Re-expression of p16 INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression Sandra P Frizelle 1 , Jon Grim 2 , Joan Zhou 1 , Pankaj Gupta 1 , David T Curiel 2 , Joseph Geradts 3 and Robert A Kratzke 1 Department of 1 Medicine, Section of Hematology/Oncology, Minneapolis Veterans Aairs Medical Center and the University of Minnesota Medical School, Minneapolis, Minnesota 55417, the 2 Gene Therapy Program, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the 3 Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA Absence of expression of the p16 IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16 INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16 INK4a expressing adenovirus (Adp16), over-expression of p16 INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16 INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelio- ma xenografts demonstrated inhibition of tumor forma- tion, tumor growth arrest and diminished tumor size and spread. p16 INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16 INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma. Keywords: p16 INK4a ; mesothelioma; tumor suppressor Introduction Rb and p16 INK4a gene expression are inversely correlated in a variety of tumors including lung (Kratzke et al., 1996; Otterson et al., 1994; Sakaguchi et al., 1996; Shapiro et al., 1995) and bladder carcinomas (Geradts et al., 1995), as well as astrocytomas (Ueki, 1996). This inverse correlation highlights the importance of releasing Rb-mediated suppression to entry into S-phase of the cell cycle in the development of these cancers. In a similar manner, the previously reported observation that all mesothe- lioma cell lines and tumors appear to express wild- type RB protein is also correlated with the absence of p16 INK4a gene product expression in virtually all of the mesothelioma tumors and cell lines examined (Kratzke et al., 1995). However, unlike other adult onset tumors, where accumulation of acquired genetic defects are common, mesotheliomas are marked by a relative absence of abnormalities in the common molecular target genes such as p53, ras, DCC, APC, or WT1 (Metcalf et al., 1992; Pass and Mew, 1996), although frequent mutations in the NF2 gene have recently been reported (Bianchi et al., 1995; Sekido et al, 1995). Thus, while lack of p16 INK4a expression may be one the most common, and perhaps near universal, molecular defect in mesothelioma, it may also be accompanied by relatively few other abnormalities. Correction of this single molecular defect may provide an interesting and unique biologic approach to therapy in this disease. The initial clinical presentation of mesothelioma is usually con®ned within the pleural or peritoneal cavity (Pass and Mew, 1996). This limited initial disease manifestation makes mesothelioma an attractive disease to attempt to treat with gene replacement therapies. Clinical trials with gene based therapies in mesothelioma have already began using the novel and interesting concept of targeting tumors with gene products that confer selective toxicity on recipient tumor cells to an administered drug (Treat et al, 1996). Successful gene transfer in malignant pleural eusions has already been demonstrated in this disease in animal models using adenoviral vectors (Smythe et al., 1994). In view of the near absolute lack of eective treatment for this disease, we have sought to develop re- expression of p16 INK4a in mesothelioma as a potential gene based treatment of this disease. Results Mesothelioma cells are easily transduced by the Adp16 vector (Figures 1 and 2) at high eciency in vitro. High levels of p16 INK4a expression were achieved after even 1 hour exposure to virus. Expression of p16 INK4a in the previously p16 INK4a negative mesothelioma cells resulted in marked hypophosphorylation of pRB (Figure 3), correlating with cell cycle arrest at the G 1 \S transition as expected (DeCaprio et al., 1992) (Table 1). Interestingly, the level of pRB expression was diminished in p16 INK4a re-expressing cells consistent with previous observations in other cells types (Sandig et al., 1997). This cell cycle arrest was followed by evidence of cell death when analysed in two of the mesothelioma cell lines tested (Table 2). The percentage of dead cells as measured by propidium iodide staining was increased over twofold in H2373 cells, and over ®vefold in H2461 cells, while having minimal increase in the H2087 (p16 positive/pRB positive) lung cancer cells. Cell death was seen primarily in the mesothelio- ma cells and correlated with growth suppression (Figure 4). The phenomenon of cell death following re-expression of p16 INK4a in malignant cells expressing Correspondence: RA Kratzke Received 9 September 1997; revised 19 January 1998; accepted 20 January 1998 Oncogene (1998) 16, 3087 ± 3095 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc