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-thioguanine

Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis c olorectal cancer, has recently been linked to a number of sporadic human ca ncers as well, Deficiency in this repair process renders cells resistant to many clinically arrive chemotherapy agents. As a result, it is of relevanc e to find an agent that selectively targets MMR-deficient cells. We have re cently shown that the halogenated thymidine (dThd) analogues iododeoxyuridi ne (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue -1 (MLH1)-deficient human cancer cells for radiosensitization. The levels o f IdUrd and BrdUrd in cellular DNA directly correlate with the ability of t hese analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediat ed MMR status impacts dThd analogue DNA levels, and consequently, analogue- induced radiosensitization. Here, we have extended these studies and show t hat, both in human and murine cells, MutS homologue-2 (MSH2) is also involv ed in processing dThd analogues In DNA. Using both E1A-transformed Msh2(+/) and Msh2(-/-) murine embryonic stem (ES)-derived cells (throughout this r eport we use Msh2(+/+) and Msh2(-/-) to refer to murine ES-derived cell lin es that are wild type or mutant, respectively, for the murine Msh2 gene) an d human endometrial cancer cells differing in MSH2 status,,ve see the class ic cytotoxic response to B-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 contra st, 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 Id Urd, High-performance liquid chromatography analysis of IdUrd and BrdUrd le vels in DNA suggests that this differential cytotoxicity may he due to lowe r analogue levels in MSH2(+) murine and human tumor cells. The DNA levels o f IdUrd and BrdUrd continue to decrease over time in Msh2(+/+) cells follow ing incubation in drug-free medium, whereas they remain high in Msh2(-/-) c ells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2-4 (hMsh2-corrected) cells. As a re sult of higher analogue levels in DNA, Msh2(-/-) cells are selectively targ eted for radiosensitization by IdUrd, Fluorescence-activated cell-sorting a nalysis of Msh2(+/+) and Msh2(-/-) cells shows that selective toxicity of t he halogenated nucleotide analogues is not correlated with a G(2)-M cell cy cle arrest and apoptosis, as Is found for selective killing of Msh2(+/+) ce lls by 6-TG. Together, these data demonstrate MSH2 involvement in the proce ssing 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.