trichostatin-a and Acute-Disease

The onset of acute pancreatitis (AP) is characterized by early protease activation followed by inflammation and organ damage, but the mechanisms are poorly understood.. We hypothesized that histone deacetylase (HDAC) inhibition might exert protective effects on AP and investigated the role of HDAC in trypsin activation, inflammation, and tissue damage in severe AP.. Male C57Bl/6 mice were treated i.p. with the HDAC inhibitor trichostatin A (2 mg/kg) prior to retrograde infusion of taurocholic acid (5 %) into the pancreatic duct. Serum levels of amylase and interleukin (IL)-6, pancreatic levels of macrophage inflammatory protein-2 (MIP-2) as well as tissue morphology and myeloperoxidase activity in the pancreas and lung were determined 24 h after taurocholate challenge. Trypsin activation was analyzed in isolated acinar cells. Quantitative RT-PCR was used to examine the expression of pro-inflammatory mediators in the pancreas.. Pretreatment with trichostatin A decreased amylase levels by 70 % and protected against tissue injury in the pancreas. Moreover, HDAC inhibition reduced systemic IL-6 by more than 95 % and pulmonary myeloperoxidase activity by 75 %. Notably, inhibition of HDAC abolished taurocholate-induced gene expression of cyclooxygenase-2, MIP-2, monocyte chemotactic protein-1, IL-6, and IL-1β in the pancreas. In addition, HDAC inhibition reduced cerulein-induced trypsinogen activation in isolated acinar cells.. Our findings show that HDAC regulates trypsin activation, inflammation, and tissue damage in AP. Thus, targeting HDAC could serve as novel therapeutic approach in the management of severe AP.

Topics: Acute Disease; Amylases; Animals; Anti-Inflammatory Agents; Ceruletide; Chemokine CXCL2; Cytoprotection; Disease Models, Animal; Enzyme Activation; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-6; Lung; Male; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Signal Transduction; Taurocholic Acid; Trypsin

Acute graft-versus-host disease (aGVHD) is a rare but serious and life-threatening complication of liver transplantation (LTx). Previously, we have demonstrated that the development of aGVHD after LTx (LTx-aGVHD) is associated with a decreased percentage of regulatory T cells (Tregs) in the peripheral blood of recipients. Histone deacetylase inhibitors promote the production of Tregs and some, such as suberoylanilide hydroxamic acid and trichostatin A (TSA), are used to treat autoimmune diseases, including GVHD after bone marrow transplantation.. In this study, LTx-aGVHD rats were treated with TSA continuously for 7 days from day 8 to 14 after LTx. Subsequently, splenic T cells were used for in vitro investigations of the mechanism of action of transplantation.. All LTx-aGVHD rats developed typical LTx-aGVHD symptoms after TSA treatment and died from LTx-aGVHD. The percentage frequency of Tregs in peripheral blood mononuclear cells was slightly up-regulated after TSA treatment, whereas TSA dramatically down-regulated Foxp3 protein and mRNA levels both in vivo and in vitro. Furthermore, TSA impaired T-cell proliferation and production of proinflammatory and anti-inflammatory cytokines in vitro.. TSA does not abrogate LTx-aGVHD in rats due to down-regulation of Tregs.

Topics: Acute Disease; Animals; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Forkhead Transcription Factors; Graft Rejection; Graft vs Host Disease; Histone Deacetylase Inhibitors; Hydroxamic Acids; Liver Transplantation; Male; Postoperative Complications; Rats; Rats, Inbred BN; Rats, Inbred Lew; RNA, Messenger; Spleen; T-Lymphocytes, Regulatory; Treatment Failure

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

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2',3'-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.

Topics: Acetylation; Acute Disease; Animals; Apoptosis; Bacterial Toxins; Cell Line; Colon; Cytokines; Enteritis; Enterotoxins; Enzyme Activation; Epithelial Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Intestinal Mucosa; Mice; Microtubules; Protein Processing, Post-Translational; rho GTP-Binding Proteins; Tubulin; Uridine Diphosphate

Histone deacetylase inhibitors (HDACi) are being studied in clinical trials with the aim to induce cellular differentiation, growth arrest, and apoptosis of tumor cells. Recent reports suggest that the multidrug resistance-1 (MDR1) gene is regulated by epigenetic mechanisms. To investigate whether additional drug transporters are regulated by HDACi and how this affects cytotoxicity, acute myeloid leukemia (AML) cells were examined.. AML cells were cultured in the presence of phenylbutyrate, valproate, suberoylanilide hydroxamic acid, or trichostatin A and analyzed for drug transporter expression and function as well as sensitivity to anticancer drugs.. MDR1, breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRP) 7 and 8 were induced in a dose- and time-dependent manner as shown by semiquantitative PCR. The pattern of gene induction was cell line specific. Phenylbutyrate induced P-glycoprotein and BCRP expression and the efflux of drugs as determined with labeled substrates. KG-1a cells treated with phenylbutyrate developed resistance to daunorubicin, mitoxantrone, etoposide, vinblastine, paclitaxel, topotecan, gemcitabine, and 5-fluorouracil; as a result drug-induced apoptosis was impaired. Chromatin immunoprecipitation revealed the hyperacetylation of histone proteins in the promoter regions of MDR1, BCRP, and MRP8 on valproate treatment. Furthermore, an alternative MRP8 promoter was induced by HDACi treatment.. Exposure of AML cells to HDACi induces a drug resistance phenotype broader than the "classic multidrug resistance," which might negatively affect treatment effectiveness.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; ATP-Binding Cassette Transporters; Biological Transport; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Immunoblotting; K562 Cells; Leukemia, Myeloid; Multidrug Resistance-Associated Proteins; Pharmaceutical Preparations; Phenotype; Phenylbutyrates; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Valproic Acid; Vorinostat

Interstitial loss of all or part of the long arm of chromosome 5, or del(5q), is a frequent clonal chromosomal abnormality in human myelodysplastic syndrome (MDS, a preleukemic disorder) and acute myeloid leukemia (AML), and is thought to contribute to the pathogenesis of these diseases by deleting one or more tumor-suppressor genes. Although a major commonly deleted region (CDR) has been delineated on chromosome band 5q31.1 (refs. 3-7), attempts to identify tumor suppressors within this band have been unsuccessful. We focused our analysis of gene expression on RNA from primitive leukemia-initiating cells, which harbor 5q deletions, and analyzed 12 genes within the CDR that are expressed by normal hematopoietic stem cells. Here we show that the gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with AML or MDS with a 5q deletion than in individuals with MDS or AML lacking a 5q deletion or in normal hematopoietic stem cells. Analysis of HL-60 cells, a myeloid leukemia line with deletion of the 5q31 region, showed that the CTNNA1 promoter of the retained allele is suppressed by both methylation and histone deacetylation. Restoration of CTNNA1 expression in HL-60 cells resulted in reduced proliferation and apoptotic cell death. Thus, loss of expression of the alpha-catenin tumor suppressor in hematopoietic stem cells may provide a growth advantage that contributes to human MDS or AML with del(5q).

Topics: Acute Disease; alpha Catenin; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromosome Deletion; Chromosomes, Human, Pair 5; DNA Methylation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; HL-60 Cells; Humans; Hydroxamic Acids; In Situ Hybridization, Fluorescence; K562 Cells; Leukemia, Myeloid; Mutation; Myelodysplastic Syndromes; Myeloid Progenitor Cells; Reverse Transcriptase Polymerase Chain Reaction; Transfection; U937 Cells

The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine, can reverse p15INK4B promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies.. We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples.. Zebularine treatment of AML193, which has a densely methylated p15INK4B promoter, results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation, arrests cells at G(2)/M, and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis.. Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies.

Topics: Acetylation; Acute Disease; Apoptosis; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p15; Cytidine; DNA Methylation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G2 Phase; Gene Expression Profiling; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia, Myeloid; Phosphorylation; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

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

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

Differentiation-inducing therapy with the DNA-methylation inhibitor Decitabine (5'-aza-deoxycytidine) and histone deacetylase (HDAC) inhibitors are now considered in acute myelogenous leukemia (AML). We investigated the in vitro effects of Decitabine and two structurally unrelated HDAC inhibitors (Sodium 4-phenyl butyrate, Tricostatin A) on clonogenic AML cells. Based on morphological criteria we identified four major colony types: (i) non-erythroid colonies, (ii) erythroid colonies that were detected only for a subset of patients and could be further sub classified into mature and immature forms, and (iii) intermediate colonies. Erythroid differentiation was associated with low CD34 expression. The colonies showed differences in morphology, viability, cell cycle distribution and expression of differentiation markers. Both Decitabine and the two HDAC inhibitors altered AML cell expression of differentiation markers, whereas the drugs did not have any major influence on cell cycle distribution. However, the pharmacological effects differed between the four colony subsets, and differences were also detected between the two HDAC inhibitors. We conclude that clonogenic AML cells can be classified into well-defined subsets based on their differentiation, and these subsets differ in their biological characteristics as well as their response to pharmacological targeting of epigenetic regulation.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; Azacitidine; Cell Cycle; Cell Differentiation; Cell Proliferation; Decitabine; DNA, Mitochondrial; Enzyme Inhibitors; Epigenesis, Genetic; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid; Male; Middle Aged; Myelodysplastic Syndromes; Phenylbutyrates; Thymidine; Tumor Cells, Cultured; Tumor Stem Cell Assay

