trichostatin-a and Cell-Transformation--Neoplastic

Small cell lung cancer (SCLC) accounts for nearly 15% of human lung cancers and is one of the most aggressive solid tumors. The SCLC cells are thought to derive from self-renewing pulmonary neuroendocrine cells by oncogenic transformation. However, whether the SCLC cells possess stemness and plasticity for differentiation as normal stem cells has not been well understood thus far. In this study, we investigated the expressions of multilineage stem cell markers in the cancer cells of SCLC cell line (NCI-H446) and analyzed their clonogenicity, tumorigenicity, and plasticity for inducing differentiation. It has been found that most cancer cells of the cell line expressed multilineage stem cell markers under the routine culture conditions and generated single-cell clones in anchorage-dependent or -independent conditions. These cancer cells could form subcutaneous xenograft tumors and orthotopic lung xenograft tumors in BALB/C-nude mice. Most cells in xenograft tumors expressed stem cell markers and proliferation cell nuclear antigen Ki67, suggesting that these cancer cells remained stemness and highly proliferative ability in vivo. Intriguingly, the cancer cells could be induced to differentiate into neurons, adipocytes, and osteocytes, respectively, in vitro. During the processes of cellular phenotype-conversions, autophagy and apoptosis were two main metabolic events. There is cross-talking between autophagy and apoptosis in the differentiated cancer cells. In addition, the effects of the inhibitor and agonist for Sirtuin1/2 on the inducing osteogenic differentiation indicated that Sirtuin1/2 had an important role in this process. Taken together, these results indicate that most cancer cells of NCI-H446 cell line possess stemness and plasticity for multilineage differentiation. These findings have potentially some translational applications in treatments of SCLC with inducing differentiation therapy.

Topics: Adipogenesis; Animals; Apoptosis; Autophagy; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Cell Transformation, Neoplastic; Humans; Hydroxamic Acids; Ki-67 Antigen; Lung Neoplasms; Mice; Mice, Nude; Neoplastic Stem Cells; Neurogenesis; Osteogenesis; Sirtuin 1; Sirtuin 2; Small Cell Lung Carcinoma; Transplantation, Heterologous

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.

Topics: Animals; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Neoplastic Stem Cells; RNA, Small Interfering; Spheroids, Cellular; Ubiquitin; Up-Regulation; Uterine Cervical Neoplasms

This study identified LTBP-2 as a pleiotropic tumor suppressor in nasopharyngeal carcinoma, which safeguards against critical malignant behaviors of tumor cells. LTBP-2 expression was significantly decreased or lost in up to 100% of NPC cell lines (7/7) and 80% of biopsies (24/30). Promoter hypermethylation was found to be involved in LTBP-2 silencing. Using a tetracycline-regulated inducible expression system, we unveiled functional roles of LTBP-2 in suppressing colony formation, anchorage-independent growth, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. Three-dimensional culture studies suggested the involvement of LTBP-2 in maintenance of tumor cell dormancy in a growth factor favorable microenvironment.

Topics: Azacitidine; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Decitabine; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Latent TGF-beta Binding Proteins; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neovascularization, Pathologic; Promoter Regions, Genetic; Tumor Microenvironment; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A

Histone deacetylase inhibitors (HDACi) are actively explored as new-generation epigenetic drugs but have low efficacy in cancer monotherapy. To reveal new mechanism for combination therapy, we show that HDACi induce cell death but simultaneously activate tumor-progressive genes to ruin therapeutic efficacy. Combined treatments to target tumorigenesis and HDACi-activated metastasis with low toxic modalities could develop new strategies for long-term cancer therapy.. Because metastasis is the major cause of cancer mortality, we measured cell migration activity and profiled metastasis-related gene expressions in HDACi-treated cancer cells. We developed low toxic combination modalities targeting tumorigenesis and HDACi-activated metastasis for preclinical therapies in mice.. We showed that cell migration activity was dramatically and dose dependently enhanced by various classes of HDACi treatments in 13 of 30 examined human breast, gastric, liver, and lung cancer cell lines. Tumor metastasis was also enhanced in HDACi-treated mice. HDACi treatments activated multiple PKCs and downstream substrates along with upregulated proapoptotic p21. For targeting tumorigenesis and metastasis with immediate clinical impact, we showed that new modalities of HDACi combined drugs with PKC inhibitory agent, curcumin or tamoxifen, not only suppressed HDACi-activated tumor progressive proteins and cell migration in vitro but also inhibited tumor growth and metastasis in vivo.. Treatments of different structural classes of HDACi simultaneously induced cell death and promoted cell migration and metastasis in multiple cancer cell types. Suppression of HDACi-induced PKCs leads to development of low toxic and long-term therapeutic strategies to potentially treat cancer as a chronic disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Combined Modality Therapy; Curcumin; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Neoplasm Metastasis; Neoplasms; Protein Kinase C; Tamoxifen

