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Sensitization of Nitrosourea-resistant Mer⁺ Human Tumor Cells to N-(2-Chloroethyl)-N'-cyclohexyl-N-nitrosourea by Mild (41°C) Hyperthermia¹

Departments of Human Oncology [R. T. M., J. J. G.] and Statistics [M. A. T.], University of Wisconsin Clinical Cancer Center, University of Wisconsin, Madison, Wisconsin 53792

Experiments were designed to determine whether heat treatment could sensitize nitrosourea-resistant human tumor cell lines expressing a repair system (O⁶-alkylguanine DNA alkyltransferase; Mer⁺) capable of removing monoadducts from the DNA of treated cells prior to the formation of lethal interstrand cross-links. Effects of temperatures compatible with systemic hyperthermia were of particular interest, and, consequently, the effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) exposure in vitro for 4 h at 37°C was compared with that for 1 h at 41°C followed by 3 h at 37°C. CCNU toxicity was significantly enhanced by heat treatment in the Mer⁺ HT-29 human colon carcinoma, and in HeLa-S3 and HeLa-CCL2 cell lines [thermal enhancement factor (ratio of CCNU doses required to reduce cell survival to 0.001 at 37° and 41°C) = 1.3–1.4]. Pharmacokinetic studies indicated that the effect of heat treatment on CCNU toxicity was not attributable to exposure to increased concentrations of reactive species, nor was the enhancement due to a direct effect of heat and/or drug on alkyltransferase activity. A similar enhancement of CCNU toxicity was also observed in a Mer^- line, HeLa-MR (thermal enhancement factor = 1.3). Heat-sequencing experiments clearly demonstrate that heat and CCNU must be administered concurrently. Alkaline elution experiments were designed to examine DNA-DNA cross-link formation in Mer⁺ and Mer^- cells exposed to CCNU at 37° and 41°C, but quantitation of cross-link formation was not possible owing to the persistence of single strand breaks in the DNA of drug-treated cells. Nevertheless, collectively the data indicate that thermal enhancement of CCNU toxicity is independent of effects on alkyltransferase activity and indicate that hyperthermia could provide an effective strategy for improving the nitrosourea response of resistant Mer⁺ tumors.

¹ This work was supported by USPHS Grants CA42325 and CA44381 awarded by the National Cancer Institute, Department of Health and Human Services. Presented in part at the 35th Annual Meeting of the Radiation Research Society, Atlanta, GA, February 21–26, 1987.

² To whom requests for reprints should be addressed, at Department of Human Oncology, University of Wisconsin Clinical Cancer Center, 600 Highland Avenue, K4/334, Madison, WI 53792.

Received 6/22/87. Revised 9/18/87. Accepted 11/30/87.