trichostatin-a and hexamethylene-bisacetamide

Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis-acting elements responsible for induction of stage-specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady-state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear run-on analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on nuclear run-on transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.

Topics: 3T3 Cells; Acetamides; Animals; Apoptosis; Cell Differentiation; Dimethyl Sulfoxide; Enzyme Inhibitors; Gene Expression Regulation; Genes, Reporter; Globins; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Hydroxamic Acids; Mice; Transcription, Genetic; Transfection

Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys(20)) trimethylation. An increase in H4-Lys(20) trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys(20) is localized to heterochromatin, the increase in H4-Lys(20) trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys(20) trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys(20) is therefore the most suitable substrate for H4-Lys(20) trimethylase. Summarizing, histone H4-Lys(20) hypotrimethylation correlates with H4 hyperacetylation and H4-Lys(20) hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin.

Topics: Acetamides; Acetylation; Amino Acid Sequence; Animals; Antineoplastic Agents; Blotting, Western; Butyrates; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Chromatin; Chromatography, High Pressure Liquid; Chromatography, Liquid; Dimethyl Sulfoxide; Enzyme Inhibitors; Heterochromatin; Histones; Hydroxamic Acids; Leukemia, Erythroblastic, Acute; Lysine; Mass Spectrometry; Metalloendopeptidases; Methylation; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Peptides; Protein Processing, Post-Translational; Sodium Oxybate; Time Factors; Transcription, Genetic

Tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1/gp75) and dopachrome tautomerase (DCT/TYRP2) belong to a family of melanocyte-specific gene products involved in melanin pigmentation. During melanocyte development expression of tyrosinase family genes is thought to be orchestrated in part by the binding of a shared basic helix-loop-helix transcription factor MITF to the M box, a regulatory element conserved among these genes. In transformed melanocytes, expression of tyrosinase and TYRPs is highly variable. Whereas TYR expression in melanoma cells is regulated by both transcriptional and post-translational mechanisms, TYRP1/gp75 transcription is often completely extinguished during melanoma tumor progression. In this study, we investigated the mechanisms of selective repression of TYRP1 transcription. Interestingly, in early stage melanoma cells TYRP1 mRNA could be induced by inhibition of protein synthesis. Transient transfection experiments with a minimal TYRP1 promoter showed that the promoter activity correlates with expression of the endogenous TYRP1 gene. Nucleotide deletion analysis revealed novel regulatory sequences that attenuate the M box-dependent MITF activity, but which are not involved in the repression of TYRP1. Gel mobility shift analysis showed that binding of the transcription factor MITF to the TYRP1 M box is selectively inhibited in TYRP1(-) cells. These data suggest that protein factors that modulate the activity of MITF in melanoma cells repress TYRP1 and presumably other MITF target genes.

Topics: Acetamides; Azacitidine; Binding Sites; Blotting, Northern; Cells, Cultured; Cycloheximide; Decitabine; DNA-Binding Proteins; Down-Regulation; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Humans; Hydroxamic Acids; Luciferases; Melanocytes; Membrane Glycoproteins; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Mutation; Oxidoreductases; Promoter Regions, Genetic; Protein Binding; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Sequence Deletion; Transcription Factors; Tumor Cells, Cultured

Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.

Topics: Acetamides; Animals; Carcinoma; Cell Differentiation; Cell Line, Transformed; Cell Transformation, Neoplastic; Cinnamates; Drug Resistance; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leukemia, Erythroblastic, Acute; Malonates; Mice; Urinary Bladder Neoplasms; Vorinostat

The herpes simplex virus type 1 (HSV-1) mutant in1814 encodes an altered form of the virion protein VP16 that is unable to transactivate immediate-early (IE) transcription. As a consequence of the mutation, in1814 initiates productive replication inefficiently after infection of tissue culture cells. Previous studies showed that this defect could be overcome by the inclusion in the culture medium of hexamethylene bisacetamide (HMBA), a compound that promotes the differentiation of murine erythroleukemia cells (MELCs). The effects of additional agents known to induce differentiation of MELCs were investigated. N'-Methylnicotinamide, at concentrations optimal for the induction of MELCs, complemented the replication of in1814 and stimulated IE gene expression. Suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid, which induce differentiation of MELCs at micromolar concentrations, did not complement in1814 but specifically blocked the action of HMBA. The histone deacetylase inhibitor trichostatin A, which also induces differentiation of MELCs, antagonized the effect of HMBA in a manner similar to that of suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid. The results demonstrate that the requirement for VP16 activity is dependent on the metabolic state of the host cell and that the pathways leading to complementation of in1814 and differentiation of MELCs are overlapping but not identical.

Topics: Acetamides; Animals; Cell Differentiation; DNA Damage; Genes, Immediate-Early; Genes, Viral; Genetic Complementation Test; Herpes Simplex Virus Protein Vmw65; Herpesvirus 1, Human; Hydroxamic Acids; Mice; Mutation; Niacinamide; Promoter Regions, Genetic; Tumor Cells, Cultured; Virus Replication; Vorinostat