Adenoviral-mediated mda-7 expression suppresses DNA repair capacity and radiosensitizes non-small-cell lung cancer cells Takashi Nishikawa 1 , Anupama Munshi 1 , Michael D Story 1 , Sheikh Ismail 2 , Craig Stevens 2 , Sunil Chada 3 and Raymond E Meyn* ,1 1 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; 2 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; 3 Introgen Therapeutics Inc., Houston, TX 77030, USA The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentia- tion. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) path- way of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells – A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radio- sensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electro- phoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs. Oncogene (2004) 23, 7125–7131. doi:10.1038/sj.onc.1207917 Published online 26 July 2004 Keywords: adenovirus; mda-7; radiation; lung cancer; DNA repair; nonhomologous end-joining Introduction Lung cancer is the leading cause of death among all malignancies in the Western world. In particular, non- small-cell lung cancer (NSCLC) accounts for 75–80% of all lung cancers (Parker et al., 1997). The only curative treatment currently available for NSCLC is surgical resection. Patients with nonresectable tumors typically undergo palliative chemotherapy or radiotherapy, but the expected survival rate in patients with stage III B–IV NSCLC is still o20% at 2 years. Thus, alternate treatments for patients with advanced NSCLC are sorely needed. This situation has stimulated investiga- tion of novel strategies, including those involving gene therapy. Although a number of gene therapy strategies for NSCLC are currently being studied, adenoviral- mediated expression of the melanoma differentiation- associated (mda-7) gene (Ad-mda7) may be particularly promising. Using a modified subtraction hybridization scheme, Jiang et al. (1995) isolated mda-7 from human melanoma cells that had been induced into terminal differentiation. The demonstration in this initial report that mda-7 is expressed in normal melanocytes during the early stages of melanoma but lost during melanoma progression suggested that mda-7 is a novel tumor suppressor gene. In subsequent investigations carried out by this group, induction of growth suppression and apoptosis was apparent in cancer cells from diverse origins, such as those in the breast, lung, colon, cervix, and prostate, when mda-7 was overexpressed using a replication-incompetent adenovirus (Ad-mda7) (Jiang et al., 1996). These effects were not evident in normal human cells. These early reports have prompted more detailed evaluations of the use of Ad-mda7 as a gene therapy strategy for cancer. In recent investigations, Ad-mda7 has been shown to inhibit the growth of NSCLC cells both in vitro and in vivo (Saeki et al., 2000; Saeki et al., 2002). Furthermore, in the anticipation that Ad-mda7 therapy may be combined with radiotherapy, we demonstrated that Ad-mda7 sensitized NSCLC cell lines to ionizing radiation by enhancing the apoptosis pathway (Kawabe et al., 2002). It was also recently reported that both Ad- mda7 and purified MDA-7 protein sensitize malignant glioma cells to ionizing radiation (Su et al., 2003). The mda-7 gene has been mapped to chromosome 1q32, where an interleukin 10-related gene cluster containing four genes (IL-10, IL-19, IL-20, and mda-7) is also located (Blumberg et al., 2001; Mhashilkar et al., 2001). Received 24 February 2004; revised 21 May 2004; accepted 24 May 2004; published online 26 July 2004 *Correspondence: RE Meyn, Department of Experimental Radiation Oncology, Unit 66, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA; E-mail: rmeyn@mdanderson.org Oncogene (2004) 23, 7125–7131 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc