p73b-expressing recombinant adenovirus: a potential anticancer agent Sanjeev Das, 1 Srikanth Nama, 1 Sini Antony, 1 and Kumaravel Somasundaram 1 1 Department of Microbiology and Cell Biology, Indian Institute of Science, Sir CV Raman Road, Bangalore 560012, India. Tumor suppressor p53-based gene therapy strategy is ineffective in certain conditions. p73, a p53 homologue, could be a potential alternative gene therapy agent as it has been found to be an important determinant of chemosensitivity in cancer cells. Previously, we have reported the generation of a replication-deficient adenovirus expressing p73b (Ad-p73). In this study, we evaluated the therapeutic potential of Ad-p73 against a panel of cancer cells (n ¼ 12) of different tissue origin. Ad-p73 infected all the cell lines tested very efficiently resulting in several-fold increase in p73b levels, which is also functional as it activated the known target gene p21 WAF1/CIP1 . Infection with Ad-p73 resulted in potent cytotoxicity in all the cell lines tested. The mechanism of p73-induced cytotoxicity in these cell lines is found to be due to a combination of cell cycle arrest and induction of apoptosis. In addition, exogenous overexpression of p73 by Ad-p73 infection increased the chemosensitivity of cancer cells by many fold to commonly used drug adriamycin. Moreover, Ad-p73 is more efficient than Ad-p53 in enhancing the chemosensitivity of mutant p53 harboring cells. Furthermore, Ad-p73 infection did not induce apoptosis in human normal lung fibroblasts (HEL 299) and human immortalized keratinocytes (HaCaT). These results suggest that Ad-p73 is a potent cytotoxic agent specifically against cancer cells and could be developed as a cancer gene therapy agent either alone or in combination with chemotherapeutic agents. Cancer Gene Therapy (2005) 12, 417–426. doi:10.1038/sj.cgt.7700803 Published online 28 January 2005 Keywords: p73; adenovirus; cell cycle arrest; apoptosis; chemosensitivity T he p53 tumor suppressor gene encodes a transcription factor, which plays an important role in cell cycle control, angiogenesis, apoptosis and genomic stability through the transactivation of its target genes. It is one of the most frequently inactivated genes in human tumors. Inactivation of p53 not only results in the development of tumor, it also results in chemoresistance in many types of cancer. Therefore, introduction of wild-type p53 gene has been pursued as a potential therapeutic strategy against various types of cancers. This strategy relies on the ability of p53 to induce cell cycle arrest and apoptosis in cancer cells. However, p53- based gene therapy is not suitable in certain cases where functional inactivation of p53 takes place, like amplification of mdm-2, mutational inactivation of p14 ARF making p53 mediated tumor suppression inoperative. 1–7 Moreover, in clinical trials the restoration of wild type p53 gene does not always lead to tumor regression or tumor growth inhibition suggesting resistance of some tumors to exogenous p53. 8–10 p73 is a member of p53 gene family and encodes proteins, which have significant homology with p53 both structurally and functionally. p73 can bind to p53 responsive elements and upregulate some of the p53 target genes suggesting that it has the potential for functional overlap with p53. When exogenously overexpressed, p73b, one of the p73 isoforms, can transactivate some of the p53 target genes leading to induction of growth arrest and apoptosis. 11,12 Although, initially it was reported that p73 is not induced by DNA damage, subsequent studies reported that p73 is indeed induced by a wide variety of DNA-damage-inducing drugs like adriamycin, etoposide, cisplatin, etc. 11,13–18 It was also reported that both p73 and p63 (another p53 family member) play a crucial role in DNA-damage-induced p53-mediated apoptosis. 19 It was shown that combined loss of p63 and p73 results in failure of cells containing wild-type p53 to undergo apoptosis in response to DNA damage. A recent study has also linked chemosensitivity of cancer cells to functional p73 irrespec- tive of p53 status. 18 It was shown that abrogation of p73 function in mutant p53 background leads to chemoresis- tance. These results suggest that p73 is a determinant of chemosensitivity and could be used as a candidate for cancer gene therapy. We have recently shown that p73 overexpression by infecting cells with a replication-deficient adenovirus expressing p73b specifically inhibits the growth of cancer cells expressing human papilloma virus E6 oncogene. 20 Another independent study demonstrated the potential Received May 31, 2004. Address correspondence and reprint requests to: Dr Kumaravel Somasundaram, Department of Microbiology and Cell Biology, Indian Institute of Science, Sir CV Raman Road, Bangalore 560012, India. E-mail: skumar@mcbl.iisc.ernet.in Cancer Gene Therapy (2005) 12, 417–426 r 2005 Nature Publishing Group All rights reserved 0929-1903/05 $30.00 www.nature.com/cgt