Cancer Therapy: Preclinical Differential Cellular Responses to Prolonged LDR-IR in MLH1-Proficient and MLH1-Deficient Colorectal Cancer HCT116 Cells

Purpose: MLH1 is a key DNA mismatch repair (MMR) protein involved in maintaining genomic stability by participating in the repair of endogenous and exogenous mispairs in the daughter strands during S phase. Exogenous mispairs can result following treatment with several classes of chemotherapeutic drugs, as well as with ionizing radiation. In this study, we investigated the role of the MLH1 protein in determining the cellular and molecular responses to prolonged low–dose rate ionizing radiation (LDR-IR), which is similar to the clinical use of cancer brachytherapy. Experimental Design: An isogenic pair of MMR (MLH1) and MMR (MLH1) human colorectal cancer HCT116 cells was exposed to prolonged LDR-IR (1.3-17 cGy/h × 24-96 h). The clonogenic survival and gene mutation rates were examined. Cell cycle distribution was analyzed with flow cytometry. Changes in selected DNA damage repair proteins, DNA damage response proteins, and cell death marker proteins were examined with Western blotting. Results: MLH1 HCT116 cells showed greater radiosensitivity with enhanced expression of apoptotic and autophagic markers, a reduced HPRT gene mutation rate, and more pronounced cell cycle alterations (increased late-S population and a G2/M arrest) following LDR-IR compared with MLH1 HCT116 cells. Importantly, a progressive increase in MLH1 protein levels was found in MLH1 cells during prolonged LDR-IR, which was temporally correlated with a progressive decrease in Rad51 protein (involved in homologous recombination) levels. Conclusions: MLH1 status significantly affects cellular responses to prolonged LDRIR. MLH1 may enhance cell radiosensitivity to prolonged LDR-IR through inhibition of homologous recombination (through inhibition of Rad51). (Clin Cancer Res 2009;15(22):6912–20) Ionizing radiation is a well-known carcinogenic hazard to humans. There are many natural and man-made sources of LDR-IR affecting humans, including occupational, medical, and environmental exposures. The carcinogenic effects of LDR-IR on humans are recently reviewed (1). However, LDR-IR is also commonly used as an effective radiotherapeutic strategy (termed brachytherapy) for some cancers, including prostate, gynecologic, lung, breast, head and neck, anal/rectal, and esophageal cancers, as well as soft tissue sarcomas. Indeed, the use of permanent LDR brachytherapy is a common treatment approach to low-risk, early-stage prostate cancer, wherein the radioactivity of the permanently implanted iodine or palladium seeds decays over several months within the gland. LDR brachytherapy is also sometimes used in the treatment of coronary artery disease to prevent restenosis after angioplasty. Hence, a better understanding of the cellular response to LDR-IR is of clinical interest. Ionizing radiation induces a complex spectrum of DNA damage, including double-strand breaks, single-strand breaks, DNA cross-links, and oxidized bases. Clearly, the ability of a cell to repair ionizing radiation–induced DNA damage will affect the overall cellular response to ionizing radiation. All five major DNA repair pathways, including homologous recombination, nonhomologous end joining, nucleotide excision repair, base excision repair, and mismatch repair (MMR) are involved in the repair of the ionizing radiation–induced DNA damages (2–5). These DNA repair pathways are tightly interrelated with ionizing radiation–induced cell cycle checkpoint arrest pathways, which allow time to complete DNA repair, as well as Authors' Affiliation: Department of Radiation Oncology and the Case Integrative Cancer Biology Program, Case Comprehensive Cancer Center, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine, Cleveland, Ohio Received 7/1/09; revised 8/13/09; accepted 8/14/09; published OnlineFirst