Histone deacetylase inhibitors (HDIs) are a new class of drugs with significant antileukemic activity. To explore mechanisms of disease-specific HDI activity in acute myeloid leukaemia (AML), we have characterised expression of all 18 members of the histone deacetylase family in primary AML blasts and in four control cell types, namely CD34+ progenitors from umbilical cord, either quiescent or cycling (post-culture), cycling CD34+ progenitors from GCSF-stimulated adult donors and peripheral blood mononuclear cells. Only SIRT1 was consistently overexpressed (>2 fold) in AML samples compared with all controls, while HDAC6 was overexpressed relative to adult, but not neo-natal cells. HDAC5 and SIRT4 were consistently underexpressed. AML blasts and cell lines, exposed to HDIs in culture, showed both histone hyperacetylation and, unexpectedly, specific hypermethylation of H3 lysine 4. Such treatment also modulated the pattern of HDAC expression, with strong induction of HDAC11 in all myeloid cells tested and with all inhibitors (valproate, butyrate, TSA, SAHA), and lesser, more selective, induction of HDAC9 and SIRT4. The distinct pattern of HDAC expression in AML and its response to HDIs is of relevance to the development of HDI-based therapeutic strategies and may contribute to observed patterns of clinical response and development of drug resistance.

Topics: Acetylation; Acute Disease; Adult; Antigens, CD34; Butyrates; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leukemia, Myeloid; Myeloid Cells; Tumor Cells, Cultured; Valproic Acid; Vorinostat

Retroviral insertional mutagenesis in BXH2 mice commonly induces myeloid leukemias. One of the most frequently involved genes in experimental studies is Meis 1. In contrast to other genes in murine models, Meis 1 has not been affected by recurrent chromosomal translocations or point mutations in human leukemias. We found a constant downregulation of the Meis 1 gene mRNA in AML1-ETO acute myeloid leukemias and in those cases harboring in frame mutations in the bZIP domain of CEBPalpha. The absence of the Meis 1 mRNA was not caused by inactivating point mutations in the coding sequence. Promoter hypermethylation was present in more than half of the cases (9/14), including samples obtained from the widely employed Kasumi-1 cell line. Double treatment with 5-Aza-2'-deoxycytidine and trichostatin A of the Kasumi-1 cell line partially reverses Meis 1 inhibition. HoxA9 levels were also low. In a cell line model (U937 Tet AML1-ETO), AML1-ETO expression was not associated with Meis 1 suppression at 72 h. Nevertheless, Meis 1 repression is dependent on the AML1-ETO transcript levels in treated leukemic patients. Chimeric products that arise from chromosomal translocations may be associated with locus-specific epigenetic inactivation. It remains to be investigated when this methylation process is acquired and which are the basic mechanisms underlying these molecular events in AML1-ETO and CEBPalpha-mutated AML.

Topics: Acute Disease; Adult; Azacitidine; Bone Marrow; Case-Control Studies; Cell Line, Tumor; Core Binding Factor Alpha 2 Subunit; Decitabine; DNA Methylation; Down-Regulation; Homeodomain Proteins; Humans; Hydroxamic Acids; Leukemia, Myeloid; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; RNA, Messenger; RUNX1 Translocation Partner 1 Protein; Transcription Factors

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

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

During the development of leukemia, genes that suppress growth and induce differentiation can be silenced by aberrant DNA methylation and by changes in chromatin structure that involve histone deacetylation. It has been reported that a positive interaction between DNA methylation and histone deacetylation takes place to inhibit transcription. Based on this observation, our working hypothesis was that a combination of inhibitors of these processes should produce an enhancement of their antineoplastic activity on leukemic cells. The cytosine nucleoside analog, 5-aza-2'-deoxycytidine (5AZA), is a potent inhibitor of DNA methylation, which can activate tumor suppressor genes in leukemic cells that have been silenced by aberrant methylation. In clinical trials, 5AZA was demonstrated to be an active antileukemic agent. Histone deacetylase inhibitors (HDI) can also activate gene expression in leukemic cell lines by producing changes in chromatin configuration, and show antineoplastic activity in preclinical studies. In this report, we investigated the in vitro antineoplastic activity of 5AZA, alone and in combination with the HDI, trichostatin A (TSA) and depsipeptide (FR901228, depsi), on the human myeloid leukemic cell lines, HL-60 and KG1a. The results showed that the combination of 5AZA with TSA or depsi produced a greater inhibition of growth and DNA synthesis and a greater loss of clonogenicity than either agent alone. These results suggest that 5AZA used in combination with HDI may be an interesting chemotherapeutic regimen to investigate in patients with acute myeloid leukemia that is resistant to conventional chemotherapy.

