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Selective Radiosensitization of Drug-resistant MutS Homologue-2 (MSH2) Mismatch Repair-deficient Cells by Halogenated Thymidine (dThd) Analogues: Msh2 Mediates dThd Analogue DNA Levels and the Differential Cytotoxicity and Cell Cycle Effects of the dThd Analogues and 6-Thioguanine¹

Department of Radiation Oncology, Case Western Reserve University, School of Medicine, and University Hospitals of Cleveland/Ireland Cancer Center, Cleveland, Ohio 44106 [S. E. B., T. W. D., J. E. S., H-S. H., T. J. K.]; Department of Human Oncology, University of Wisconsin, Madison, Wisconsin 53792 [S. E. B., T. J. K.]; and Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, 2333 AL Leiden, the Netherlands [N. d. W.]

Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis colorectal cancer, has recently been linked to a number of sporadic human cancers as well. Deficiency in this repair process renders cells resistant to many clinically active chemotherapy agents. As a result, it is of relevance to find an agent that selectively targets MMR-deficient cells. We have recently shown that the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue-1 (MLH1)-deficient human cancer cells for radiosensitization. The levels of IdUrd and BrdUrd in cellular DNA directly correlate with the ability of these analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediated MMR status impacts dThd analogue DNA levels, and consequently, analogue-induced radiosensitization. Here, we have extended these studies and show that, both in human and murine cells, MutS homologue-2 (MSH2) is also involved in processing dThd analogues in DNA. Using both E1A-transformed Msh2^+/+ and Msh2^-/- murine embryonic stem (ES)-derived cells (throughout this report we use Msh2^+/+ and Msh2^-/- to refer to murine ES-derived cell lines that are wild type or mutant, respectively, for the murine Msh2 gene) and human endometrial cancer cells differing in MSH2 status, we see the classic cytotoxic response to 6-thioguanine (6-TG) in Msh2^+/+ and human HEC59/2–4 (MSH2⁺) MMR-proficient cells, whereas Msh2^-/- cells and human HEC59 (MSH2^-/-) cells are tolerant (2-log difference) to this agent. In contrast, there is very little cytotoxicity in Msh2^+/+ ES-derived and HEC59/2–4 cells to IdUrd, whereas Msh2^-/- and HEC59 cells are more sensitive to IdUrd. High-performance liquid chromatography analysis of IdUrd and BrdUrd levels in DNA suggests that this differential cytotoxicity may be due to lower analogue levels in MSH2⁺ murine and human tumor cells. The DNA levels of IdUrd and BrdUrd continue to decrease over time in Msh2^+/+ cells following incubation in drug-free medium, whereas they remain high in Msh2^-/- cells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2–4 (hMsh2-corrected) cells. As a result of higher analogue levels in DNA, Msh2^-/- cells are selectively targeted for radiosensitization by IdUrd. Fluorescence-activated cell-sorting analysis of Msh2^+/+ and Msh2^-/- cells shows that selective toxicity of the halogenated nucleotide analogues is not correlated with a G₂-M cell cycle arrest and apoptosis, as is found for selective killing of Msh2^+/+ cells by 6-TG. Together, these data demonstrate MSH2 involvement in the processing of IdUrd and BrdUrd in DNA, as well as the differential cytotoxicity and cell cycle effects of the halogenated dThd analogues compared with 6-TG. Therefore, IdUrd and BrdUrd may be used clinically to selectively target both MLH1- and MSH2-deficient, drug-resistant cells for radiosensitization.

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