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Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway


The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirus-mediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.

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This work was sponsored by Cancer Research UK. We are thankful to the following for supplying reagents and technical advice: David Beach (LinXE and LinXA), Gordon Peters (pWZL Eco receptor neo, pBabe hTERT puro), Moshe Oren (pBabe p53DD puro), Thomas Wolfel (pcDNA3 CDK4R24C), Eiji Hara (pcDNA3 Id-1 hygro), Darren Tomlinson (pFB-hygro). We are grateful to Paul Roberts (Cytogenetics Department, St James's University Hospital, Leeds, UK) and Stuart Pepper (Patterson Institute, Manchester, UK) for performing G-banded karyotype analyses and Affymetrix SNP array analyses, respectively.

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Correspondence to M A Knowles.

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Chapman, E., Hurst, C., Pitt, E. et al. Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway. Oncogene 25, 5037–5045 (2006).

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