The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti‐cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4‐methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose‐dependent switch to anti‐survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC‐mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC‐induced DNA damage but impacts signalling processes upstream of apoptosis execution level.     

人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

人端粒酶逆转录酶核酶抑制端粒酶活性

为有效切割端粒酶逆转录酶mRNA以降低端粒酶活性 ,从而使肿瘤细胞生长变慢 ,凋亡增加。设计并合成了针对端粒酶逆转录酶mRNA的锤头状核酶基因 ,构建了该核酶基因的体外转录和真核表达质粒。检测了该核酶对端粒酶逆转录酶mRNA的体外切割效力。并将该核酶基因转染至肿瘤细胞中 ,检测其对肿瘤细胞端粒酶活性和生物学性状的影响。结果表明 ,该核酶在体外和细胞内均能有效切割端粒酶逆转录酶mRNA ;在细胞内能明显抑制端粒酶活性 ,使细胞生长变慢 ,倍增时间延长。因而 ,该核酶可望成为有效的端粒酶抑制剂 ,在抑制肿瘤生长中发挥作用     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.     

Detection of Telomerase Activity in Tissues and Primary Cultured Lymphoid Cells of <Emphasis Type="Italic">Penaeus japonicus</Emphasis>

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG)_n repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.     

Telomerase with mutated catalytic motifs has dominant negative effects on telomerase activity and inhibits cell growth

Telomerase catalytic subunit (TERT) seems a key factor controlling telomerase activity, telomere length, and cell growth. To further address this issue, we forced expression of a catalytically inactive mutant human TERT (hTERT) in hTERT-immortalised sheep fibroblasts to examine its effects. Expression of mutant hTERT compromised telomerase activity reconstituted by wild-type hTERT in a manner directly attributable to mutant hTERT expression level. High levels of mutant hTERT expression inhibited cell growth with a subset of cells entering replicative senescence. Furthermore, significant telomere attrition was evident in two of three clones with high levels of mutant hTERT expression. Our findings are consistent with the notion that hTERT homodimers are necessarily required to form a functional telomerase complex at the telomere substrate. We also highlight the requirement of a more thorough understanding of telomerase- and telomere-associated factors to understand fully the interplay that governs telomere homeostasis in vitro and in vivo.     

Discovery of new chromen-4-one derivatives as telomerase inhibitors through regulating expression of dyskerin

A series of new trimethoxyphenyl-4H-chromen derivatives as telomerase inhibitors through regulation dyskerin were designed and synthesised. The anticancer activity assay in vitro showed that compound 5i 3-(4-(4-isonicotinoylpiperazin-1-yl)butoxy)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one exhibited high activity against Hela, SMMC-7721, SGC-7901, U87 and HepG2 cell lines. Compound 5i also showed potent inhibitory activity against telomerase. The further results confirmed this title compound could significantly improve pathological changes induced rat hepatic tumor in vivo. Preliminary mechanisms showed that compound 5i inhibited telomerase activity through decrease expression of dyskerin.