Expression of P-glycoprotein associated protein in Ehrlich fractionated and multidrug resistance ascites tumor cells after irradiation

Purpose: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possib le role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. Methods and Materials: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis w as performed for determination of PGP and Mrp1, reverse transcriptase-polym erase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semi quantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to inve stigate sensitivity, whereas the steady-state drug accumulation of daunorub icin (DNR), H-3-vincristine (VCR), and H-3-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determini ng of ATPase activity, the release of inorganic phosphate from ATP was quan tified using a colorimetric method. Results: Compared with EHR2, the irradiated cell line EHR2/irr showed incre ased expression of PCP (threefold), Mr-pi (eightfold), and Mrp1 mRNA (sixfo ld), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR 2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resista nce to VP16, twofold resistance to vincristine, but remained sensitive to D NR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathi one by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/ irr. In EHR2/irr, the steady-state accumulation of H-3-VCR and H-3-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepare d from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPa se activity was neither stimulated by vinblastine nor VER. Conclusion: Irradiation induced a multidrug-resistant phenotype in sensitiv e tumor cells. This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a + b mRNA. The in fluence of irradiation on PGP and Mrp1 expression seemed to be different. ( C) 2001 Elsevier Science Inc.