SHORT REPORT Effects of loss of p53 and p16 function on life span and survival of human urothelial cells Nicola J. Shaw 1 , Nikolaos T. Georgopoulos 2 , Jennifer Southgate 1 * and Ludwik K. Trejdosiewicz 2 1 Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, United Kingdom 2 Cancer Research UK Clinical Centre, St James’s University Hospital, Leeds, United Kingdom Human urothelial cell carcinoma evolves via the accumulation of numerous genetic alterations, with loss of p53 and p16 function representing important stages in the development of superficial lesions and their progression to malignant disease. To investigate the effects of disabling either or both proteins in otherwise normal human urothelial cells, we performed retroviral transductions with a dominant negative p53 miniprotein and/or mutant cyclin- dependent kinase 4 (CDK4 R24C ) in 3 independent cell lines. Although cells with disabled p53 function showed a higher prolif- eration rate, inactivation of neither p53 nor p16 function resulted in any extension of life span and the double-transductants failed to flourish, demonstrating that further genetic alterations are required to attain an immortalised phenotype. However, CDK4 R24C transductants showed a marked increase in apoptotic susceptibility to membrane-presented CD40 ligand, being inter- mediate between normal cells (nonsusceptible) and transformed cells (highly susceptible). By contrast, loss of p53 function alone only slightly increased the apoptotic susceptibility of urothelial cells. These results demonstrate that loss of p16 function, while insufficient to immortalise urothelial cells, nevertheless renders the cells more vulnerable to apoptosis induced by CD40 ligation. ' 2005 Wiley-Liss, Inc. Key words: apoptosis; CD40; bladder; p53; p16; cancer; urothelium In developed countries, bladder cancer is a common malig- nancy, 90% of cases being UCCs arising from the urothelium, the transitional epithelium that lines the urinary tract. 1 The develop- ment of malignant bladder cancer is a multistep process associated with the accumulation of multiple genetic alterations. Studies mapping genetic alterations to clinicopathologic characteristics have made considerable progress in developing and refining model pathways of bladder cancer development and progression. 1–7 Common alterations include stage- and grade-independent loss of heterozygosity from chromosome 9p21, which has been associated with loss of the p16 INK4a CDK inhibitor protein, and loss of p53 tumour-suppressor protein from chromosome 17p, which has been correlated with progression to invasive disease. 1,2,4,5,8 These stud- ies suggest that the nature, timing and combination of particular genetic alterations will influence both tumour phenotype and sub- sequent course of behaviour. To an extent, these models have been supported by transgenic studies where mice have been engineered to express oncogenes in a urothelium-specific manner. 9–12 How- ever, little is known about the functional consequences of individ- ual genetic alterations on the biology of urothelial cells and, there- fore, about how they alone, or in combination, contribute to the development and progression of cancer. One approach to address this issue would be to disable specific gene function in NHU cells and investigate how this affects cell phenotype and behaviour. Many of the genes implicated in bladder cancer are involved in normal cell cycle regulation, particularly at the G 1 /S transition. These include both the p16 and p53 tumour-suppressor pro- teins. 13,14 In response to DNA damage, p53 protein is stabilised and modulates the expression of numerous genes involved in cell cycle arrest, DNA repair and/or apoptosis. 13 In particular, p53 transactivates expression of the CDK inhibitor p21 WAF1/Cip1 , which regulates progression of cells past the G 1 /S checkpoint by inhibiting the cyclin D1–CDK4/6 and cyclin E–CDK2-mediated phosphorylation of Rb protein–E2F complexes. 15 p21 therefore induces arrest in G 1 phase and initiates either DNA repair or apop- tosis if the DNA damage is irreparable. p16 has also been impli- cated in G 1 arrest in response to DNA damage, suggesting that the p53 pathway is not the only mechanism of cellular response to genotoxic insult. 16 p16 accumulation is also thought to contribute to the onset of cellular senescence, a state of permanent growth arrest encountered after prolonged culture of various types of nor- mal human epithelial cell, including urothelial cells, keratinocytes and mammary epithelial cells. 17–19 p16 acts by binding CDK4/6, thereby preventing the latter from interacting with cyclin D1 to phosphorylate Rb–E2F. 14 This also leads to inhibition of cell cycle progression at the G 1 /S checkpoint. Because progression to invasive disease is associated with immortalisation and commonly involves changes in apoptotic sus- ceptibility, we examined these 2 critical aspects of urothelial cell tumour progression as a function of inactivation of p53, p16 or both. Using our well-characterised system of NHU cell cul- ture, 20,21 we produced ‘‘paramalignant’’ urothelial cells by means of retroviral transduction, introducing a dominant negative p53 miniprotein (p53DD) 22 and a p16-insensitive CDK4 mutant (CDK4 R24C ) 23 either alone or in combination. Material and methods Production of p53- and p16-disabled human urothelial cell lines Retroviral vectors. The pLXSN-p53DD retroviral vector, 24 containing the coding sequence for the p53DD miniprotein, a C- terminal dominant negative fragment of p53, 22 was a kind gift from Dr M. Oren (Rehovot Weizmann Institute, Israel). The p16- insensitive CDK4 R24C 23 was a generous gift from Dr T. W€ olfel (Mainz University, Mainz, Germany). CDK4 R24C cDNA was subcloned into the EcoRI site of pLXSP, thus generating pLXSP- CDK4 R24C . The pLXSP vector was generated by replacing the neomycin resistance gene of pLXSN (Clontech, supplied by BD Biosciences, Oxford, UK) with a puromycin resistance gene. Transfection of the packaging cell line. RetroPack PT67 pack- aging cells (Clontech, Palo Alto, CA) were transfected with Grant sponsor: York Against Cancer; Grant sponsor: Biotechnology and Biological Sciences Research Council; Grant sponsor: Cancer Research UK. The first 2 authors contributed equally to the work. *Correspondence to: Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5YW, United Kingdom. Fax: 1441904328704. E-mail: js35@york.ac.uk Received 29 September 2004; Accepted after revision 24 January 2005 DOI 10.1002/ijc.21114 Published online 11 April 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: CDK, cyclin-dependent kinase; CHX, cycloheximide; CMF, cell multiplication factor; HPV, human papillomavirus; hTERT, human telomerase reverse transcriptase; MAb, monoclonal antibody; MMC, mitomycin C; NHU, normal human urothelium; PD, population doubling; Rb, retinoblastoma; TNF, tumour necrosis factor; UCC, urothelial cell carcinoma. The authors certify that they have not entered into any agreement that could interfere with their access to the data on the research or upon their ability to analyse the data independently, to prepare manuscripts and to publish them. Int. J. Cancer: 116, 634–639 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer