Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression
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Su Hang1,2, Agnes F.Y. Tiwari1, Hextan Y.S. Ngan3, Yim-Ling Yip4, Annie L.M. Cheung4, Sai Wah Tsao4, Wen Deng1
1School of Nursing, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China
2College of Forensic Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shanxi Province, P.R. China
3Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, SAR, China
4School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China
Wen Deng, email: [email protected]
Keywords: cervical epithelial cells, immortalization, p16INK4a, telomerase, HPV-negative
Received: March 13, 2016 Accepted: May 29, 2016 Published: June 17, 2016
Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer.
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