Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity Nand K. Sah, 1 Anupama Munshi, 1 Takashi Nishikawa, 1 Tapas Mukhopadhyay, 2 Jack A. Roth, 2 and Raymond E. Meyn 1 1 Departments of Experimental Radiation Oncology and 2 Thoracic and Cardiovascular Surgery, University of Texas MD Anderson Cancer Center, Houston, TX Abstract Functional inactivation of the p53 gene and robust DNA repair capacity may be among the salient causes of radio- resistance in tumor cells. We expressed the wild-type (wt) p53 gene in a p53-mutant human epidermoid carcinoma cell line, A431, using an adenoviral vector [adenovirus-p53 (Ad- p53), INGN 201], examined its radiosensitivity, and corre- lated p53 status and radiosensitivity with cellular repair functions. Using clonogenic survival assays and the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, we demonstrated that preirradiation treatment with Ad-p53 significantly increased the radiosen- sitivity of A431 cells over controls. Induction of p53 expression using a construct where p53 expression was under the control of an inducible promoter also significantly increased radiosensitivity of H1299 lung tumor cells, which are otherwise null for p53. These results did not correlate with radiation-induced apoptosis but did correlate with functional impairment of DNA repair and suppressed expression of several repair-related genes, such as Ku70, DNA-dependent protein kinase, ataxia telangiectasia mutat- ed, and X-ray-sensitive complementation group 4. Normal human fibroblast MRC-9 cells showed no impairment in the repair capability due to Ad-p53 despite the suppression of some repair genes. Expression of Ku70, which is known to mediate diverse cellular functions, correlated with the differential effects of p53 on radiosensitivity in the normal and tumor cells. (Mol Cancer Ther. 2003;2:1223– 1231) Introduction Radiotherapy continues to be a frontline treatment for cancer despite the risk of normal tissue complications. Enhancing the antitumor effects by combining radiation with other agents often allows lower doses to be used, thereby minimizing side effects. Gene therapy in combi- nation with radiation is one such promising strategy (1 – 6). It has been estimated that about 50% of all tumors have mutations in p53, and the p53 pathway may be nonfunc- tional for other reasons in many more. A number of investigators have shown that exogenous expression of wild-type (wt) p53 sensitizes human tumor cells to radiation in vitro and in vivo (7 – 16). p53 is well known for its role in monitoring genomic stability. The mechanisms underlying this function of p53 are not fully understood (17). Nevertheless, it is known that genetic insults activate p53, which in turn induce down- stream repair genes including GADD45, p48XPE, and XPC (17 – 19) that are involved in the nucleotide excision repair (20) and base excision repair (21, 22) processes. Genetic insults may also lead to DNA double-strand breaks (DSB) that are repaired by interchromosomal and intrachromoso- mal homologous recombination (HR) and nonhomologous end joining (NHEJ). The NHEJ pathway is especially im- portant for repairing radiation-induced DSBs. Factors that are known to participate in NHEJ include Ku70, Ku80, DNA- dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs), Artemis, X-ray-sensitive complementation group 4 (XRCC4), and DNA ligase IV (23, 24). Deficiency of any of these factors also results in defects in V(D)J recombination, leading to severe combined immunodefi- ciency. There are conflicting reports in the literature re- garding the role of wt-p53 in the regulation of NHEJ. Several reports show a positive involvement of wt-p53 in accentuating NHEJ (25 – 28), but others have demonstrat- ed down-regulation of NHEJ-mediated DSB repair by wt- p53 (29). The multiple functions of wt-p53 are tightly regulated by several molecular processes including post- translation modification (30, 31), multiple-site phosphory- lation (32–34), acetylation (35), multiple protein-DNA interactions (36), conformational changes (37), and sumoy- lation and glycosylation (36) depending on the cell type. We therefore hypothesized that p53 may play distinct roles in governing DNA repair capacity in normal and trans- formed cells. To test whether radiosensitization of human tumor cells by the gene therapy vector adenovirus-p53 (Ad-p53) is due to suppression of NHEJ, we treated human p53- mutant epidermoid carcinoma A431 cells and examined the expression of proteins that participate in NHEJ. We found that Ad-p53 radiosensitized A431 cells and that this effect correlated with a down-modulation of proteins involved in NHEJ. Normal human fibroblasts were not radiosensitized by Ad-p53, which suggests that Ad-p53 has a differential effect on DNA repair in tumor cells versus normal cells. Received 5/15/03; revised 8/8/03; accepted 8/28/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: P01 CA78778, P01 CA06294, and P30 CA 16672 from the National Cancer Institute. Present address: Dr. Nand K. Sah, Department of Botany, RD College, Sheikhpura 811105, India. Requests for Reprints: Raymond E. Meyn, Department of Experimental Radiation Oncology, Unit 066, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3424; Fax: (713) 794-5369. E-mail: rmeyn@mdanderson.org Molecular Cancer Therapeutics 1223