• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.1070-1076
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• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.4
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• pp.907-915
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• 2007

In this study the effect of water extract of Sophora tonkinensis Gapnep (RST) was investigated on the growth of human lung carcinoma A549 cells. Exposure of A549 cells to RST resulted in the growth inhibition in a dose-dependent manner as measured by MTT assay. The antiproliferative effect by RST treatment in A549 cells was associated with morphological changes such as membrane shrinking and cell rounding up. RST treatment did not induce the cell cycle arrest and the levels of tumor suppressor p53 as well as cyclin-dependent kinase inhibitor p21(WAF1/CIP1). It was found that RST treatment decreased the levels of cyclooxygenase (COX) -2 mRNA and protein expression without significant changes in the expression of COX-1 and inducible nitric oxide synthase (iNOS), which was correlated with a decrease in prostaglandin E2 (PGE2) synthesis. RST treatment also slightly inhibited the levels of human telomerase reverse transcriptase (hTERT) mRNA and protein expression, and the activity of telomerase. Taken together, these findings suggested that RST-induced inhibition of human lung carcinoma A549 cell growth was aoosciated with the inhibition of COX-2 expression and PGE2 production. These results provided important new insights into the possible molecular mechanisms of the anti-cancer activity of RST.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.6
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• pp.1437-1449
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• 2007

In this study, the effect of extract of Corydalis yanhusuo (ECT) used in Oriental medicine therapy was investigated on the cell growth and apoptosis of HepG2 human hepatoma cells. It was found that ECT could inhibit the cell growth effectively in a dose-dependent manner, which was associated with morphological change and apoptotic cell death such as formation of apoptotic bodies, DNA fragmentation and increased populations of apoptotic-sub G1 phase. And we observed the effects of ECT on loss of mitochondrial membrane potential (MMP), using the JC-1 probe by DNA flow cytometric analysis. Apoptosis of HepG2 cells by ECT was associated with a down-regulation of anti apoptotic Bcl-2 expression, inhibitor of apoptosis proteins (IAPs) expression and proteolytic activation of caspase-3 and caspase-9. However, ECT did not affect the pro-apoptotic Bax expression and activity of caspase-8. ECT treatment also concomitant degradation and /or inhibition of poly (ADP-ribose) polymerase (PARP), phospholipase C-1 ($PLC{\gamma}1$). Furthermore, ECT treatment caused a dose-dependent inhibition of iNOS and cyclooxygenase-2 (Cox-2). Additionally ECT have been implicated in the regulation of telomerase expression. ECT treatment induced the down-regulation of telomerase reverse transcriptase mRNA (hTERT) expression of HepG2 cells. Taken together, these findings suggest that ECT may be a potential chemotherapeutic agent for the control of HepG2 human hepatoma cells.

• BMB Reports
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• v.44 no.8
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• pp.512-516
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• 2011

Establishment of immortalized human dermal papilla cells (DPCs) retaining the characteristics of DPCs would be a great help for hair researchers. We recently established a simian virus 40T (SV40T)-transformed human DP cell line (SV40TDPC). However, the cell line senesced around passage 25 and ceased proliferation. In this study, we introduced the human telomerase reverse transcriptase (hTERT) gene into SV40T-DPC and established an immortalized human DP cell line. The cell line, SV40T-hTERT-DPC, did not induce tumors when inoculated into nude mice. SV40T-hTERT-DPC maintained morphology of early passage DPCs, expressed markers of DPCs, and retained responses to Wnt/${\beta}$-catenin and bone morphogenic protein (BMP) signaling pathways known to be required for hair-inducing activity of DPCs. The data strongly suggest that SV40T-hTERT-DPC retains many characteristics of human DPCs in vivo without malignant transformation.

• Mycobiology
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• v.43 no.1
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• pp.37-42
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• 2015

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

• Genomics & Informatics
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• v.6 no.2
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• pp.84-86
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• 2008

The Tetrahymena group I intron has been shown to employ a trans-splicing reaction and has been modified to specifically target and replace human telomerase reverse transcriptase (hTERT) RNA with a suicide gene transcript, resulting in the induction of selective cytotoxicity in cancer cells that express the target RNA, in animal models as well as in cell cultures. In this study, we evaluated the target RNA specificity of trans-splicing phenomena by the group I intron in mice that were intraperitoneally inoculated with hTERT-expressing human cancer cells to validate the anti-cancer therapeutic applicability of the group I intron. To this end, an adenoviral vector that encoded for the hTERT-targeting group I intron was constructed and systemically injected into the animal. 5'-end RACE-PCR and sequencing analyses of the trans-spliced cDNA clones revealed that all of the analyzed products in the tumor tissue of the virus-infected mice resulted from reactions that were generated only with the targeted hTERT RNA. This study implies the in vivo target specificity of the trans-splicing group I intron and hence suggests that RNA replacement via a trans-splicing reaction by the group I intron is a potent anti-cancer genetic approach.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3483-3488
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• 2012

Objective: Several studies have demonstrated associations between the TERT rs2736098 single nucleotide polymorphisms (SNPs) and susceptibility to cancer development. However, there are conflicting results. A systematic meta-analysis was therefore performed to establish the cancer risk associated with the polymorphism. Methods: In this meta-analysis, a total of 6 case-control studies, including 5,567 cases and 6,191 controls, were included. Crude odds ratios with 95% confidence intervals were used to assess the strength of associations in several genetic models. Results: Our results showed no association reaching the level of statistical significance for overall risk. Interestingly, in the stratified analyses (subdivided by ethnicity), significantly increased risks were found in the Asian subgroup which indicates the TERT rs2736098 polymorphism may have controversial involvement in cancer susceptibility. Conclusions: Overall, this meta-analysis indicates that the TERT rs2736098 polymorphism may have little involvement in cancer susceptibility.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.431-435
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• 2012

In this study, we describe a novel approach to achieve replicative selectivity of conditionally replicative adenovirus that is based upon trans-splicing ribozyme-mediated replacement of cancer-specific RNAs. We developed a specific ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to induce adenoviral E1A gene expression selectively in cancer cells that express the RNA. Western blot analysis showed that the ribozyme highly selectively triggered E1A expression in hTERT-expressing cancer cells. RT-PCR and sequencing analysis indicated that the ribozyme-mediated E1A induction was caused via a high fidelity trans-splicing reaction with the targeted residue in the hTERT-expressing cells. Moreover, reporter activity under the control of an E1A-dependent E3 promoter was highly transactivated in hTERT-expressing cancer cells. Therefore, adenovirus containing the hTERT RNA-targeting trans-splicing ribozyme would be a promising anticancer agent through selective replication in cancer cells and thus specific destruction of the infected cells.

• Genomics & Informatics
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• v.3 no.4
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• pp.172-174
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• 2005

Recent anti-cancer approaches have been based to target tumor-specifically associated and/or causative molecules such as RNAs or proteins. As this specifically targeted anti-cancer modulator, we have previously described a novel human cancer gene therapeutic agent that is Tetrahymena group I intron-based trans-splicing ribozyme which can reprogram and replace human telomerase reverse transcriptase (hTERT) RNA to selectively induce tumor-specific cytotoxicity in cancer cells expressing the target RNA. Moreover, the specific ribozyme has been shown to efficiently retard tumor tissues in xenograft mice which had been inoculated with hTERT-expressing human cancer cells. In this study, we assessed specificity of trans-splicing reaction in cells to evaluate the therapeutic feasibility of the specific ribozyme. In order to analyze the trans-spliced products by the specific ribozyme in hTERT-positive cells, RT, 5'-end RACE-PCR, and sequencing reactions of the spliced RNAs were employed. Then, whole analyzed products resulted from reactions only with the hTERT RNA. This study suggested that the developed ribozyme perform highly specific RNA replacement of the target RNA in cells, hence trans-splicing ribozyme will be one of specific agents for genetic approach to revert cancer.

• Proceedings of the KACD Conference
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• 2003.11a
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• pp.546.2-546
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• 2003

Dental follicle is the mesenchymal tissue which surrounds developing tooth germ. During tooth root development, periodontal components such as cementum, periodontal ligament and alveolar bone are considered to be created by progenitors present in the dental follicle. However, little is known about these progenitors. Previously we observed that cultured bovine dental follicle cells (BDFC) contained putative cementoblast progenitors. To further analyze the biology of these cells, we have attempted to immortalize BDFC by expression of the polycomb group protein Bmi-1 and human telomerase reverse transcriptase (hTERT). The BDFC expressing Bmi-1 and hTERT showed extended life span by 90 population doublings more than normal BDFC, and still contained cells with potential to differentiate into cementoblasts upon implantation into immunodeficiency mice. Among them, we established a clonal cell line designated as BCPb8, which formed cemetum-like mineralized tissue reactive to anti-cementum specific monoclonal antibody, 3G9, and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin and type I collagen upon implantation. Thus with the combination of hTERT and Bmi-1, we succeeded in immortalization of cementoblast progenitor in BDFC without affecting differentiation potential. The BCPb8 progenitor cell line could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for periodontitis.