trichostatin-a and Lymphoma

DNA methylation and histone deacetylation play important roles in the occurrence and development of cancers by inactivating the expression of tumor suppressors, including p16(INK4a), a cyclin-dependent kinase inhibitor. The present study investigated the effect of epigallocatechin-3-gallate (EGCG) alone or in combination with trichostatin A (TSA) on p16(INK4a) gene expression and growth in human malignant lymphoma CA46 cells. CA46 cell viability and cell cycle were analyzed; methylation of the p16(INK4a) gene was assessed by nested methylation-specific PCR (n-MSP). p16(INK4a )mRNA and protein expression was determined by real-time quantitative PCR and western blot analyses, respectively. Both EGCG and TSA alone inhibited CA46 cell proliferation; the combined treatment (6 µg/ml EGCG and 15 ng/ml TSA) significantly reduced CA46 cell proliferation from 24 to 96 h (all P<0.001). Cells treated with 24 µg/ml EGCG or the combination treatment (6 µg/ml EGCG and 15 ng/ml TSA) had lower proliferative indices when compared to the other groups. Co-treatment with EGCG and TSA decreased p16(INK4a) gene methylation, which coincided with increased p16(INK4a) mRNA and protein expression. Thus, EGCG and TSA synergistically reactivate p16(INK4a) gene expression in part through reducing promoter methylation, which may decrease CA46 cell proliferation.

Topics: Catechin; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Drug Synergism; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Lymphoma; Promoter Regions, Genetic

MK-0457 and MK-5108 are novel aurora kinase inhibitors (AKi) leading to G(2)-M cell-cycle arrest. Growth and survival of multiple lymphoma cell lines were studied with either drug alone or in combination with vorinostat, a histone deacetylase inhibitor (HDACi), using MTS and Annexin V assays, followed by molecular studies. Either of the AKi alone at 100 to 500 nmol/L resulted in approximately 50% reduced cell growth and 10% to 40% apoptosis. Addition of vorinostat reactivated proapoptotic genes and enhanced lymphoma cell death. Quantitative PCR and immunoblotting revealed that epigenetic and protein acetylation mechanisms were responsible for this activity. The prosurvival genes Bcl-X(L) and hTERT were downregulated 5-fold by combination drug treatment, whereas the proapoptotic BAD and BID genes were upregulated 3-fold. The p53 tumor suppressor was stabilized by an increased acetylation in response to vorinostat and a reduced Ser315 phosphorylation in response to aurora kinase A. Vorinostat or trichostatin A decreased MYC mRNA and protein as well as c-Myc-regulated microRNAs. MYC is a critical gene in these responses, as MYC knockdown combined with the expression of the c-Myc antagonist MXD1 raised cell sensitivity to the effects of either AKi. Thus, the HDACi vorinostat leads to both transcriptional and posttranscriptional changes to create a proapoptotic milieu, sensitizing cells to mitosis-specific agents such as AKis.

Topics: Antineoplastic Combined Chemotherapy Protocols; Aurora Kinase A; Aurora Kinases; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cell Survival; Cyclohexanecarboxylic Acids; Drug Synergism; Gene Expression Regulation, Neoplastic; Genes, myc; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Lymphoma; MicroRNAs; Piperazines; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Telomerase; Thiazoles; Vorinostat

To better understand the mechanisms underlying the role of the immunoglobulin heavy-chain gene (IgH) 3' enhancers on bcl-2 transcriptional deregulation in t(14;18) lymphoma, we characterized the physical interactions of the IgH 3' enhancer region with the bcl-2 promoters. Using the chromosome conformation capture technique, we found that the IgH 3' enhancers physically interact with the bcl-2 promoter region over a 350 kb genomic region in t(14;18) lymphoma cells. No interactions of the bcl-2 promoter region with sequences distant to the IgH enhancers were observed. The physical interactions of the IgH enhancers with the bcl-2 5' region are functionally involved in the transcriptional control of bcl-2. The histone deacetylase inhibitor, trichostatin A, repressed bcl-2 transcription and decreased the IgH enhancer-bcl-2 promoter region interactions. We showed by chromatin immunoprecipitation assay and small interference RNA transfection studies that the POU2 family transcription factor Oct-2 and its cofactor Bob-1 have an important function in mediating the IgH enhancer-bcl-2 promoter region interactions. This study reveals a new aspect of the regulatory role of the IgH 3' enhancers on bcl-2 transcription in t(14;18) lymphomas.

Topics: Cell Line, Tumor; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; Enhancer Elements, Genetic; Enzyme Inhibitors; Genes, bcl-2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoglobulin Heavy Chains; Lymphoma; Octamer Transcription Factor-2; Promoter Regions, Genetic; RNA, Small Interfering; Trans-Activators; Transcription, Genetic; Translocation, Genetic

The glucocorticoid receptor (GR) is mainly expressed as nine-exon alternatively spliced variants, encoding functional GRalpha and nonfunctional GRbeta. Overexpression of GRbeta splice variant was found in glucocorticoid-resistant patients with some autoimmune diseases and hematological malignancies. Employing reverse transcription, real-time quantitative PCR, and western blot analysis, we determined an effect of trichostatin A (TSA), sodium butyrate (NaBu) and 5-aza-2'-deoxycytidine (5-dAzaC) on GRalpha and GRbeta expression in Hut-78 T- and Raji B-lymphoma cell lines. We found that TSA, NaBu, and 5-dAzaC significantly increase the expression of GRalpha transcript and protein, whereas GRbeta transcript and protein expression was profoundly decreased in Hut-78 T- and Raji B- lymphoma cell lines. Our observation suggests that changes of epigenetic milieu inside cells may alter the expression of GRalpha and GRbeta isoforms.

Topics: Antineoplastic Agents; Azacitidine; Blotting, Western; Butyrates; Cell Line, Tumor; Decitabine; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Lymphoma; Polymerase Chain Reaction; Receptors, Glucocorticoid; Reverse Transcription

The tumor suppressor Chk2 kinase plays crucial roles in regulating cell-cycle checkpoints and apoptosis following DNA damage. We investigated the expression levels of the genes encoding Chk2 and several cell-cycle regulators in nine cell lines from lymphoid malignancies, including three Hodgkin's lymphoma (HL) lines. We found that all HL cell lines exhibited a drastic reduction in Chk2 expression without any apparent mutation of the Chk2 gene. However, expression of Chk2 in HL cells was restored following treatment with the histone deacetylase inhibitors trichostatin A (TsA) and sodium butyrate (SB), or with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC). Chromatin-immunoprecipitation (Chip) assays revealed that treatment of HL cells with TsA, SB or 5Aza-dC resulted in increased levels of acetylated histones H3 and H4, and decreased levels of dimethylated H3 lysine 9 at the Chk2 promoter. These results indicate that expression of the Chk2 gene is downregulated in HL cells via epigenetic mechanisms.

Topics: Acetylation; Apoptosis; Azacitidine; Butyrates; Cell Line, Tumor; Checkpoint Kinase 2; Decitabine; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histones; Hodgkin Disease; Humans; Hydroxamic Acids; Lymphoma; Methylation; Protein Serine-Threonine Kinases; RNA, Messenger