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Effects of Streptozotocin/Bis-chloroethylnitrosourea Combination Therapy on O⁶-Methylguanine DNA Methyltransferase Activity and mRNA Levels in HT-29 Cells in Vitro¹

Section of Hematology/Oncology, Department of Medicine [R. O. P.], Department of Pharmacology [B. W. F., L. C. E.], and Program in Molecular Biology [Q. D., L. C. E.], Stritch School of Medicine, Loyola University Medical Center, Maywood, Illinois 60153

Treatment of chloroethylnitrosourea-resistent cells with streptozotocin (STZ) prior to bis-chloroethylnitrosourea (BCNU) exposure has been shown to result in a depletion of O⁶-methylguanine DNA methyltransferase (MGMT) activity, increased BCNU-induced interstrand cross-linking, and a 2–3 log enhancement of BCNU cytotoxicity in vitro. The current study was undertaken to define the kinetics of repletion of MGMT activity following the STZ/BCNU combination and to assess at the molecular level the effects of the combination on MGMT mRNA expression. Results demonstrate that MGMT activity can be depleted by >90% relative to untreated controls using an optimized STZ/BCNU combination regimen and that >50% depletion can be maintained for at least 24 h. This depletion appears to be independent of effects at the mRNA level because neither STZ alone nor the STZ/BCNU combination significantly altered steady state levels of MGMT mRNA. Cytotoxicity studies are consistent with MGMT repletion data and demonstrate that, as the interval between STZ and BCNU exposures increases, the degree of enhanced cytotoxicity induced by the combination relative to BCNU alone decreases. These results suggest that the enhanced cytotoxicity induced by the STZ/BCNU combination over BCNU treatment alone is favored by both the lack of induction of expression of MGMT mRNA and by slow reappearance of MGMT activity.

¹ This study was supported in part by USPHS Grant RO1 CA45628 to L.C.E. and USPHS National Research Service Award Fellowship F32 CA08685 to R.O.P.

² To whom requests for reprints should be addressed, at Hematology/Oncology, Loyola University of Chicago, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153.

³ Present address: Arizona Cancer Center, University of Arizona, Tuscon, AZ 85724.

Received 8/23/90. Accepted 1/ 3/91.

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