Project description:The TERT and HKR3 mechanism regulating cell cycle related expression changes remains unknown during HCC progress. We evaluated the relationship between hTERT expression and human kruppel-related 3 (HKR3) and cell cycle-related factors in Hep3B cell lines. Overall design: NT-Control, HKR3 overexpression and TERT kncokdown in Hep3B cells
Project description:Hep3B and Huh7 are two types of human hepatoma cell lines (HCC). In our laboratory, we cultured their stem-like cancer cells (HCSCs), Hep3B-C and Huh7-C. And we have demonstrated that these cells had enhanced stem cell properties, drug resistance, properties of EMT, and stronger tumor-initiating capabilities. To explore functionally crucial miRNAs in HCSCs, 2 samples of HCSCs and 2 samples of HCCs were sequenced by the Illumina Genome Analyzer II. Through differential expression analysis, we finally identified 9 up- and 9 down-regulated miRNAs which were consistently up- and down-regulated in two stem cells compared to the cancer cells. Expression analysis using total RNAs extracted from 2 HCSC cell lines (Hep3B-C and Huh7-C), and 2 HCC cell lines (Hep3B and Huh7).
Project description:Hep3B and Huh7 are two types of human hepatoma cell lines (HCC). In our laboratory, we cultured their stem-like cancer cells (HCSCs), Hep3B-C and Huh7-C. And we have demonstrated that these cells had enhanced stem cell properties, drug resistance, properties of EMT, and stronger tumor-initiating capabilities. To explore functionally crucial mRNAs in HCSCs, 2 samples of HCSCs and 2 samples of HCCs were sequenced by the Illumina Genome Analyzer II. Through differential expression analysis, we finally identified 115 up- and 402 down-regulated miRNAs which were consistently up- and down-regulated in two stem cells compared to the cancer cells. Expression analysis using total RNAs extracted from 2 HCSC cell lines (Hep3B-C and Huh7-C), and 2 HCC cell lines (Hep3B and Huh7).
Project description:<b><i>Background:</i></b> Telomerase reverse transcriptase (<i>TERT</i>) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. <i>TERT</i> promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in <i>TERT</i> regulation. <b><i>Methods:</i></b> Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the <i>TERT</i> promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors <i>GABPA</i> (GA binding protein transcription factor subunit alpha) and <i>MYC</i>, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). <b><i>Results:</i></b> We found the mutant <i>TERT</i> promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous <i>TERT</i> mutant cell lines. We demonstrated that the transcriptional activators <i>GABPA</i> and <i>MYC</i> bind only to the mutant <i>TERT</i> allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, <i>TERT</i> exhibits <i>MAE</i> from the mutant allele only. <b><i>Conclusions:</i></b> In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous <i>TERT</i> mutations.
Project description:BACKGROUND:All-trans retinoic acid (ATRA) is currently being used to treat hematological malignancies, given the ability to inhibit cell proliferation. This effect seems to be related to epigenetic changes of the TERT (Telomerase Reverse Transcriptase) promoter. When hypomethylated, ATRA-inducible TERT repressors can bind the promoter, repressing transcription of TERT, the rate-limiting component of telomerase. Ovarian carcinomas are heterogeneous tumors characterized by several aberrantly methylated genes among which is TERT. We recently found a hypomethylation of TERT promoter in about one third of serous carcinoma, the most lethal histotype. Our aim was to investigate the potential role of ATRA as an anticancer drug in a sub-group of ovarian carcinoma where the TERT promoter was hypomethylated. METHODS:The potential antiproliferative and cytotoxic effect of ATRA was investigated in seven serous ovarian carcinoma and one teratocarcinoma cell lines and the results were compared to the methylation status of their TERT promoter. RESULTS:The serous ovarian carcinoma cell line OVCAR3, harboring a hypomethylated TERT promoter, was the best and fastest responder. PA1 and SKOV3, two cell lines with an intermediate methylated promoter, revealed a weaker and delayed response. On the contrary, the other 5 cell lines with a highly methylated promoter did not respond to ATRA, indicative of ATRA-resistant cells. CONCLUSIONS:Our results demonstrate an inverse correlation between the methylation level of TERT promoter and ATRA efficacy in ovarian carcinoma cell lines. Although these results are preliminary, ATRA treatment could become a new powerful, personalized therapy in serous ovarian carcinoma patients, but only in those with tumors harboring a hypomethylated TERT promoter.
Project description:Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.
Project description:Telomerase reverse transcriptase (TERT) is pathologically expressed in the vast majority of human cancers, but the epigenetic regulation of its expression is only beginning to be understood. In particular, the active TERT gene in cancer cells has been characterized as having a hypermethylated CpG island, opposite to the general association of DNA methylation with gene repression. Here, we analyzed TERT promoter CpG methylation in 833 human cancer cell lines representing 23 different tissue types and found hypermethylation of the upstream portion of the CpG island and more conserved hypomethylation of a region including the proximal TERT promoter and exon 1. In cell lines with monoallelic expression of TERT, we found allelic methylation of the proximal TERT promoter. This included cell lines with the -124 or -146 activating promoter mutation as well as wild-type TERT cancer lines. In these cell line types, decreased proximal promoter methylation is associated with the active allele. Compared to cells with monoallelic expression of TERT, lines with biallelic expression of TERT had even lower methylation in the proximal TERT promoter. Thus, in cell lines from cancers of many different tissues, the TERT proximal promoter has canonical DNA methylation, with low methylation correlating with increased TERT expression.
Project description:PMA has both mitogenic and antiproliferative effects on human hepatoma Hep3B cells. In response to low PMA concentration (10 nM), Hep3B cells displayed an increasing proliferation potentiation. At high PMA concentration (1 microM) Hep3B cells exhibited modest cytostatic effects. Determinations of protein kinase C (PKC) activity in PMA-treated cells revealed that alterations in PKC activity are associated with proliferative capacity. The decrease in PKC activity mediated by a high dose of PMA was accompanied by cell growth inhibition. Increases in PKC activity mediated by a low dose of PMA were consistent with proliferation stimulation. Immunoblot analysis showed that there are at least six PKC isoenzymes: alpha, delta, epsilon, mu, zeta and iota/lambda, constitutively expressed in Hep3B cells. Cellular fractionation and immunocytochemical staining results demonstrated that both 10 nM and 1 microM PMA treatments induced a marked translocation of PKC-alpha from cytosol to membrane or nuclear fraction within 5-30 min. At the same time PKC-delta and epsilon were translocated from the membrane to nuclear fraction. In addition, prolonged treatment with 1 microM PMA, but not with 10 nM PMA, selectively mediated the down-regulation of these three PKC isoenzymes. The distinct effects of different concentrations of PMA on cell proliferation and PKC-alpha, delta and epsilon isoenzyme modulation support the involvement of these three PKC isotypes in the mechanism of action of Hep3B cells in cell growth events.
Project description:The mechanism of telomerase re-activation in cancer had remained elusive until the discovery of frequent mutations in the promoter of the TERT gene that encodes the catalytic reverse transcriptase subunit of telomerase. We investigated the regulation of TERT expression in melanoma cell lines and our results show that promoter mutations render TERT expression dependent on MAPK activation due to oncogenic BRAF or NRAS mutations. Mutations in the TERT promoter create binding sites for ETS transcription factors. ETS1, expressed in melanoma cell lines, undergoes activating phosphorylation by ERK at Thr38 residue as a consequence of constitutively activated MAPK pathway. We demonstrate that ETS1 binds on the mutated TERT promoter leading to the re-expression of the gene. The inhibition of ETS1 resulted in reduced TERT expression. We provide evidence that the TERT promoter mutations provide a direct link between TERT expression and MAPK pathway activation due to BRAF or NRAS mutations via the transcription factor ETS1.
Project description:Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread).We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection.We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.