The Short-Term Effects Of HTERT Silence On Cellular Proliferation And Apoptosis In Ovarian Cancer Cells With Different P53 State

Background : Telomerase is well-known as a cellular reverse transcriptase that synthesizes telomeric (TTAGGG) repeats at the ends of chromosomes. It permits cancer cells to overcome the progressive loss of telomere during each round of cell division thus plays a critical role in cell immortality. Telomerase expression is predominantly regulated at the transcriptional level of the human telomerase reverse transcriptase (hTERT), one of the main components of telomerase. Because of its preferential expression in malignant tumor and absence in most normal tissues, it has been widely accepted that hTERT is a potential target of a cancer therapy which would be more cancer specific and perhaps with fewer cytotoxic side effects, as compared with currently approved therapies.It is believed that 'pure' telomerase inhibitors require a lag time before telomeres shorten to a critical length that could trigger senescence, and this process must be slow and depends on the initial telomere length and the cell division cycle. Interestingly, many present studies targeting hTERT and telomerase showed dramatic short-term effects leading to rapid cell death of the tumor cells,which was apparently not dependent on telomere loss, suggesting that some occult influence may present immediately in the absence of telomerase,which lead to a 'fast-track' resulting in rapid cell death without obvious telomere loss. As the anti-tumor mechanism of hTERT inhibition remains unclear, its effects are diverse in different tumor cell lines according to existing reports. It is necessary to test the anti-tumor effects of hTERT inhibition with different histological types.As a critical tumor suppressor implicated in several diverse growth-related pathways, including apoptosis, cell cycle arrest, and senescence, p53 is proved to down regulate the hTERT transcription, which is likely to be mediated by inhibition of the Sp1 binding to the hTERT promoter. Not until recently has it been found that the overexpression or repression of hTERT may have an immediate negative or positive effect on the transcription of p53 in some human cells before telomeric changes are detectable, suggesting that there may be a feedback loop between hTERT and p53, and hTERT may exert an extra-telomeric effect on its own regulatory gene components independent of telomere length. But whether the consequent change of p53 expression level is associated with the 'fast-track' of telomerase has not been elucidated yet. Due to the close relationship between p53 and apoptosis, we hypothesized that the possible rapid cell death caused by the knockdown of hTERT may be partially attributed to altered functional p53.Objective:In this study, we constructed two recombinant plasmids of short hairpin RNA(shRNA) targeting hTERT named pG1 and pG2 respectively. Another clone, pGCR, which expresses a shRNA scrambled sequence, was also constructed as a control. Three telomerase-positive human epithelial ovarian cancer cell lines: wild type-p53(wt-p53) A2780, mutant type-p53(mt-p53) OVCAR3 and p53-null SKOV3 were transfected with pG1, pG2 and pGCR respectively both in vitro and in vivo, to test the possibility of rapid growth arrest and apoptosis induced by hTERT gene silence, and to determine whether this phenomenon is related to the activation of p53 expression. We sought to determine the mode of short-term effects induced by hTERT inhibitors in human ovarian carcinoma cells that harbor different status of p53, and we hoped an improved understanding of this process could provide insight into mechanisms of sensitivity and/or resistance to hTERT inhibitors, thus uncover new targets for ovarian cancer therapy.Methods: A2780, OVCAR3 and SKOV3 were transfected with pG1, pG2 and pGCR in vitro respectively, and untreated cells were also used as one of controls. The efficiency of hTERT inhibition was confirmed with real-time RT-PCR and western blot, and telomerase activity was evaluated with TRAP assay. Cell proliferation was measured with CCK-8 assay, and cell circle analysis was performed by Flow cytometric. Apoptosis was detected with Annexinâ…¤-PE/7AAD and TUNEL assay, and expression of p53 and p21 were measured with western blot.A2780 and SKOV3 cells were injected into hypodermic tissue to construct implanted-tumor model in nude mice. After xenograft tumor formed, recombinant plasmids of pG2, pGCR and saline were injected intratumorally every other day respectively for two weeks.The size of tumor was measured at the same time. Tumors were excised, weighted, sectioned and assessed for mophplogy by HE, and apoptosis was detected with TUNEL and electron microscope after treatment. The efficiency of hTERT inhibition was confirmed with western blot, and expression of p53 and p21 were measured with immunohistochemistry.Results: 1. Restriction enzyme mapping and sequencing demonstrated that pG1, pG2 and pGCR were constructed successfully. Real-time RT-PCR, western blot and TRAP assay showed shRNA directed against hTERT could efficiently silence hTERT expression and inhibit telomerase activity in ovarian cancer cells.2. knock-down of hTERT induced rapid growth reduction in vitro in all three cell lines of ovarian cancer, and cell cycle analysis showed that growth inhibited by hTERT silence is mediated by S-phase arrest. Two cell lines, A2780 and OVCAR3, with enhancive apoptosis under the influence of hTERT suppression, both presented accumulation of p53 and p21 proteins. In contrast, the knock down of hTERT in p53-null SKOV3 cells resulted in a rapid decreased expression of p21 and did not cause obvious cell apoptosis.3. Hypodermic tumor-implanted model were established successfully by 100% with A2780 and SKOV3 cells. As compared with pGCR and saline-treated mice, recombinant plasmid pG2 could significantly slow down tumor growth, decrease tumor volume and tumor weight in vivo in both two xenograft tumor. Massive apoptosis and necrosis presented in A2780-implanted tumor after treatment, with reinforcement of p53 and p21 expression. But only necrosis presented in SKOV3-implanted tumor, and the expression of p21 appeared to be unaffected. No obvious side effects were observed in treated mice.Conclusions: Our data demonstrated that recombinant plasmids of shRNA could efficiently and specifically knock-down hTERT expression, decrease telomerase activity, and inhibit tumor cell growth immediately both in vitro and in vivo, but the mechanism of cell death is differ in ovarian cancer cells with different p53 state, and apoptosis induced immediately by inhibition of hTERT may be due, in part, to up-regulation of growth inhibitory genes p53 and p21. We confirmed once again that, besides the 'classical' mechanism by which inhibition of telomerase could lead to telomere shortening preceding cell death, an 'fast - track' not requiring telomere shortening also exist in cancer cells. This is meaningful for the anticancer therapy targeting telomerase and hTERT.