過去研究指出，肺癌細胞的抗藥性與腫瘤抑癌基因之功能缺損有關。本研究證實，以不同劑量之賽默芬 (GCV)，處理已轉殖疱疹病毒胸腺嘧啶激酶 (HSV-tk) 質體之p53點突變 (R267P) 的非小細胞肺癌 (NSCLC) 細胞株H1437產生胞殺性 (cytotoxicity)，讓腫瘤細胞生長停滯，以至死亡。進一步探討HSV-tk表現與胞殺性的關係，其療效得以放大的原因是利用細胞間物質流通所引起的「旁觀者效應」，在這也說明離體細胞或是活體研究上何以有效抑制腫瘤細胞的生長。另外，利用流式細胞儀分析H1437轉殖株於GCV誘導後，結果顯示，細胞停滯於Ｓ及G2/M細胞週期，再利用PKH67染色法，進一步證實細胞的生長確有停滯。研究中也發現，細胞老化的重要指標 (SA-β-gal) 呈現正反應，確認轉殖H1437細胞於GCV處理生長受到抑制，主要是由於細胞老化所導致。繼續探究導致老化的分子機制發現：(a) 隨著GCV處理的時間增加，端粒酶蛋白 (TERT) 的表現下降，再以TRAP assay分析端粒酶的活性也隨GCV增加而減弱，(b) 內生性細胞週期調控分子，如cyclin A及cyclin B1有明顯增加，腫瘤抑制蛋白p21下降，因此證實，轉殖H1437細胞生長和分裂的停滯與GCV所引發端粒酶活性降低有關。本研究的結論也知，p53突變之人類非小細胞肺癌細胞中，HSV-tk/GCV基因治療系統造成腫瘤細胞的死亡，主要是由於細胞發生老化所導致，而此方式若能再積極探討，對未來在臨床上癌症的治療可以提供一個新方向。

In view of the functional loss of tumor suppressors being associated to drug resistance in most of lung cancers, we have demonstrated dose-dependent ganciclovir (GCV)-induced cytotoxicity following delivery of recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA in highly metastatic and tumorigenic human non-small-cell-lung cancer (NSCLC) cells H1437 with mutant p53 (R267P). It is found that cells with acquisition of HSV-tk cDNA conferred GCV susceptibility to the selected clones and the effect proved proportional to the level of HSV-TK expressed both in vivo and in vitro. The inhibition of cell proliferation in culture and the regression of xenograft tumors in nude mice were demonstrated caused by triggering senescence program as a result of GCV exposure in the selected HSV-tk clones. The potent bystander effects both in vivo and in vitro systems was found correlated with HSV-TK expressed accordingly. In addition, H1437 cells underwent ecotopic transfer of HSV-tk lead to S- and G2/M-phase arrest after GCV induction prior to emergence of senescence-like phenotype that expression of the senescence marker SA-β-gal and enlarged and fattened morphology. And we also observed that transfectant cells were growth-retarded after drug treatment by PKH67 fluorescence detection. To address more in-depth mechanism leading to senescence phenotype, we identified two steps prior to cell death as triggered by senescence progression: (a) decreased expression and activity of telomerase (b) escalation of endogenous cell cycle modulators, cyclin A and cyclin B1. Taken all the work together, we concluded that HSV-tk/GCV system constitutes an effective treatment in human NSCLC cells with mutated p53 and the pathway leading to cell death through senescence activation ought to become an alternative and effective approach in restraining growth of human NSCLC cells lacking functional p53.

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