trichostatin-a and Teratocarcinoma

Retinoic acid (RA) causes differentiation of mouse F9 embryonic carcinoma cell line into primitive and parietal (with dibutiril-cAMP) endoderm. The role of AP-1 transcription factor during RA-induced differentiation was studied in F9 cell line. It was shown that differentiated cells acquired protein complexes, which are specifically bound to well characterized AP-1 32P-labeled binding sites from collagenase (Col-AP-1) and c-jun (Jun2-AP-1) promoters. These complexes contain c-Fos/c-Jun with Col-AP-1 site and c-Jun/ATF-2 with Jun2-AP-1 site as revealed by supershift analysis. DNA-binding activity of these complexes is high in parietal endoderm but low-detectable in undifferentiated cells. DNA-binding activity of AP-1 transcription factor correlates with increased expression of c-fos and c-jun genes. RT-PCR analysis showed an increase in steady-state level of c-fos and c-jun gene transcription at the stage of parietal endoderm (terminally differentiated F9 cells). Transcription of immediate early c-fos and c-jun genes and DNA-binding activity of c-Fos/c-Jun complex are serum dependent. The rate of c-fos and c-jun gene transcription and DNA-binding activity of c-Fos/c-Jun complex decreased in serum-starved cells, but was rapidly induced upon stimulation with serum. Undifferentiated F9 cells contain a very low level of c-fos mRNA, with may be a consequence of repressive chromatin structure in promoter region. Histone deacetylase (HDAC) activity is necessary to restrict expression of specific number of genes, also HDAC inhibitors are well known inductors of differentiation and anticancer agents. Frow cytometry analysis showed a decreased rate of proliferation of F9 cells after their incubation with HDAC inhibitors, sodium butirate and trichostatin A. Also, these ihibitors induced the transcription of c-fos gene. So, we conclude that HDAC activity may be necessary to sustain a high proliferative rate of undifferentiated F9 cells.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Hydroxamic Acids; Mice; Protein Binding; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teratocarcinoma; Transcription Factor AP-1; Transcription, Genetic; Tretinoin

Synaptophysin, one of the major proteins on synaptic vesicles, is ubiquitously expressed throughout the brain. Synaptophysin and synapsin I, another synaptic vesicle protein, are also expressed by retinoic acid-induced neuronally differentiated P19 teratocarcinoma cells. Here, we show that inhibition of histone deacetylase activity in P19 cells is sufficient to activate transcription of the synaptophysin and synapsin I genes, indicating that neuronal differentiation and impairment of histone deacetylases results in a similar gene expression pattern. The transcription factor REST, a repressor of neuronal genes in non-neuronal tissues, has been shown to function via recruitment of histone deacetylases to the transcription unit, indicating that modulation of the chromatin structure via histone deacetylation is of major importance for REST function and neuron-specific gene transcription. Furthermore, REST has been shown to be the major regulator of neuronal expression of synapsin I. Here, we have identified a functional binding site for REST in the first intron of the human synaptophysin gene indicating that REST blocks human synaptophysin gene transcription through an intronic neuron-specific silencer element. The synaptophysin promoter is, however, devoid of neuron-specific genetic elements and directs transcription in both neuronal and non-neuronal cells. Using a dominant-negative approach we have identified the transcription factor Sp1 as one of the regulators responsible for constitutive transcription of the human synaptophysin gene.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cells, Cultured; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydroxamic Acids; Introns; Male; Mice; Molecular Sequence Data; Neurons; Promoter Regions, Genetic; Repressor Proteins; Sp1 Transcription Factor; Synapsins; Synaptophysin; Teratocarcinoma; Testicular Neoplasms; Transcription Factors; Transcription, Genetic; Tretinoin

Telomerase is active in about 90% of cancers and contributes to the immortality of cancer cells by maintaining the lengths of the ends of chromosomes. Undifferentiated embryonic human teratocarcinoma (HT) cells were found to express high levels of hTERT, the catalytic subunit of telomerase, and the hTERT promoter was unmethylated in these cells. Retinoic acid (RA)-induced differentiation led to hTERT gene silencing and increased methylation of the hTERT promoter. Treatment with trichostatin A, a histone deacetylase inhibitor, resulted in hTERT reactivation only in very early differentiating HT cells. After methylation patterns had been established within the hTERT promoter region in late differentiating cells, 5-azacytidine, a common demethylating agent, activated the hTERT gene but trichostatin A had no effect on hTERT transcription. These studies suggest that histone deacetylation is involved in early hTERT gene down-regulation and that DNA methylation may maintain silencing of the hTERT gene in these cells.

Topics: Antineoplastic Agents; Azacitidine; Catalytic Domain; Cell Differentiation; DNA (Cytosine-5-)-Methyltransferases; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Hydroxamic Acids; Methylation; Promoter Regions, Genetic; Telomerase; Teratocarcinoma; Tretinoin; Tumor Cells, Cultured

Permissiveness for human cytomegalovirus (HCMV) infection is dependent on the state of cellular differentiation and has been linked to repression of the viral major immediate early promoter (MIEP). We have used conditionally permissive cells to analyze differential regulation of the MIEP and possible mechanisms involved in latency. Our data suggest that histone deacetylases (HDACs) are involved in repression of the MIEP in non-permissive cells as inhibition of HDACs induces viral permissiveness and increases MIEP activity. Non-permissive cells contain the class I HDAC, HDAC3; super-expression of HDAC3 in normally permissive cells reduces infection and MIEP activity. We further show that the MIEP associates with acetylated histones in permissive cells, and that in peripheral blood monocytes the MIEP associates with heterochromatin protein 1 (HP1), a chromosomal protein implicated in gene silencing. As monocytes are believed to be a site of viral latency in HCMV carriers and reactivated virus is only observed upon differentiation into macrophages, we propose that chromatin remodeling of the MIEP following cellular differentiation could potentially play a role in reactivation of latent HCMV.

Topics: Acetylation; Cell Differentiation; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Cytomegalovirus; Enzyme Inhibitors; Gene Expression Regulation, Viral; Genes, Immediate-Early; Genes, Reporter; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immediate-Early Proteins; Macrophages; Models, Genetic; Monocytes; Promoter Regions, Genetic; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Teratocarcinoma; Transfection; Tumor Cells, Cultured; Viral Proteins; Virus Activation; Virus Latency

Trapoxin (cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8- oxo-9,10-epoxy-decanoyl)) is a fungal product that induces morphological reversion from transformed to normal in sis-transformed NIH3T3 fibroblasts. Trapoxin was found to cause accumulation of highly acetylated core histones in a variety of mammalian cell lines. In vitro experiments using partially purified mouse histone deacetylase showed that a low concentration of trapoxin irreversibly inhibited deacetylation of acetylated histone molecules. Chemical reduction of an epoxide group in trapoxin completely abolished the inhibitory activity, suggesting that trapoxin binds covalently to the histone deacetylase via the epoxide. In contrast, inhibition by trichostatin A, a known potent inhibitor of histone deacetylase, was reversible. Despite the different mode of inhibition, trapoxin and trichostatin A induced almost the same biological effects on the cell cycle and differentiation. These results strongly suggest that the in vivo effects commonly induced by these agents can be attributed to histone hyperacetylation resulting from the inhibition of histone deacetylase.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antifungal Agents; Antineoplastic Agents; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; G1 Phase; Histone Deacetylase Inhibitors; Hydroxamic Acids; Kinetics; Mammary Neoplasms, Experimental; Mice; Molecular Sequence Data; Molecular Structure; Oncogenes; Peptides; Teratocarcinoma; Tumor Cells, Cultured