trichostatin-a and Uterine-Cervical-Neoplasms

The aim of this study was to investigate the effects of trichostatin A (TSA) on cervical cancer and the related mechanisms.. The HeLa and Caski cervical cancer cell lines were treated with different concentrations of TSA. Cell viability was measured by MTT assays. Cell apoptosis was analysed using flow cytometry. Expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6), protein arginine methyltransferase 5 (PRMT5), and stanniocalcin 1 (STC1) was determined by qRT-PCR and Western blotting. Protein levels of LC3 II/I, beclin1, p62, JNK, and p-JNK were detected by Western blotting.. Treatment with TSA significantly decreased HeLa and Caski cell viability and enhanced the apoptosis rate in a dose-dependent manner. TSA markedly elevated beclin1 protein levels and the LC3 II/I ratio and significantly reduced p62 levels in a dose-dependent manner. In addition, TSA (1 μM) significantly suppressed PRMT5 and TRPV6 levels and enhanced STC1 and p-JNK levels. The lysosomal inhibitor bafilomycin-A1 synergistically enhanced the TSA-mediated increase in autophagic flux. Either the overexpression of TRPV6 or the inhibition of JNK signalling markedly enhanced cell viability, inhibited apoptosis, and autophagy and reduced p-JNK levels in TSA-treated cells. The inhibition of STC1 significantly increased TRPV6 protein levels and reduced p-JNK levels. Overexpression of PRMT5 dramatically decreased STC1 and p-JNK protein levels and increased TRPV6 levels.. TSA suppresses cervical cancer cell proliferation and induces apoptosis and autophagy through regulation of the PRMT5/STC1/TRPV6/JNK axis.

Topics: Apoptosis; Autophagy; Calcium Channels; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Enzyme Inhibitors; Female; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; JNK Mitogen-Activated Protein Kinases; Macrolides; Protein-Arginine N-Methyltransferases; Signal Transduction; TRPV Cation Channels; Uterine Cervical Neoplasms

Persistent infection with the high-risk of human papillomavirus (HR-HPVs) is the primary etiological factor of cervical cancer; HR-HPVs express oncoproteins E6 and E7, both of which play key roles in the progression of cervical carcinogenesis. Zinc Finger Nucleases (ZFNs) targeting HPV E7 induce specific shear of the E7 gene, weakening the malignant biological effects, hence showing great potential for clinical transformation.. Our aim was to develop a new comprehensive therapy for better clinical application of ZFNs. We here explored the anti-cancer efficiency of HPV targeted ZFNs combined with a platinum-based antineoplastic drug Cisplatin (DDP) and an HDAC inhibitor Trichostatin A (TSA).. SiHa and HeLa cells were exposed to different concentrations of DDP and TSA; the appropriate concentrations for the following experiments were screened according to cell apoptosis. Then cells were grouped for combined or separate treatments; apoptosis, cell viability and proliferation ability were measured by flow cytometry detection, CCK-8 assays and colony formation assays. The xenograft experiments were also performed to determine the anti-cancer effects of the combined therapy. In addition, the HPV E7 and RB1 expressions were measured by western blot analysis.. Results showed that the combined therapy induced about two times more apoptosis than that of ZFNs alone in SiHa and HeLa cells, and much more inhibition of cell viability than either of the separate treatment. The colony formation ability was inhibited more than 80% by the co-treatment, the protein expression of HPV16/18E7 was down regulated and that of RB1 was elevated. In addition, the xenografts experiment showed a synergistic effect between DDP and TSA together with ZFNs.. Our results demonstrated that ZFNs combined with DDP or TSA functioned effectively in cervical cancer cells, and it provided novel ideas for the prevention and treatment of HPV-related cervical malignancies.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Proliferation; Cisplatin; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Therapy, Combination; Female; Humans; Hydroxamic Acids; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Neoplasms, Experimental; Structure-Activity Relationship; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Zinc Finger Nucleases