To investigate the effect of monoamine oxidase inhibitor tranylcypromine (TCP) on the differentiation of human U251 glioma cells, we treated U251 cells with TCP and/or 100 nmol/L histone deacetylase inhibitor trychostatin A (TSA). The differentiation of U251 cells was observed with inverted microscopy. The cell proliferation and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Apoptosis was observed by Hoechst 33258 staining. The levels of differentiation-related genes were assessed by real-time PCR and Western blotting. TCP-induced differentiation was characterized by typical morphological changes, inhibition of cellular proliferation, accumulation of cells in the G1 phase of the cell cycle, decreased expression of the pluripotency transcription factors Oct4 and Sox2, and increased expression of glial fibrillary acid protein (GFAP). The combination of TCP and TSA treatment also triggered an over-expression of GFAP. These findings suggest that TCP may induce differentiation of U251 glioma cells, and the differentiation process may be promoted by histone deacetylase inhibitor TSA.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Glioma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Monoamine Oxidase Inhibitors; Tranylcypromine

Polycomb group (PcG) proteins such as Enhancer of zeste homolog 2 (EZH2) are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. Targets of PcG include cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. Senescence is a characteristic of melanocytic nevi, benign melanocytic proliferations that can be precursors of malignant melanoma. In this study, we report that EZH2, which we find absent in melanocytic nevi but expressed in many or most metastatic melanoma cells, functionally suppresses the senescent state in human melanoma cells. EZH2 depletion in melanoma cells inhibits cell proliferation, restores features of a cellular senescence phenotype, and inhibits growth of melanoma xenografts in vivo. p21/CDKN1A is activated upon EZH2 knockdown in a p53-independent manner and contributes substantially to cell cycle arrest and induction of a senescence phenotype. EZH2 depletion removes histone deacetylase 1 (HDAC1) from the CDKN1A transcriptional start site and downstream region, enhancing histone 3 acetylation globally and at CDKN1A. This results in recruitment of RNA polymerase II, leading to p21/CDKN1A activation. Depletion of EZH2 synergistically activates p21/CDKN1A expression in combination with the HDAC inhibitor trichostatin A. Since melanomas often retain wild-type p53 function activating p21, our findings describe a novel mechanism whereby EZH2 activation during tumor progression represses p21, leading to suppression of cellular senescence and enhanced tumorigenicity.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Melanoma; Mice; Nevus, Pigmented; Polycomb Repressive Complex 2; RNA Polymerase II; Skin Neoplasms; Transcription Factors

The accumulation of reactive oxygen species and subsequent oxidative DNA damage underlie the development of Barrett's oesophagus (BO) and its progression to Barrett's dysplasia (BD) and adenocarcinoma (BAC).. The promoter regions of 23 genes of the glutathione S-transferase (GST) and glutathione peroxidase (GPX) families were systematically analysed. Quantitative bisulfite pyrosequencing, real-time RT-PCR, western blot and immunohistochemical (IHC) analysis methods were utilised in this study.. 14 genes were identified that have CpG islands around their transcription start sites: GSTs (GSTM2-M5, GSTA4, GSTP1, GSTZ1, GSTT2, GSTO1 and GSTO2) and GPXs (GPX1, GPX3, GPX4 and GPX7). Analysis of an initial set of 20 primary samples demonstrated promoter DNA hypermethylation and mRNA downregulation of GPX3, GPX7, GSTM2, GSTM3 and GSTM5 in more than half of the BAC samples. Further analysis of 159 primary human samples (37 normal, 11 BO, 11 BD and 100 BACs) indicated frequent hypermethylation (>or=10% methylation) of GPX3 (62%), GPX7 (67%), GSTM2 (69.1%) and GSTM3 (15%) in BACs. A significant inverse correlation between DNA methylation and mRNA expression level was shown for GPX3 (p<0.001), GPX7 (p = 0.002), GSTM2 (p<0.001) and GSTM5 (p = 0.01). Treatment of oesophageal cancer cell lines with 5-aza-2'-deoxycytidine and trichostatin-A led to reversal of the methylation pattern and re-expression of these genes at the mRNA and protein levels. The IHC analysis of GPX3, GPX7 and GSTM2 on a tissue microarray that contained 75 BACs with normal squamous oesophageal samples demonstrated an absent to weak staining in tumours (52% for GPX3, 57% for GPX7 and 45% for GSTM2) and a moderate to strong immunostaining in normal samples.. Epigenetic inactivation of members of the glutathione pathway can be an important mechanism in Barrett's tumourigenesis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Azacitidine; Barrett Esophagus; Cell Transformation, Neoplastic; CpG Islands; Decitabine; Disease Progression; DNA Methylation; DNA, Neoplasm; Down-Regulation; Epigenesis, Genetic; Esophageal Neoplasms; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Glutathione Transferase; Humans; Hydroxamic Acids; Middle Aged; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

High-mobility group box 1 (HMGB1) is a nuclear protein that acts as a ligand of the receptor for advanced glycation end products (RAGE) and its expression enhances progression of cancer. However, the mechanism underlying HMGB1 secretion is still unclear. In this study, we examined the effect of deoxycholic acid (DCA), a promoter of colon carcinogenesis, on HMGB1 secretion.. We used an in vitro transformation model comprised of IEC6 intestinal epithelial cells treated with azoxymethane (AOM) and/or DCA. HMGB1 expression and secretion were examined by Western and Northern blot analyses, and ELISA. Intracellular translocation of HMGB1 was examined by protein fractionation.. AOM + DCA-treated IEC6 cells showed upregulation of HMGB1 mRNA expression and increased level of HMGB1 protein in culture medium, but decreased level of HMGB1 protein in the nucleus. AOM + DCA treatment increased level of histone H4 acetylation, which induced translocation of HMGB1 from the nucleus to the cytoplasm and increased HMGB1 secretion. Leptomycin B inhibited extranuclear translocation and secretion of the HMGB1 protein.. These findings suggest that DCA affects intracellular localization and secretion of HMGB1.

Topics: Acetylation; Animals; Antibiotics, Antineoplastic; Azoxymethane; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cholagogues and Choleretics; Colon; Deoxycholic Acid; Enzyme Inhibitors; Epithelial Cells; Fatty Acids, Unsaturated; Histones; HMGB1 Protein; Hydroxamic Acids; Intestinal Mucosa; Male; Rats; Rats, Inbred F344; RNA, Messenger; Up-Regulation

Humans are exposed to arsenicals through many routes with the most common being in drinking water. Exposure to arsenic has been associated with an increase in the incidence of cancer of the skin, lung and bladder. Although the relationship between exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation state of 13 000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation occur during arsenical-induced malignant transformation that are linked to the expression state of the associated gene. DNA hypermethylation was detected in hypoacetylated promoters in the select cases analyzed. These epigenetic changes occurred frequently in the same promoters whether the selection was performed with arsenite [As(III)] or with monomethylarsonous acid, suggesting that these promoters were targeted in a non-random fashion, and probably occur in regions important in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by altering the epigenetic terrain of select genes.