Topics: Acetylation; Acute Disease; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Azacitidine; Cell Division; Decitabine; Depsipeptides; DNA Methylation; DNA Replication; DNA, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia, Myeloid; Neoplasm Proteins; Peptides, Cyclic; Protein Processing, Post-Translational; Tumor Cells, Cultured; Tumor Stem Cell Assay

Primary acute myeloid leukaemia (AML) blasts can be induced to differentiate into dendritic-like leukaemia cells (DLLC) by culture with certain cytokine combinations. DLLC offer potential for use as autologous vaccines based on their ability to present putative leukaemia-specific antigens to T cells. It has been reported, however, that in around 30-50% of AML cases the leukaemia cells are not capable of undergoing DLLC differentiation. The purpose of this study was to identify the features that represent successful DLLC differentiation and, for those cases shown to be resistant to cytokine-induced differentiation, to use differentiating agents in an attempt to overcome the differentiation block. Leukaemia cells derived from 42 patients with AML were cultured in vitro with cytokines GM-CSF, IL-4 and TNFalpha/CD40L. In 22 cases the leukaemic cells underwent DLLC differentiation based on characteristic morphological changes and expression of costimulatory and dendritic cell-associated molecules. Four cases were not evaluable because of poor viability over the culture period. The remaining 16 cases failed to show evidence of DLLC differentiation. Many of these differentiation resistant cases were associated with poor risk karyotypic features. Nine of the resistant cases were selected for further study. Differentiating agents trichostatin (TSA), azacytidine (AZA) and bryostatin (BRYO) were used in combination with cytokines for the first 96 h of the culture period. Bryostatin (BRYO) alone was shown to be capable of overcoming differentiation resistance and allowing DLLC differentiation to proceed.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Azacitidine; Bryostatins; CD40 Ligand; Cell Differentiation; Child; Cytokines; Dendritic Cells; Drug Resistance; Drug Synergism; Female; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-D Antigens; Humans; Hydroxamic Acids; Immunophenotyping; In Situ Hybridization, Fluorescence; Interleukin-12; Interleukin-4; Karyotyping; Lactones; Leukemia, Myeloid; Lymphocyte Culture Test, Mixed; Macrolides; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The (8;21) translocation, found in 12% of acute myeloid leukemia (AML), creates the chimeric fusion product, AML1-ETO. Previously, we demonstrated that the ETO moiety recruits a transcription repression complex that includes the histone deacetylase (HDAC1) enzyme. Here, we used inhibitors of HDAC1 to study the pathophysiology of AML1-ETO. Both the potent inhibitor, trichostatin (TSA), and the well-known but less specific inhibitor, phenylbutyrate (PB), could partially reverse ETO-mediated transcriptional repression. PB was also able to induce partial differentiation of the AML1-ETO cell line, Kasumi-1. With the intention of developing a clinically useful protocol, we combined PB with a number of other agents that induced differentiation and apoptosis of Kasumi-1 cells. In summary, transcriptional repression mediated by AML1-ETO appears to play a mechanistic role in the t(8;21) AML, and relief of repression using agents such as PB (alone or in combination) may prove to be therapeutically useful.

Topics: 3T3 Cells; Acute Disease; Animals; Apoptosis; Cell Differentiation; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Core Binding Factor Alpha 2 Subunit; DNA-Binding Proteins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid; Mice; Neoplasm Proteins; Phenylbutyrates; Proto-Oncogene Proteins; Recombinant Fusion Proteins; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Transcription, Genetic; Translocation, Genetic; Tumor Cells, Cultured

We investigated the effect of the histone deacetylase inhibitors (HDIs), trichostatin A and trapoxin A on leukemia cells and cell lines from the viewpoint of differentiation induction. TSA induced differentiation in erythroid cell lines by itself, whereas it synergistically enhanced the differentiation that was directed by all-trans retinoic acid (ATRA) or vitamin D3 in U937, HL60 and NB4 cells. The combined treatment of HDI with ATRA induced differentiation in ATRA-resistant HL60 and NB4 cells. The transcriptional expression during the treatment with HDI was examined in HL60, U937 and MEG-O1. Cell cycle-regulator genes (p21waf1 and p16INK4A) were upregulated or constantly expressed, erythroid-specific genes (GATA-1, beta-globin) were silent or downregulated, and housekeeping genes (beta-actin and GAPDH) were constantly expressed. Twelve of 35 (34%) clinical samples from AML patients ranging from M0 to M7 also displayed both phenotypical and morphological changes by the treatment with TSA alone. HDIs are thus the potent inducer or enhancer of differentiation in acute myeloid leukemia and regulate transcription in an ordered manner.

Topics: 3T3 Cells; Acute Disease; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Cell Differentiation; Cell Line; Enzyme Inhibitors; Erythroid Precursor Cells; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Hydroxamic Acids; Leukemia, Myeloid; Megakaryocytes; Mice; Peptides; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured