trichostatin-a and Myelodysplastic-Syndromes

Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors.. Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents.. PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.

Topics: Apoptosis; Azacitidine; Benzamides; Butyrates; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Decitabine; DNA Modification Methylases; DNA Repair; Drug Synergism; Flow Cytometry; Fluorescent Antibody Technique; Fluorobenzenes; Histone Acetyltransferases; HL-60 Cells; Humans; Hydroxamic Acids; Immunohistochemistry; K562 Cells; Leukemia, Myeloid; Myelodysplastic Syndromes; Phenanthrenes; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Pyridines; U937 Cells

The study was purposed to explore the effect and mechanisms of decitabine and/or Trichostatin A (TSA) on SKM-1 cells in vitro. The effect of decitabine and/or TSA on proliferation of SKM-1cells was analyzed with trypan blue exclusion; the differentiation of SKM-1 cells was detected by nitro-blue tetrazolium (NBT) reduction and flow cytometry; the apoptosis of cells was measured by Annexin V-FITC; the mRNA expression of Fas, survivin and P15(INK4B) in cells treated with decitabine and/or TSA was evaluated by RT-PCR. The results showed that decitabine and/or TSA were capable of inhibiting SKM-1 cell growth and promoting cell differentiation; they stimulated the expression of CD14 and CD11b and inhibited HLA-DR expression; meanwhile and decitabine or/and TSA could induce cell apoptosis, up-regulate mRNA expression of Fas and P15(INK4B), and down-regulate survivin mRNA expression. It is concluded that decitabine can induce apoptosis/differentiation of SKM-1 cells, whose mechanisms may related to the expression of Fas, survivin and P15(INK4B). Decitabine has the synergistic effect with TSA.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Decitabine; Drug Synergism; fas Receptor; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Myelodysplastic Syndromes; Survivin

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

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