The SLC5A8 gene encodes Na monocarboxylate transporter 1, which is epigenetically inactivated in various tumour types. This has been attributed to the fact that it prevents the entry of histone deacetylase (HDAC) inhibitors and favours the metabolic reprogramming of neoplastic cells. Nevertheless, its expression and regulation in cervical cancer (CC) have not been elucidated to date. The aim of the present study was to investigate whether SLC5A8 expression is silenced in CC and if epigenetic mechanisms are involved in its regulation. Using RNA and DNA from human CC cell lines and tumour tissues from patients with CC, the expression of SLC5A8 was analysed by reverse transcription polymerase chain reaction and the methylation status of its CpG island (CGI) by bisulphite‑modified sequencing. Additionally, SLC5A8 reactivation was examined in the CC cell lines following treatment with DNA methylation (5‑aza‑2'‑deoxycytidine) and HDAC inhibitors (trichostatin A and pyruvate). All the CC cell lines and a range of tumour tissues (65.5%) exhibited complete or partial loss of SLC5A8 transcription. The bisulphite‑sequencing revealed that hypermethylation of the CGI within SLC5A8 first exon was associated with its downregulation in the majority of cases. The transporter expression was restored in the CC cell lines following exposure to 5‑aza‑2'‑deoxycytidine alone, or in combination with trichostatin A or pyruvate, suggesting that DNA methylation and histone deacetylation contribute to its inhibition in a cell line‑dependent manner. Together, the results of the present study demonstrate the key role of DNA hypermethylation in the repression of SLC5A8 in CC, as well as the involvement of histone deacetylation, at least partially. This allows for research focused on the potential function of SLC5A8 as a tumour suppressor in CC, and as a biomarker or therapeutic target in this malignancy.

Topics: Adult; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Down-Regulation; Epigenetic Repression; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Hydroxamic Acids; Middle Aged; Monocarboxylic Acid Transporters; Papillomavirus Infections; Pyruvic Acid; Sequence Analysis, DNA; Uterine Cervical Neoplasms; Young Adult

Recent studies from our group and many others have shown the ability of histone deacetylase (HDAC) inhibitors for retarding the growth of carcinomas of cervix, colon and rectum in vitro. A search for naturally occurring HDAC inhibitors continues due to the adverse effects associated with known HDAC inhibitors like SAHA and TSA. Therefore in the current study, naturally occurring cinnamic acids derivatives were screened for HDAC inhibitory effect using in silico docking method which identified cinnamic acids as potential candidates. Cinnamic acids (CA) are naturally occurring phenolic compounds known to exhibit anticancer properties. However, it is not clearly known whether the anticancer properties of CA derivatives are due to the inhibition of oncogenic HDACs, if so how the efficacy varies among various CA derivatives. Hence, the HDAC inhibitory potential of CA derivatives containing increasing number of hydroxylic groups or methoxy moieties was determined using Discovery Studio software and the most potent CA derivatives tested ex vivo (biochemical assay) as well as in vitro (using cell based assay). Among CA derivatives tested, dihydroxy cinnamic acid (DHCA, commonly known as caffeic acid) exhibited better interactions with HDAC2 (compared to other isoforms) in silico and inhibited its activity ex vivo as well as in vitro. Targeted reduction of HDAC activity using DHCA induced death of cancer cells by (a) generating reactive oxygen species, (b) arresting cells in S and G2/M phases; and (c) induction of caspase-3 mediated apoptosis. In conclusion, we demonstrated that DHCA inhibited cancer cell growth by binding to HDAC followed by the induction of apoptosis.

Topics: Apoptosis; Binding Sites; Butyric Acid; Cell Line, Tumor; Cinnamates; Colorectal Neoplasms; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Uterine Cervical Neoplasms

Kallikrein-related peptidase 7 (KLK7) is a serine protease encoded within the kallikrein gene cluster located on human chromosome region 19q13.3-13.4. KLK7 is overexpressed in human pancreatic ductal adenocarcinomas (PDACs), but not in normal pancreas. Examination of KLK7 mRNA levels in pancreatic cancer cell lines revealed that it is readily detected in MIA PaCa-2 and PK-1 cells, but not in Panc-1 cells. Treatment of Panc-1 cells with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) significantly induced KLK7 mRNA expression. Similarly, KLK7 is highly expressed in cervical cancer cells, but its expression in the human cervical cancer cell line HeLa is only detected following TSA treatment. Promoter deletion analysis revealed that the proximal -238 promoter region, containing a putative Sp1-binding site, was sufficient for TSA activation of luciferase reporter activity, which was abrogated by the disruption of the Sp1-binding sequence. Consistent with the notion that TSA induced KLK7 expression via Sp1, co-expression of Sp1 with the KLK7-promoter/luciferase construct produced a significant increase in reporter activity. Chromatin immunoprecipitation (ChIP) analysis revealed enriched Sp1 occupancy on the KLK7 promoter following TSA treatment. Similarly, ChIP analysis showed the histone active mark, H3K4Me3, in the KLK7 promoter region was significantly increased after exposure to TSA.

Topics: Cell Line, Tumor; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Kallikreins; Pancreatic Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Sp1 Transcription Factor; Transcription, Genetic; Uterine Cervical Neoplasms

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Cell Survival; Enzyme Activation; Female; HeLa Cells; Histone Deacetylases; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; Nanoparticles; Oxidative Stress; Palladium; Uterine Cervical Neoplasms

Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein.

Topics: Animals; Cell Line, Tumor; Female; HeLa Cells; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Mice, Transgenic; Papillomavirus E7 Proteins; Papillomavirus Infections; Receptors, Retinoic Acid; Tumor Suppressor Protein p53; Up-Regulation; Uterine Cervical Neoplasms

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

Dimethylcelecoxib, a non-COX-2 inhibiting derivative of celecoxib, inhibits PGE(2) synthesis by transcriptional inhibition of mPGES-1. Previously we demonstrated that DMC downregulates EGR1 expression and increases nuclear NF-κB in human cervical cancer cells (HeLa). Both transcription factors are important regulators of mPGES-1 expression. Here we show that treatment of HeLa cells with DMC inhibits EGR1 promoter activity by influencing the transactivation activity of NF-κB. Mutation of the NF-κB motif as well as downregulation of NF-κB(p65)RelA using siRNA repealed the inhibitory effect of DMC on the EGR1 promoter. The transactivation activity of NF-κB is regulated by various co-activators or co-repressors. One of these co-repressors is HDAC1. DMC did not influence HDAC1 expression, but the HDAC activity was enhanced under DMC influence. After DMC treatment NF-κB co-immunoprecipitated with HDAC1. Electromobility shift assays depicted an increased interaction between NF-κB-HDAC1 and DNA containing NF-κB binding motives. Performing CHIP-assays we finally demonstrated the interaction of NF-κB and HDAC1 at the EGR1 promoter that was in part reversed by the HDAC1 inhibitor trichostatin A. Using siRNA against HDAC1 we could repeal the inhibitory effect of DMC on the EGR1 promoter. In conclusion we demonstrated that treatment of HeLa cells with DMC leads to an enhanced formation of a complex consisting of NF-κB and HDAC1 that binds to the EGR1 promoter resulting in downregulation of EGR1 expression which plays a major role for transcriptional inhibition of mGPES-1 expression. How these effects of DMC may contribute to a potential therapeutical benefit of various diseases is discussed.

Topics: Celecoxib; Dinoprostone; Early Growth Response Protein 1; Electrophoretic Mobility Shift Assay; Female; HeLa Cells; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoprecipitation; Intramolecular Oxidoreductases; Methylation; Promoter Regions, Genetic; Prostaglandin-E Synthases; Protein Binding; Pyrazoles; RNA, Small Interfering; Signal Transduction; Sulfonamides; Transcription Factor RelA; Transcription, Genetic; Transcriptional Activation; Uterine Cervical Neoplasms

Histone deacetylase (HDAC) inhibitors play an important role in inducing growth arrest, differentiation, and/or apoptosis in cancer cells. Given their ability to disrupt critical biological processes in cancer cells, these agents are emerging as potential therapeutics for cancer. Recently, it has been identified that HDAC inhibitors can also efficiently enhance the radiation sensitivity of cells, both in vitro and in vivo. In this study, we investigated whether the potent HDAC inhibitor, Trichostatin A, modulates the radiation sensitivity of the human cervical carcinoma cell line Hela under hypoxic conditions. We concluded that TSA could significantly inhibit the proliferation of Hela cells in a dose-and time-dependent manner under normoxic and hypoxic conditions. Hypoxia resulted in the cervical carcinoma Hela cells resistant to TSA. The findings from clonogenic survival assays indicate that incubation with TSA for 24 hours prior to irradiation enhances the radiation sensitivity of Hela cells under hypoxic conditions. More generally, we found Hela cells under hypoxic conditions treated with TSA could significantly down-regulate the expressions of HIF-1alpha and VEGF proteins. Taken together, our results demonstrated that TSA acts as a powerful radiosensitizer in Hela cells under hypoxic conditions probably by down-regulated expression of HIF-1alpha and VEGF proteins.

Topics: Cell Hypoxia; Cell Proliferation; Cell Survival; Down-Regulation; Female; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Radiation Tolerance; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A

Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.. Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase (DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B.. HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L group with untreated control with t-test. There was no significance between 1 µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.. DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.

Topics: Apoptosis; Cell Line; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3B; Female; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Uterine Cervical Neoplasms

Histone deacetylase inhibitors (HDACi) show promise as a novel class of antitumoral agents and have shown the ability to induce apoptosis of tumor cells. To gain a better understanding of the action of HDACi, we conducted a functional gene screen approach named suppression of mortality by antisense rescue technique to identify the key genes responsible for the tumor-selective killing trichostatin A. Over 20 genes associated with HDACi-induced mortality were identified. One of the confirmed positive hits is LIV1, a putative zinc transporter. LIV1 is significantly induced by treatment with HDACi in a number of tumor cells, but not in normal cells. Knockdown of LIV1 suppressed apoptosis induced by HDACi in tumor cells. Although HDACi induced a slight increase in the free intracellular zinc concentration, knockdown of LIV1 significantly enhanced the intracellular zinc level, which was associated with resistance to apoptosis. On the other hand, pretreatment of the cells with a specific zinc chelator TPEN reversed the apoptosis resistance conferred by knockdown of LIV1. However, the biological effects of TPEN were abolished by addition of physiologic concentrations of zinc. Taken together, the present study identifies LIV1 as a critical mediator responsible for HDACi-induced apoptosis. The effect of LIV1 is, at least in part, mediated by affecting intracellular zinc homeostasis, which may be related to alteration of the catalytic activity of the Caspase 3 and expression of some BCL-2 family genes. As such, these findings highlight a novel mechanism underlying the action of HDACi that could be potentially useful in the clinical setting.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cation Transport Proteins; Cell Death; Cell Growth Processes; Cell Line, Tumor; DNA, Antisense; Drug Synergism; Female; Gene Knockdown Techniques; HeLa Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Transfection; Uterine Cervical Neoplasms; Zinc

The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Azacitidine; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Death-Associated Protein Kinases; Decitabine; DNA Methylation; DNA Modification Methylases; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Protein Biosynthesis; Protein Synthesis Inhibitors; RNA, Messenger; Uterine Cervical Neoplasms

Transcriptional silencing of the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT) in a proportion of transformed cell lines is associated with methylated CpG hotspots in the MGMT 5' flank. The goal of the study was to evaluate the mechanism by which CpG methylation of theMGMT promoter region influenced silencing of the gene. Analysis of histone acetylation status in two regions of the promoter using chromatin immunoprecipitation assay showed that a higher level of histone acetylation was associated with expression in three MGMT-expressing cell lines (HeLa CCL2, HT29, and Raji) compared with three MGMT-silenced cell lines (HeLa S3, BE, and TK6). To determine how the modulation of CpG methylation and histone acetylation influenced MGMT expression, we exposed the cells to 5-aza-2'deoxycytidine (5-Aza-dC), inhibitor of DNA methylation, which strongly up-regulated MGMT expression in three MGMT-silenced cell lines whereas trichostatin A, inhibitor of histone deacetylase, weakly induced MGMT. However, combined treatment with 5-Aza-dC and trichostatin A significantly up-regulated MGMT RNA expression to a greater extent than in cells treated with either agent alone suggesting that histone deacetylation plays a role in MGMT silencing but that CpG methylation has a dominant effect. Consistent with enhanced MGMT expression, 5-Aza-dC increased the association of acetylated histone H3 and H4 bound to the MGMT promoter. Chromatin immunoprecipitation analysis of methyl-CpG binding domain containing proteins detected a greater amount of MeCP2, MBD1, and CAF-1 bound to the MGMT promoter in MGMT-silenced cells. Our findings implicate specific MBD proteins in methylation-mediated transcriptional silencing of MGMT.

Topics: Acetylation; Azacitidine; Cell Line, Transformed; Cell Line, Tumor; DNA Methylation; DNA-Binding Proteins; Female; Gene Expression Regulation, Enzymologic; Gene Silencing; HeLa Cells; Histones; Humans; Hydroxamic Acids; O(6)-Methylguanine-DNA Methyltransferase; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Uterine Cervical Neoplasms

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase holoenzyme as well as the rate-limiting component of the telomerase enzyme complex. However, the role of the hTERT in apoptosis induced by histone deacetylase inhibitor has only been marginally addressed. For the first time, our study demonstrated that trichostatin A (TSA) briefly activated the proliferation of cervical cancer cell lines, HeLa and SiHa, within 12 h, but then inhibited cell growth after that time point. In response to TSA, hTERT expression, telomerase activity, and telomere length also underwent similar changes during the same time frame. Furthermore, the data in our study showed that cells transfected with dominant negative hTERT were more likely to undergo apoptosis induced by TSA than cells transfected with wild-type hTERT. The cyclin/cdk inhibitor p21waf1 was down-regulated by hTERT without changing the expression of p53. Results from this study suggest that the hTERT might be a primary target of TSA and the anti-apoptosis effect of hTERT might be carried out through a p21waf1-dependent and p53-independent pathway.

Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; RNA, Messenger; Telomerase; Telomere; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Histone deacetylase (HDAC) inhibitors have shown promise in clinical cancer therapy and to consistently induce p21WAF1/CIP1 expression in a p53-independent manner and via increased acetylation of the chromatin at the Sp1 sites in the p21WAF1/CIP1 promoter region. However, the exact mechanism by which HDAC inhibitors induce p21WAF1/CIP1 remains unclear. In this study, we observed that Trichostatin A (TSA), a HDAC inhibitor, induced strikingly p21WAF1/CIP1 expression in human cervical cancer (HeLa) cells, and this induction correlated with downregulation of c-myc expression. Coincident with this observation, knock down of c-myc with a c-myc specific small interfering RNA dramatically induced expression of p21WAF1/CIP1 in these cancer cells. These data suggest that c-myc may play a critical role in repression of p21WAF1/CIP1 expression in HeLa cells. More importantly, using chromatin immunoprecipitation assay, we observed for the first time that c-myc bound to the endogenous p21WAF1/CIP1 promoter in untreated HeLa cells, but not in TSA-treated cells. Taken together, TSA induced c-myc downregulation and release from the endogenous p21WAF1/CIP1 promoter contributes, at least partially, to transcriptional activation of the p21WAF1/CIP1 in HeLa cells.

Topics: Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Female; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sp1 Transcription Factor; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3' end of hmi-er1 produces two major isoforms, hMI-ER1alpha and hMI-ER1beta, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1alpha and hMI-ER1beta in the regulation of transcription. Using fusion proteins of hMI-ER1alpha or hMI-ER1beta tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1alpha and hMI-ER1beta occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Caenorhabditis elegans Proteins; Carcinoma; Conserved Sequence; DNA-Binding Proteins; Enzyme Inhibitors; Female; Helminth Proteins; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Molecular Sequence Data; Protein Isoforms; Protein Structure, Tertiary; Recombinant Proteins; Repressor Proteins; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Uterine Cervical Neoplasms