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Adenovirus-Mediated Wild-Type P53 Radiosensitizes Human Tumor Cells by Suppressing DNA Repair Capacity

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Adenovirus-Mediated Wild-Type P53 Radiosensitizes Human Tumor Cells by Suppressing DNA Repair Capacity Molecular Cancer Therapeutics 1223 Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity Nand K. Sah,1 Anupama Munshi,1 Enhancing the antitumor effects by combining radiation Takashi Nishikawa,1 Tapas Mukhopadhyay,2 with other agents often allows lower doses to be used, Jack A. Roth,2 and Raymond E. Meyn1 thereby minimizing side effects. Gene therapy in combi- 1 2 nation with radiation is one such promising strategy (1–6). Departments of Experimental Radiation Oncology and Thoracic It has been estimated that about 50% of all tumors have and Cardiovascular Surgery, University of Texas MD Anderson Cancer Center, Houston, TX 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 Abstract wild-type (wt) p53 sensitizes human tumor cells to Functional inactivation of the p53 gene and robust DNA radiation in vitro and in vivo (7–16). repair capacity may be among the salient causes of radio- p53 is well known for its role in monitoring genomic resistance in tumor cells. We expressed the wild-type (wt) stability. The mechanisms underlying this function of p53 p53 gene in a p53-mutant human epidermoid carcinoma cell are not fully understood (17). Nevertheless, it is known that line, A431, using an adenoviral vector [adenovirus-p53 (Ad- genetic insults activate p53, which in turn induce down- p53), INGN 201], examined its radiosensitivity, and corre- stream repair genes including GADD45, p48XPE, and XPC lated p53 status and radiosensitivity with cellular repair (17–19) that are involved in the nucleotide excision repair functions. Using clonogenic survival assays and the terminal (20) and base excision repair (21, 22) processes. Genetic deoxynucleotidyl transferase-mediated nick end labeling insults may also lead to DNA double-strand breaks (DSB) assay for apoptosis, we demonstrated that preirradiation that are repaired by interchromosomal and intrachromoso- treatment with Ad-p53 significantly increased the radiosen- mal homologous recombination (HR) and nonhomologous sitivity of A431 cells over controls. Induction of p53 end joining (NHEJ). The NHEJ pathway is especially im- expression using a construct where p53 expression was portant for repairing radiation-induced DSBs. Factors that under the control of an inducible promoter also significantly are known to participate in NHEJ include Ku70, Ku80, DNA- increased radiosensitivity of H1299 lung tumor cells, which dependent protein kinase (DNA-PK) catalytic subunit are otherwise null for p53. These results did not correlate (DNA-PKcs), Artemis, X-ray-sensitive complementation with radiation-induced apoptosis but did correlate with group 4 (XRCC4), and DNA ligase IV (23, 24). Deficiency functional impairment of DNA repair and suppressed of any of these factors also results in defects in V(D)J expression of several repair-related genes, such as Ku70, recombination, leading to severe combined immunodefi- DNA-dependent protein kinase, ataxia telangiectasia mutat- ciency. There are conflicting reports in the literature re- ed, and X-ray-sensitive complementation group 4. Normal garding the role of wt-p53 in the regulation of NHEJ. human fibroblast MRC-9 cells showed no impairment in the Several reports show a positive involvement of wt-p53 in repair capability due to Ad-p53 despite the suppression of accentuating NHEJ (25–28), but others have demonstrat- some repair genes. Expression of Ku70, which is known to ed down-regulation of NHEJ-mediated DSB repair by wt- mediate diverse cellular functions, correlated with the p53 (29). The multiple functions of wt-p53 are tightly differential effects of p53 on radiosensitivity in the normal regulated by several molecular processes including post- and tumor cells. (Mol Cancer Ther. 2003;2:1223–1231) translation modification (30, 31), multiple-site phosphory- lation (32–34), acetylation (35), multiple protein-DNA Introduction interactions (36), conformational changes (37), and sumoy- lation and glycosylation (36) depending on the cell type. Radiotherapy continues to be a frontline treatment for We therefore hypothesized that p53 may play distinct roles cancer despite the risk of normal tissue complications. in governing DNA repair capacity in normal and trans- formed cells. Received 5/15/03; revised 8/8/03; accepted 8/28/03. To test whether radiosensitization of human tumor cells The costs of publication of this article were defrayed in part by the by the gene therapy vector adenovirus-p53 (Ad-p53) is payment of page charges. This article must therefore be hereby due to suppression of NHEJ, we treated human p53- marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mutant epidermoid carcinoma A431 cells and examined Grant support: P01 CA78778, P01 CA06294, and P30 CA 16672 the expression of proteins that participate in NHEJ. We from the National Cancer Institute. found that Ad-p53 radiosensitized A431 cells and that this Present address: Dr. Nand K. Sah, Department of Botany, RD College, effect correlated with a down-modulation of proteins Sheikhpura 811105, India. involved in NHEJ. Normal human fibroblasts were not Requests for Reprints: Raymond E. Meyn, Department of Experimental radiosensitized by Ad-p53, which suggests that Ad-p53 Radiation Oncology, Unit 066, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) has a differential effect on DNA repair in tumor cells 792-3424; Fax: (713) 794-5369. E-mail: [email protected] versus normal cells. Downloaded from mct.aacrjournals.org on September 27, 2021. © 2003 American Association for Cancer Research. 1224 p53 Suppresses Repair and Radiosensitizes Tumor Cells Materials and Methods culture dishes in two sets of triplicates for each dose of Cell Culture radiation; sufficient numbers were seeded to ensure that about 50–100 macroscopic colonies would appear in each The human epidermoid carcinoma cell line A431 was plate at the end of 8–10 days. Colonies were stained with obtained from American Type Culture Collection (Rock- 0.5% gentian violet solution and counted. The percent ville, MD). The cells were maintained in DMEM-F12 growth plating efficiency and surviving fraction were calculated medium supplemented with 10% FCS, 10-mM glutamine, for each radiation dose. The percent plating efficiency for and 100-unit/ml penicillin G + 100-Ag/ml streptomycin. each dish was calculated by dividing the number of Cells were grown at 37jC in 95% air/5% CO . The H1299 2 colonies by the number of cells plated and multiplying by non-small cell lung cancer cell line carrying a stably 100. The surviving fraction for each radiation dose was then transfected ecdysone-inducible p53 construct (38) was calculated by dividing the plating efficiency determined for maintained in RPMI 1640 with 10% FCS, 10-mM glutamine, that treatment by the plating efficiency for the appropriate 100-unit/ml penicillin G, 100-Ag/ml streptomycin, 200-Ag/ unirradiated control. The survival curves shown were ml G418 (Life Technologies, Inc., Rockville, MD), and normalized for the toxicity of the vectors by using the 100-Ag/ml zeocin (Invitrogen, Carlsbad, CA). The MRC-9 treatments with vector alone as the appropriate unirradi- normal human fibroblast cells were cultured in a-MEM ated controls for the vector + radiation survival curves. The containing 10% FCS, 10-mM glutamine, 100-unit/ml peni- width of the shoulder region of the survival curves, a cillin G, and 100-Ag/ml streptomycin supplemented with classical indication of the relative repair capacity of the cells nonessential amino acids and vitamins. (39), was calculated by graphical analysis as the quasi- Adenovirus threshold dose or Dq (40). h h The E1-deleted adenovirus- -gal (Ad- -gal), Ad-p53, Western Blot and adenovirus-luciferase (Ad-luc) vectors were obtained Cells were harvested after treatment with Ad-p53 or Ad- from Introgen Therapeutics (Houston, TX). Ad-luc was luc and lysed by vortexing in a lysis buffer containing used as a control vector for all experiments. 50-mM HEPES (pH 7.0), 150-mM NaCl, 1.5-mM MgC2, 1-mM Antibodies EGTA, 100-mM NaF, 10-mM sodium pyrophosphate, 10% Antibodies to p21 and Ku70 were obtained from Santa glycerol, and 1% Triton X-100. The buffer was fortified with Cruz Biotechnologies, Inc. (Santa Cruz, CA). Antibody to 1-mM sodium orthovanadate, 2-mM phenylmethylsulfonyl p53 was purchased from DAKO Corp. (Carpinteria, CA). fluoride, 10-Ag/ml aprotinin, and 10-Ag/ml leupeptin at h Antibodies to -actin and Ku80 were obtained from Sigma the time of use. The lysate was incubated on ice for 20 min (St. Louis, MO). Antibodies to DNA-PKcs, XRCC4, and and centrifuged at 12,000 rpm at 4jC for 15 min to remove ataxia telangiectasia mutated (ATM) were purchased from any cellular debris. Protein concentration of the lysates was GeneTex (San Antonio, TX). determined by the Bio-Rad protein assay method. Equal Gene Delivery amounts of the protein were resolved on 7–12% polyacryl- A431 cells (0.5 Â 106) were plated in 100-mm culture amide gels and transferred to Immobilon membrane dishes. Forty-eight hours after plating, cells were infected (Millipore, Billerica, MA). The membrane was washed in with vector for 1 h in 1 ml of serum-free medium. After Tris (20 mM)-buffered saline (150 mM; pH 7.4) + 0.05–0.1% infection with the appropriate vector, complete medium Tween (TBS-T) and blocked in a blocking buffer as with 10% FBS was added to the cells. Cells were incubated suggested by the manufacturer for 1 h at room temperature for an additional 48 h. Infectivity of the cells was tested (RT) or at 4jC overnight. After being washed with TBS-T using Ad-h-gal followed by h-gal expression. For this, again, the membrane was incubated with primary antibody 25 Â 103 cells/well were seeded in each well of a six-well in a suitable buffer for 1 h at RT or overnight at 4jC.
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