell lines. Of value is that the results of this study show that the evaluation of the acetylation levels of target proteins may possibly have the potential of being used as additional indicators of the responsiveness or sensitivity of different cancer cell types to this continuously growing class of anticancer agents.

Topics: Acetylation; Apoptosis; Cell Line, Tumor; DNA Fragmentation; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Hydroxamic Acids; K562 Cells; Leukemia; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Tubulin

In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV-positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8-fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV-positive AGS/BX1 cells when compared with that of EBV-negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus-targeted drug for treatment of EBV-associated epithelial malignancies.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Burkitt Lymphoma; Butyrates; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Survival; Fluorescent Antibody Technique; Herpesvirus 4, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Polymerase Chain Reaction; Stomach Neoplasms; Valproic Acid; Vorinostat

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Chemical Fractionation; Chromatin; Deoxyribonucleases; Histones; Humans; Hydroxamic Acids; Immunoprecipitation; Leukemia; Poly-ADP-Ribose Binding Proteins; Protein Transport; Proteins; Substrate Specificity

Arsenic trioxide (ATO) is an effective therapeutic agent for acute promyelocytic leukemia (APL) and other hematopoietic malignancies. We found that ATO down-regulated the global DNA methylation level in HL-60 cells with high-performance capillary electrophoresis (HPCE) assay. Using combination index method of Chou and Talalay, interactions between ATO and epigenetic therapeutic agents were analyzed in three human leukemia cell lines (HL-60, U937, and K562). A synergistic interaction was observed in HL-60 cells between ATO and 5-Aza-2'-Deoxycytidine (DAC), while an antagonistic interaction was found in U937 cells between ATO and valproic acid (VPA). The combination of ATO with trichostatin A (TSA) caused an antagonistic interaction in U937 and K562 cells. These results not only highlight possible diversity of the anti-leukemia mechanisms of ATO, but also provide initial guide for further investigation of leukemia therapies based on the combination of ATO with epigenetic agents.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Azacitidine; Cell Proliferation; Cell Survival; Decitabine; DNA Methylation; DNA Modification Methylases; DNA, Neoplasm; Drug Antagonism; Drug Synergism; Electrophoresis, Capillary; Growth Inhibitors; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; K562 Cells; Leukemia; Oxides; U937 Cells; Valproic Acid

The endemic species of Antrodia camphorate (AC) is a promising chemotherapeutic drug for cancer. We found that the ethanol extract from wild fruiting bodies of Antrodia camphorata (EEAC) could induce HL 60 cells apoptosis via histone hypoacetylation, up-regulation of histone deacetyltransferase 1 (HDAC 1), and down-regulation of histone acetyltransferase activities including GCN 5, CBP and PCAF in dose-dependent manner. In combination with histone deacetylase inhibitor, trichostatin A (TSA), did not block EEAC-induced apoptosis. Interestingly, combined treatment (100 nM of TSA and 100 microg/ml EEAC) caused synergistic inhibition of cell growth and increase of apoptotic induction. EEAC could effectively increase the cytotoxic sensitivity of TSA through the up-regulation of DR5 and NFkappaB activation. In this present study, bioassay-guided fractionation of EEAC led to a major active compound, zhankuic acid A, as the bioactive marker. Moreover, our findings may represent an experimental basis for developing EEAC as a potential chemotherapeutic adjuvant.

Topics: Antrodia; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; NF-kappa B; Plant Extracts; Receptors, Death Domain

Histone deacetylase was overexpressed in a variety of cancers and was closely correlated with oncogenic factors. The histone deacetylase inhibitor, trichostatin A (TSA) was shown to induce apoptosis in many cancer cells. However, the mechanism of TSA on induction of cancer cells apoptosis is poorly understood. This study was designed to characterize the global gene expression profiles before and after treatment of human leukemia cell line Molt-4 with TSA. Flow cytometry, MTT and DNA ladder were used to observe the effect of TSA on the apoptosis of MOLT-4 cells and normal human peripheral blood mononuclear cells (PBMC). Microarray, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the difference of gene and protein expressions of Molt-4 cells after incubation of the cells with TSA. The results showed that TSA could induce Molt-4 apoptosis in dose- and time-dependent manners but spared PBMCs. Microarray analysis showed that after incubation with TSA for 9 h, 310 genes were upregulated and 313 genes were deregulated. These genes regulate the growth, differentiation and survival of cells. Among these genes, STAT5A was down-regulated by 80.4% and MYC was down-regulated by 77.3%. It was concluded that TSA has definite growth-inhibiting and apoptosis-inducing effects on Molt-4 cells in time- and dose-dependent manners, with weak cytotoxic effects on PBMCs at the same time. The mechanism of TSA selectively inducing apoptosis and inhibiting growth may be ascribed to the changes of pro-proliferation genes and anti-apoptosis genes.

Topics: Apoptosis; Cell Line, Tumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Microarray Analysis; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; STAT5 Transcription Factor

Histone deacetylase inhibitors (HDACi) have demonstrated promising therapeutic potential in clinical trials for hematological malignancies. HDACi, such as SAHA/Vorinostat, Trichostatin A, and MS-275 were found to induce apoptosis of leukemic blasts through activation of the death receptor pathway and transcriptional induction of the Tumor Necrosis Factor (TNF)-related pro-apoptotic family members, TRAIL and FasL. The impact of HDACi on TNF-related costimulatory molecules such as 4-1BB ligand (4-1BBL/TNFSF9) is however not known. Following exposure to SAHA/Vorinostat, Trichostatin A, and MS-275, transcript levels were determined by real time PCR in Jurkat, Raji and U937 cells. Treatment with HDACi up-regulated TNFSF9 gene expression in the three leukemia cell lines, yet to different extend and with distinct kinetics, which did not require de novo protein synthesis and was not associated with DNAse I hypersensitive chromatin remodeling. Transcriptional activity of TNFSF9 promoter-luciferase constructs was induced up to 12 fold by HDACi, and implication of Sp1/Sp3 transcription factors binding to functional GC-box elements was evidenced by reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays. Functionality of modulated target genes was assessed in allogeneic mixed leukocyte reaction experiments. MS-275- and to a lesser extent Trichostatin A- and SAHA-treated Raji cells significantly up regulated T lymphocytes proliferation which was reduced by about 50% by a 4-1BB blocking recombinant protein, while MS-275- but neither Trichostatin A- nor SAHA-treated cells up-regulated IFNgamma secretion by T lymphocytes. Our results identify 4-1BBL/4-1BB as a downstream target of HDACi, especially of MS-275 anti-leukemia action in vitro. Thus, HDACi such as MS-275 displaying dual TNF-dependent proapoptotic and costimulatory activities might be favored for inclusion in HDACi-based anti-cancer therapeutic strategies.

Topics: 4-1BB Ligand; Antineoplastic Agents; Benzamides; Cell Proliferation; Gene Expression Regulation, Leukemic; Humans; Hydroxamic Acids; Immune System; Jurkat Cells; Leukemia; Mutagenesis, Site-Directed; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tumor Necrosis Factor Receptor Superfamily, Member 9; U937 Cells

Quercetin (QU) is recognized as a promising anticancer drug, but its mechanism remains elusive. Here we found that QU induced human leukemia cell death in a dose-dependent manner. However, it did not show a dose-dependent inhibition on ROS generation (indicated by the level of malondialdehyde, MDA) in the same cells. QU showed similar antioxidant activity at concentrations of 50, 75 and 100 microM. Consistent with that, the antioxidant, N-acetyl-cysteine (NAC) could only further decrease the ROS generation and enhance the cell death triggered by QU at the concentrations less than 50 microM. These results indicate that an additional mechanism is involved in the anticancer activity of high concentrations of QU. When the effect of QU on histone acetylation was studied, QU induced significant histone hyperacetylation at 75 and 100 microM, indicating the possible involvement of histone hyperacetylation in the anticancer activity of high concentrations of QU. This conclusion was supported by the findings that when histone acetylation in the cells treated by QU was increased by different concentrations of TSA, the cell death was significantly enhanced. Our results thus provide the first evidence that QU can induce histone hyperacetylation and this induction of histone hyperacetylation may represent an unrevealed mechanism in its anticancer activity.

Topics: Acetylation; Acetylcysteine; Cell Death; Cell Proliferation; Free Radical Scavengers; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Lipid Peroxidation; Malondialdehyde; Quercetin; Reactive Oxygen Species

Pyrrolidine dithiocarbamate (PDTC) has been considered as a potential anticancer drug due to its powerful apoptogenic effect towards cancer cells, where Cu(2+) plays a distinct yet undefined role. Here we report that Cu(2+) is critically needed for PDTC to inhibit histone acetylation in both human leukemia HL-60 cells and human hepatoma Hep3B cells. The inhibition of histone acetylation mainly resulted from the increase of intracellular Cu(2+), but was not due to the inhibition of NF-kappaB activity by PDTC-Cu(2+) since the combinations of Cu(2+) with SN50, MG132 (two known NF-kappaB inhibitors), or bathocuproine disulfonate (BCS, a specific Cu(2+) chelator that does not cross the plasma membrane), did not lead to obvious inhibition of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cells exposed to PDTC-Cu(2+) showed a comparable decrease in histone acetylation levels in HL-60 cells in the absence or presence of the HDAC inhibitors, trichostatin A (TSA) or sodium butyrate (NaBu); the inhibition rates were about 45, 44 and 43%, respectively. PDTC-Cu(2+) had no effect on the activity of HDAC in vitro, but significantly inhibited the HAT activity both in HL-60 cells and in a cell-free in vitro system. PDTC-Cu(2+) also induced HL-60 cell apoptosis, and treating cells with TSA, NaBu or BCS significantly attenuated the apoptosis induced by PDTC-Cu(2+). Collectively, these results showed that inhibition of histone acetylation represents a distinct mechanism for the cytotoxicity of PDTC in the presence of Cu(2+), where HAT is its possible molecular target.

Topics: Acetylation; Apoptosis; Butyrates; Cell Line, Tumor; Cell Proliferation; Chelating Agents; Copper; Copper Sulfate; Enzyme Inhibitors; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Phenanthrolines; Pyrrolidines; Thiocarbamates

Disturbance of the normal functions of wild-type RUNX1 resulting from chromosomal translocations or gene mutations is one of the major molecular mechanisms in human leukemogenesis. RUNX1-related chimeras generated by the chromosomal translocations repress transcriptional activity of wild-type RUNX1 by recruiting the co-repressor/histone deacetylase complex. Thus, histone deacetylase inhibitors are expected to restore normal functions of wild-type RUNX1 and thereby affect the growth and differentiation ability of leukemic cells expressing the chimera. We investigated the in vitro effects of histone deacetylase inhibitors, trichostatin A and valproic acid, on human leukemic cell lines such as SKNO-1 and Kasumi-1 expressing RUNX1/ETO, Reh expressing TEL/RUNX1 and SKH-1 co-expressing RUNX1/EVI1 and BCR/ABL. We also employed K562 cells expressing BCR/ABL without such a chimera as a control. Treatment with each inhibitor increased acetylated histone 4 in all of these cell lines. Interestingly, proliferation of SKNO-1, Kasumi-1, SKH-1 and Reh cells was significantly suppressed after 3-day culture with trichostatin A or valproic acid, when compared to that of K562 cells. We observed cell cycle arrest and apoptotic induction in the RUNX1 chimera-expressing cells by the propidium iodide staining. Up- and downregulation of cell cycle regulator genes appeared to be the molecular basis for the former, and activation of both extrinsic and intrinsic apoptotic caspases for the latter. We propose histone deacetylase inhibitors to be an attractive choice in the molecular targeting therapy of RUNX1-related leukemia.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 2 Subunit; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; K562 Cells; Leukemia; Mutant Chimeric Proteins; Valproic Acid

The objective of the present study was to investigate the effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on the cell growth and apoptosis and its effect on the telomerase activity in human leukemic cell line U937. Exposure of U937 cells to TSA resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. The increase in apoptosis was associated with the up-regulation in proapoptotic Bax expression and down-regulation of antiapoptotic Bcl-2 and Bcl-X(L). TSA treatment inhibited the levels of cIAP family members and induced the proteolytic activation of caspase-3, which was associated with concomitant degradation of poly(ADP-ribose)-polymerase and beta-catenin protein. TSA treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by TSA treatment. We therefore conclude that TSA demonstrated antiproliferative and apoptosis-inducing effects on U937 cells in vitro, and that changes in Bcl-2 family protein levels as well as telomerase activity may play an important role in its mechanism of action.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Enzyme Inhibitors; Flow Cytometry; Histone Deacetylases; Humans; Hydroxamic Acids; Leukemia; Microscopy, Fluorescence; Reverse Transcriptase Polymerase Chain Reaction; Telomerase

Previous studies have documented that while several drug-resistant cells enter apoptosis upon treatment with histone deacetylase inhibitors (iHDACs), their drug-sensitive counterparts do not. In the present study, we have investigated at the molecular level why parental drug-sensitive tumor cells do not respond to Trichostatin A and suberoylanilide hydroxamic acid, two iHDACs that promote apoptosis in drug-resistant leukaemia cells. Taking murine leukaemia L1210 cells as a model, we have determined that: (i) PKC-alpha expression is more elevated in parental L1210 than in drug-resistant L1210/R cells, (ii) activation of PKC neutralizes iHDACs-mediated apoptosis in L1210/R cells, (iii) depletion of PKC in parental L1210 cells results in a positive response to iHDACs-mediated apoptosis, and (iv) transfection of a mutant constitutively active PKC-alpha form in L1210/R cells makes the cells refractory to apoptosis induction by iHDACs. These results allow us to conclude that activation/high expression of PKC-alpha protects parental drug-sensitive L1210 cells from iHDACs-mediated apoptosis. Thus, determination of PKC-alpha levels/activity in leukaemia seems to be relevant when choosing efficient chemotherapy protocols based on the use of apoptosis-inducing anticancer drugs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Mice; Mutant Proteins; Protein Kinase C-alpha; Transfection; Tumor Cells, Cultured; Vorinostat

Ectopic expression of LMO2 occurs in approximately 45% of T-lineage acute lymphoblastic leukemias (T-ALL), sometimes in association with chromosomal translocations. Recently, a lymphoproliferative disorder developed in two participants in a gene therapy trial due to LMO2 activation via integration of the retroviral vector. To investigate these regulatory disruptions, we analyzed the promoter region and identified a tissue-specific repressor. The fragment containing this element could also produce tissue-specific suppression of transcription from the SV40 promoter. This suppression involves histone acetylation which can be relieved with Trichostatin A (TSA). The negative element is in a region consistently removed from LMO2 in the known chromosomal translocations.

Topics: Adaptor Proteins, Signal Transducing; Base Sequence; Consensus Sequence; DNA-Binding Proteins; Histone Deacetylases; Humans; Hydroxamic Acids; Jurkat Cells; K562 Cells; Leukemia; LIM Domain Proteins; Metalloproteins; Molecular Sequence Data; Organ Specificity; Proto-Oncogene Proteins; Proto-Oncogenes; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transfection

Quercetin (QU) and trichostatin A (TSA) are promising anticancer drugs. While QU mainly exerts its anticancer activity through scavenging reactive oxygen species (ROS), the anticancer activity of TSA was attributed to its inhibition on histone deacetylases (HDAC). In the present study it was investigated, whether the combination of QU and TSA could improve their anticancer activity against human leukemia cells (HL-60). The cytotoxicity of QU and TSA increased in a time and dose-dependent manner. QU (10, 20 and 40 microM) was able to diminish the ROS generation (indicated by the level of malondialdehyde, MDA) but showed no influence on the histone acetylation in HL-60 cells; on the contrary, TSA (20, 40, 80 and 160 nM) showed no inhibition on ROS generation but significantly increased the histone acetylation, indicating the possible role of both scavenging ROS and increasing histone acetylation in the induction of cell death in HL-60 cells. This conclusion was confirmed by the findings that the combinations of QU and TSA at different concentrations could not only diminish ROS generation, but also increase histone acetylation, and hence showed more significant cytotoxicity in HL-60 cells than either of its components. Collectively, the present data indicate that a combination of QU and TSA can cooperatively kill HL-60 cells through the combination of their activities of scavenging ROS and increasing histone acetylation.

Topics: Acetylation; Acylation; Antineoplastic Agents; Cell Proliferation; Cell Survival; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Malondialdehyde; Quercetin; Reactive Oxygen Species

Human chromosomal translocation t(12;21)(p12;q22) is one of the most frequent rearrangement in human leukemia, and produces the TEL/RUNX1 fusion protein. The TEL/RUNX1 fusion protein creates a transcriptional repressor that interferes in dominant fashion with RUNX1-dependent transactivation. Here, we demonstrate that the repressor activity of TEL/ RUNX1 differs from that of TEL, even though both TEL and TEL/RUNX1 interact with the nuclear hormone co-repressor (N-CoR) and histone deacetylase (mSin3A) in vivo. Co-immunoprecipitation experiments demonstrated that TEL/RUNX1 forms homodimers in vivo, and heterodimerizes with the TEL when the two proteins are expressed together. These interactions require the HLH (helix-loop-helix) region of TEL. Immunoprecipitation and immunofluorescence analysis showed that p300 interacts with TEL/RUNX1 and is sequestered in the cytoplasm by it. These results suggest that the p300-TEL/RUNX1 complex and heterodimerization of TEL/RUNX1 with TEL may be responsible for the ability of TEL/RUNX1 to inhibit RUNX1-mediated transactivation. It appears that loss of TEL function activates a pathway that cooperates with TEL/RUNX1 and sequesters coactivator(s) into nonfunctional complex in the cytoplasm thus inhibiting transcription of target genes.

Topics: Acetyltransferases; Animals; Cell Cycle Proteins; Cell Line; Chromosomes, Human, Pair 12; Core Binding Factor Alpha 2 Subunit; Dimerization; DNA-Binding Proteins; ETS Translocation Variant 6 Protein; Gene Expression Regulation; Genes, Reporter; Histone Acetyltransferases; Humans; Hydroxamic Acids; Leukemia; Oncogene Proteins, Fusion; p300-CBP Transcription Factors; Protein Conformation; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Recombinant Fusion Proteins; Repressor Proteins; Transcription Factors; Transcription, Genetic

Recurrent cytogenetic abnormalities in leukemic blasts make these an attractive source for dendritic cells (DC) to induce a leukemia-specific immune response. In this study, three leukemic cell lines were investigated: Kasumi-1 and SKNO-1 (two acute myeloid leukemia (AML) cell lines carrying the (8;21)-chromosomal translocation, resulting in the expression of the leukemia-specific fusion protein AML1-eight-twenty-one) and REH, an acute lymphoblastic leukemia cell line with the (12;21)-chromosomal translocation and expression of translocation ETS-like leukemia-AML1. These fusion proteins are implicated in the pathogenesis of the leukemic state by recruiting corepressors and histone deacetylases (HDAC), which interfere with normal cell differentiation. In vitro generation of DC was achieved using a cytokine cocktail containing tumor necrosis factor alpha, granulocyte macrophage-colony stimulating factor, c-kit ligand, and soluble CD40 ligand; yet, addition of the HDAC inhibitor (Hdi) trichostatin A enhanced DC differentiation with retention of the fusion transcripts. These leukemic DC showed high-level CD83 and human leukocyte antigen (HLA)-DR expression and had a high allostimulatory potential. Only DC generated from these cell lines after Hdi induced blast-specific cytotoxic T cell responses in HLA-A-matched T cells with a cytotoxicity of 42% in parental Kasumi-1 and 83% in parental REH cells, respectively. This model system suggests that the Hdi supports the in vitro differentiation of DC from leukemic blasts with AML1-containing fusion proteins.

Topics: Antigens, CD; CD83 Antigen; Cell Differentiation; Cell Line, Tumor; Core Binding Factor Alpha 2 Subunit; Cytokines; Dendritic Cells; DNA-Binding Proteins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; HLA-DR Antigens; Humans; Hydroxamic Acids; Immunoglobulins; Leukemia; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Membrane Glycoproteins; Oncogene Proteins, Fusion; Proto-Oncogene Proteins; T-Lymphocytes, Cytotoxic; Transcription Factors

Antioxidants and trichostatin A (TSA) are promising anticancer drugs, and are capable of enhancing the neoplastic toxicity of other chemicals that exert anticancer activity via different mechanisms. Since antioxidants and TSA (the specific inhibitor of histone deacetylase) are believed to combat cancer via different mechanisms, we sought to determine whether combining them would improve their anticancer activity in human leukemia cells (HL-60).. HL-60 cells were treated with antioxidants (ascorbic acid, AA and N-acetyl-cysteine, NAC), TSA or their combination, and cell proliferation arrest, lactate dehydrogenase (LDH) release and cell viability were measured as indicators of cell damage. Accumulation of reactive oxygen species (ROS) and the acetylation of histones were also measured.. The cytotoxicity of AA, NAC and TSA increased in a time- and dose-dependent manner. AA (1, 2 and 4 mM) and NAC (0.2, 0.5 and 1 mM) were able to diminish ROS generation but showed no influence on histone acetylation in HL-60 cells. In contrast, TSA (20, 50, 100 and 200 nM) did not inhibit ROS generation but significantly increased histone acetylation, indicating a possible role for both scavenging ROS and increasing histone acetylation in the induction of cell death in HL-60 cells. This conclusion was further confirmed by the finding that the combination of antioxidant and TSA not only diminished ROS generation, but also increased histone acetylation, and hence showed greater cytotoxicity in HL-60 cells than either component alone.. Our findings show that combining antioxidants and TSA can enhance their neoplastic toxicity at least in human leukemia HL-60 cells, providing a new approach to the design of chemotherapy strategies and the development of anticancer drugs.

Topics: Acetylation; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Histones; HL-60 Cells; Humans; Hydroxamic Acids; L-Lactate Dehydrogenase; Leukemia; Reactive Oxygen Species

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader-Willi syndrome.

Topics: Acetylation; Acetyltransferases; Gene Expression Regulation, Enzymologic; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Saccharomyces cerevisiae Proteins; Trans-Activators; Tumor Cells, Cultured

EVI-1 and its variant form, MDS1/EVI1, have been reported to act in an antagonistic manner and be differentially regulated in samples from patients with acute myeloid leukaemia and rearrangements of the long arm of chromosome 3. Here, we show that both EVI-1 and MDS1/EVI1 can repress transcription from a reporter construct containing EVI-1 binding sites and interact with histone deacetylase in mammalian cells. This interaction can be recapitulated in vitro and is mediated by a previously characterized transcription repression domain, whose activity is alleviated by the histone deacetylase inhibitor trichostatin A.

Topics: Animals; Cell Line; COS Cells; DNA-Binding Proteins; Electroporation; Enzyme Inhibitors; Female; Gene Rearrangement; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Leukemia; MDS1 and EVI1 Complex Locus Protein; Oncogene Proteins, Fusion; Pregnancy; Proto-Oncogenes; Recombinant Fusion Proteins; Transcription Factors; Transcription, Genetic

v-ErbA, a mutated thyroid hormone receptor alpha (TRalpha), is thought to contribute to avian erythroblastosis virus (AEV)-induced leukemic transformation by constitutively repressing transcription of target genes. However, the binding of v-ErbA or any unliganded nuclear receptor to a chromatin-embedded response element as well as the role of the N-CoR-SMRT-HDAC co-repressor complex in mediating repression remain hypothetical. Here we identify a v-ErbA-response element, VRE, in an intronic DNase I hypersensitive site (HS2) of the chicken erythroid carbonic anhydrase II (CAII) gene. In vivo footprinting shows that v-ErbA is constitutively bound to this HS2-VRE in transformed, undifferentiated erythroblasts along with other transcription factors like GATA-1. Transfection assays show that the repressed HS2 region can be turned into a potent enhancer in v-ErbA-expressing cells by mutation of the VRE. Differentiation of transformed cells alleviates v-ErbA binding concomitant with activation of CAII transcription. Co-expression of a gag-TRalpha fusion protein in AEV-transformed cells and addition of ligand derepresses CAII transcription. Treatment of transformed cells with the histone deacetylase inhibitor, trichostatin A, derepresses the endogenous, chromatin-embedded CAII gene, while a transfected HS2-enhancer construct remains repressed. Taken together, our data suggest that v-ErbA prevents CAII activation by 'neutralizing' in cis the activity of erythroid transcription factors.

Topics: Alpharetrovirus; Animals; Base Sequence; Carbonic Anhydrases; Cell Differentiation; Cell Transformation, Neoplastic; DNA Footprinting; Enhancer Elements, Genetic; Erythropoiesis; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Hydroxamic Acids; Introns; Leukemia; Molecular Sequence Data; Oncogene Proteins v-erbA; Protein Binding; Receptors, Thyroid Hormone; Response Elements