Topics: Acetylation; Arsenicals; Azacitidine; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Decitabine; Environmental Exposure; Histones; Humans; Hydroxamic Acids; Lung Neoplasms; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Teratogens; Urinary Bladder Neoplasms; Urothelium

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

Transcription factor haploinsufficiency plays a role in the pathogenesis of many diseases, including cancer. In a mouse model of prostate tumor initiation, loss of a single allele of the tumor suppressor Nkx3.1 stochastically inactivates the expression of a class of dosage-sensitive target genes. Here we show that dosage sensitivity is associated with the differential histone H3/H4 acetylation states of Nkx3.1 target genes. When histone acetylation is induced in Nkx3.1+/- mouse prostates with the histone deacetylase inhibitor Trichostatin A, Nkx3.1 can bind to and reactivate the expression of dosage-sensitive target genes. We incorporated our findings into a mathematical model that entails the association of Nkx3.1 with histone acetyltransferase activity. Subsequent experiments indicate that Nkx3.1 associates with and recruits the histone acetyltransferase p300/CREB-binding protein-associated factor to chromatin. Finally, we demonstrate a role for the dosage-sensitive target gene intelectin/omentin in suppressing prostate tumorigenicity. Our results reveal how the interplay between transcription factor dosage and chromatin affects target gene expression in tumor initiation.

Topics: Acetylation; Animals; Cell Transformation, Neoplastic; Chromatin; Chromatin Assembly and Disassembly; Disease Models, Animal; Gene Dosage; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Histones; Homeodomain Proteins; Hydroxamic Acids; Lectins; Loss of Heterozygosity; Male; Mice; Mice, Mutant Strains; Models, Biological; p300-CBP Transcription Factors; Prostatic Neoplasms; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Transcription Factors; Tumor Suppressor Proteins

Chemoresistance has been one of the major problems in anticancer therapy. In our effort to find a potential molecular target for overcoming the chemoresistance in prostate cancer, a promising anticancer drug trichostatin A (TSA) induced cell death was found to be compromised by enhanced NF-kappaB activation in 267B1/K-ras human prostate epithelial cancer cells. However, both the NF-kappaB activation and chemoresistance were reduced by pretreatment with proteasome inhibitor-I (ProI), accompanied by accumulations of both IkappaBalpha and p65/RelA (but not p50/NF-kappaB1) in the cytoplasm. Clonogenic cell survival and soft agar assays further confirmed the increased TSA chemosensitivity of 267B1/K-ras cells by ProI treatment. Moreover, dominant negative mutant of IKKbeta, IkappaBalpha and p65 enhanced the chemosensitization, too. Unexpectedly, using LY294002 and PD98059, phosphatidylinositol-3-kinase and mitogen-activated protein kinase were also implied in TSA chemoresistance through NF-kappaB activation, while these compounds had showed no effect on radiosensitization in the cells. On the other hand, together with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, activations of caspase-8 and caspase-3 by TSA and ProI were noticed, suggesting the involvement of apoptotic process in chemosensitization of 267B1/K-ras cells. Altogether, these results suggest that blocking the NF-kappaB activation pathway could be an efficient target for improving the TSA chemosensitization and applying to the development of anticancer therapeutics in Ki-Ras-overexpressing tumorigenic cells, including prostate cancer.

Topics: Animals; Apoptosis; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Humans; Hydroxamic Acids; Male; Mice; Mice, Nude; NF-kappa B; Prostatic Neoplasms; Protein Synthesis Inhibitors; ras Proteins; Transfection

Histone acetylase and histone deacetylase are two crucial enzymes that determine the structure of chromatin, regulating gene expression. In this study, we observed that trichostatin A (TSA), a specific histone deacetylase inhibitor, could effectively inhibit the growth of v-Src-transformed (IV5) cells and abrogate their ability to form colonies in soft agar. Further analysis demonstrated that, although TSA reduced the expression of Eps8 in a dose- and time-dependent manner, both the protein expression and kinase activity of v-Src remained constant, and the abundance and phosphotyrosine levels of Src substrates, including cortactin, focal adhesion kinase, p130(Cas), paxillin, and Shc, were not altered. Notably, removal of TSA from the medium restored not only the expression of Eps8, but also cellular growth. Northern and reverse transcription-PCR analyses revealed the significant reduction of eps8 transcripts in TSA-treated IV5 cells relative to control cells. When active Src-expressing chicken embryonic cells were forced to overexpress p97(Eps8), they became resistant to TSA-mediated anti-proliferation. Furthermore, using small interference RNA of eps8, we demonstrated the requirement for Eps8 in IV5 cell proliferation. Thus, our results highlight a critical role for p97(Eps8) in TSA-exerted growth inhibition of v-Src-transformed cells.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Agar; Animals; Blotting, Northern; Cell Division; Cell Line; Cell Transformation, Neoplastic; Chickens; Cortactin; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Histones; Hydroxamic Acids; Immunoblotting; Microfilament Proteins; Mutation; Paxillin; Phosphoproteins; Phosphotyrosine; Precipitin Tests; Protein-Tyrosine Kinases; Proteins; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; src-Family Kinases; Time Factors; Transfection

Transformation of fibroblasts with the v-fos oncogene produces a highly invasive phenotype that is mediated by changes in gene expression. Inhibition of histone deacetylase (HDAC) activity with trichostatin A (TSA) or valproic acid (VPA) at concentrations that do not affect morphology, motility, chemotaxis or proliferation, strongly inhibits invasion and results in the re-expression of a significant proportion of those genes that are downregulated in the v-Fos-transformed cells. Independent expression of three of these re-expressed genes, (Ring1 and YY1 binding protein (RYBP); protocadherin gamma subfamily C,3 (PCDHGC3); and signal transducer and activator of transcription 6 (STAT6)) in Fos-transformed cells, has no effect on morphology, motility, chemotaxis or proliferation, but strongly inhibits invasion. Therefore, we conclude that the ability of v-Fos-transformed cells to invade is dependent upon repression of gene expression through either direct or indirect HDAC activity.

Topics: Actins; Animals; Blotting, Northern; Blotting, Western; Cadherin Related Proteins; Cadherins; Cell Division; Cell Line; Cell Line, Transformed; Cell Movement; Cell Transformation, Neoplastic; Chemotaxis; Cloning, Molecular; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Enzymologic; Histone Deacetylases; Hydroxamic Acids; Microscopy, Confocal; Microscopy, Phase-Contrast; Neoplasm Invasiveness; Oncogene Proteins v-fos; Phenotype; Rats; Repressor Proteins; RNA; STAT6 Transcription Factor; Trans-Activators; Transfection; Valproic Acid

The carcinogenic process initiated by nongenotoxic carcinogens involves modulation of gene expression. Nickel compounds have low mutagenic activity, but are highly carcinogenic. In vitro both mouse and human cells can be efficiently transformed by soluble and insoluble nickel compounds to anchorage-independent growth. Because previous studies have shown that carcinogenic nickel compounds silence genes by inhibiting histone acetylation and enhancing DNA methylation, we investigated the effect of enhancing histone acetylation on cell transformation. The exposure of nickel-transformed cells to the histone deacetylase inhibitor trichostatin A (TSA) resulted in the appearance of significant number of revertants measured by their inability to grow in soft agar. Using the Affymetrix GeneChip we found that the level of expression of a significant number of genes was changed (suppressed or upregulated) in nickel-transformed clones but returned to a normal level in revertants obtained following TSA treatment. Moreover, we found that treatment of cells with TSA inhibited the ability of nickel to transform mouse PW cells to anchorage-independent growth. Treatment with TSA also inhibited the ability of nickel to transform human HOS cells, although to a lesser extent. In contrast, treatment with TSA was not able to revert established cancer cell lines as readily as the nickel-transformed cells. These data indicated that modulation of gene expression is important for nickel-induced transformation.

Topics: Animals; Anticarcinogenic Agents; Cell Line, Tumor; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression; Gene Expression Profiling; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Nickel; Oligonucleotide Array Sequence Analysis; Osteoblasts; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Epigenetic regulation of gene expression significantly influences cell growth and differentiation. Here we show that epigenetic silencing of Fas determines tumor growth in vivo and apoptotic sensitivity in vitro. In established tumors with epigenetically repressed Fas, restoration of Fas activity either by transfection of fas or treatment with Trichostatin A (TSA), an inhibitor of histone deacetylase, suppresses tumor growth and restores chemosensitivity. The TSA-dependent chemosensitivity and tumor growth control require both tumor Fas and the host NK (natural killer) cell functions. This work demonstrates the importance of epigenetic modification of Fas in tumor progression and immune evasion, and emphasizes the essential interplay between Fas and innate immunity in the control of chemoresistant tumors.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Disease Progression; Drug Resistance, Neoplasm; fas Receptor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Hydroxamic Acids; Immunity, Innate; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, SCID; Neoplasms; Time Factors; Transfection; Tumor Cells, Cultured

To elucidate the role of focal adhesion kinase (pp125FAK) in transformation, its phosphorylation in transformed fibroblasts was compared with that of detransformed fibroblasts induced by a histone deacetylase inhibitor, trichostatin A (TSA). Inhibition of histone deacetylase activity in two different ras-transformed fibroblast lines by TSA induced a morphological change into a flattened and more spread morphology, implying detransformation. These morphological changes included increased spreading ability of transformed NIH 3T3 cells on fibronectin. Of the six tyrosine phosphorylation sites in pp125FAK, phosphorylation at position 861 (Tyr-861) was clearly decreased during detransformation by TSA. It resulted from decreased activity of Src family tyrosine kinase and/or decreased amount of Src kinase interacting with pp125FAK. Furthermore, phosphorylation of Tyr-861 was reduced substantially by the Src family kinase inhibitor, PP1, while overexpression of Src kinase increased its phosphorylation, implying that Src kinase regulates phosphorylation of pp125FAK at Tyr-861. All of these findings suggest that increased phosphorylation of pp125FAK at Tyr-861 correlates with Ras-induced transformation of fibroblasts, and TSA is able to detransform them through regulation of pp125FAK phosphorylation at Tyr-861 by an Src family kinase.

Topics: 3T3 Cells; Animals; Cell Adhesion; Cell Line, Transformed; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Genes, ras; Histone Deacetylase Inhibitors; Hydroxamic Acids; Mice; Phosphorylation; Protein-Tyrosine Kinases; src-Family Kinases; Tyrosine

Although p53 inactivation is implicated as a mechanism to explain diminished apoptotic response, it is clear that tumor cells that possess transcriptionally functional p53 can also be resistant to diverse apoptotic stimuli. We hypothesize that oncogenic activation and DNA damage are sufficient stimuli to increase the p53-dependent transcription of Fas and thereby establish a situation in which cell to cell contact could be a selective pressure to either lose p53 function or inactivate components of the Fas death pathway. Examination of genetically matched tumor cell lines that possessed either wild-type or null p53 loci indicated that cells possessing functional p53 increased their surface levels of Fas and Fas ligand (FasL) in response to DNA damage. In contrast, stress induced by changes in the tumor microenvironment such as decreased oxygen did not up-regulate Fas or FasL. Cells with wild-type p53 underwent Fas-mediated killing in the presence of either FasL-expressing killer cells or activating Fas antibodies, whereas cells in which p53 was deleted or inactivated were protected from such killing. Furthermore, Fas and FasL expression and induction became transcriptionally repressed in transformed cells with wild-type p53 with increasing passage, whereas other p53 downstream targets and functions, such as p21 inducibility and cell cycle arrest, remained intact. Repression of the Fas locus could be reverted by treatment with the histone deacetylase inhibitor trichostatin A. These results support a model of tumor progression in which oncogenic transformation drives tumor cells to lose either p53 or their Fas sensitivity as a means of promoting their survival and evade immune surveillance.

Topics: Animals; Apoptosis; Cell Hypoxia; Cell Line, Transformed; Cell Transformation, Neoplastic; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kinetics; Membrane Glycoproteins; Mice; Mice, Inbred MRL lpr; RNA, Messenger; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The Fos and Jun oncoproteins form dimeric complexes that stimulate transcription of genes containing activator protein-1 regulatory elements. We found, by representational difference analysis, that expression of DNA 5-methylcytosine transferase (dnmt1) in fos-transformed cells is three times the expression in normal fibroblasts and that fos-transformed cells contain about 20 percent more 5-methylcytosine than normal fibroblasts. Transfection of the gene encoding Dnmt1 induced morphological transformation, whereas inhibition of dnmt1 expression or activity resulted in reversion of fos transformation. Inhibition of histone deacetylase, which associates with methylated DNA, also caused reversion. These results suggest that fos may transform cells through alterations in DNA methylation and in histone deacetylation.

Topics: 5-Methylcytosine; Acetylation; Animals; Cell Size; Cell Transformation, Neoplastic; Cytosine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, fos; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Proto-Oncogene Proteins c-fos; Rats; Transcription, Genetic; Transfection

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

v-ErbA, a mutated thyroid hormone receptor alpha (TRalpha), is thought to contribute to avian erythroblastosis virus (AEV)-induced leukemic transformation by constitutively repressing transcription of target genes. However, the binding of v-ErbA or any unliganded nuclear receptor to a chromatin-embedded response element as well as the role of the N-CoR-SMRT-HDAC co-repressor complex in mediating repression remain hypothetical. Here we identify a v-ErbA-response element, VRE, in an intronic DNase I hypersensitive site (HS2) of the chicken erythroid carbonic anhydrase II (CAII) gene. In vivo footprinting shows that v-ErbA is constitutively bound to this HS2-VRE in transformed, undifferentiated erythroblasts along with other transcription factors like GATA-1. Transfection assays show that the repressed HS2 region can be turned into a potent enhancer in v-ErbA-expressing cells by mutation of the VRE. Differentiation of transformed cells alleviates v-ErbA binding concomitant with activation of CAII transcription. Co-expression of a gag-TRalpha fusion protein in AEV-transformed cells and addition of ligand derepresses CAII transcription. Treatment of transformed cells with the histone deacetylase inhibitor, trichostatin A, derepresses the endogenous, chromatin-embedded CAII gene, while a transfected HS2-enhancer construct remains repressed. Taken together, our data suggest that v-ErbA prevents CAII activation by 'neutralizing' in cis the activity of erythroid transcription factors.

Topics: Alpharetrovirus; Animals; Base Sequence; Carbonic Anhydrases; Cell Differentiation; Cell Transformation, Neoplastic; DNA Footprinting; Enhancer Elements, Genetic; Erythropoiesis; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Hydroxamic Acids; Introns; Leukemia; Molecular Sequence Data; Oncogene Proteins v-erbA; Protein Binding; Receptors, Thyroid Hormone; Response Elements

During screening for inhibitors of ras-mediated differentiation of PC12 cells, trichostatin A (TSA) was isolated from the metabolites of Streptomyces as a potent inhibitor. TSA blocked both oncogenic ras- and NGF-induced neurite outgrowth from PC12 cells. However, addition of TSA 1 h after NGF-stimulation did not inhibit neuronal differentiation, suggesting that TSA affects an early step in the NGF-signaling pathway mediated by ras. Northern blotting analysis showed that TSA prolonged the maximum expression period of c=fos mRNA triggered by NGF and delayed its return to the basal level. TSA reduced c-jun mRNA induction by NGF but greatly enhanced c-myc mRNA induced by NGF. Yoshida et al. (J. Biol. Chem, 265, 17174-17179, 1990) showed that TSA inhibits histone deacetylation, which might influence the gene expression involved in cellular differentiation. In this study, we also found that TSA prevents histone deacetylation in PC12 cells as well as other cell lines, suggesting that inhibition of histone deacetylation by TSA might affect the expression of early-response genes. We also demonstrated that TSA induced reversion of oncogenic ras-transformed NIH3T3 cells to a normal morphology, suggesting that inhibitors of ras-mediated differentiation of PC12 cells may be effective as anticancer agents.

Topics: 3T3 Cells; Acetylation; Animals; Cell Differentiation; Cell Transformation, Neoplastic; Gene Expression Regulation; Genes, ras; Histones; Hydroxamic Acids; Mice; Nerve Growth Factors; Neurites; PC12 Cells; Rats; Time Factors

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

A novel compound, depudecin, induced production of the flat phenotype of Ki-ras-transformed NIH3T3 cells at the low concentration of 1 microgram/ml. This effect was reversible. Actin stress fiber was detected in these cells after depudecin treatment. Almost complete reversion to the flat phenotype was observed at 6 h after depudecin addition. The synthesis of ras-mRNA did not decrease enough with depudecin treatment at the concentration of 10 micrograms/ml to reverse the transformed morphology.

Topics: 3T3 Cells; Actins; Alkadienes; Animals; Antifungal Agents; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Epoxy Compounds; Fatty Alcohols; Gene Expression; Genes, ras; Hydroxamic Acids; Kinetics; Mice; Phenotype; RNA, Messenger