PII S0360-3016(01)01719-9 BIOLOGY CONTRIBUTION EXPRESSION OF P-GLYCOPROTEIN AND MULTIDRUG RESISTANCE ASSOCIATED PROTEIN IN EHRLICH ASCITES TUMOR CELLS AFTER FRACTIONATED IRRADIATION DORTE NIELSEN, M.D., PH.D., CHRISTIAN MAARE, M.D., PH.D., JENS ERIKSEN, M.SC., PH.D., THOMAS LITMAN, M.SC., PH.D., AND TORBEN SKOVSGAARD, M.D., PH.D. Department of Oncology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Purpose: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible 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 was performed for determination of PGP and Mrp1, reverse transcriptase–polymerase chain reaction (RT-PCR) for determination of mdr1a  b mRNA, and semiquantita- tive RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3 H-vincristine (VCR), and 3 H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. Results: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3 H-VCR and 3 H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. Conclusion: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a  b mRNA. The influence of irradiation on PGP and Mrp1 expression seemed to be different. © 2001 Elsevier Science Inc. Drug resistance, Irradiation, Multidrug resistance associated protein, P-glycoprotein, Tumor cell lines. INTRODUCTION Drug resistance has been documented in patients after treat- ment not only with anticancer drugs, but also with radio- therapy (1). Multidrug resistance (MDR) in murine model systems is conferred by at least two different integral pro- teins: P-glycoprotein (PGP) encoded by the mdr1a and b gene, and the murine multidrug resistance associated pro- tein (Mrp1) (2– 4). The primary structures of PGP and Mrp1 are quite dissimilar, sharing only 15% amino acid identity. Nevertheless, these proteins belong to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily of transport proteins (3, 5, 6) and confer resistance to impor- tant chemotherapeutic agents such as anthracyclines, vinca alkaloids, and epipodophyllotoxins. Both are considered to act as drug efflux pumps with broad specificity for drugs. A variety of environmental stresses and circumstances that affect cellular differentiation have been shown to in- duce mdr1 gene expression and increased expression of PGP (7). Previously, Hill et al. (8) have reported that in vitro exposure of mammalian cells to fractionated irradia- tion results in the expression of PGP. This expression oc- curred without concomitant increase in expression of mdr1 messenger ribonucleic acid (mRNA). Furthermore, Mc- Clean et al. (9) found increased expression of PGP in Reprint requests to: Dorte Nielsen M.D., Ph.D., Department of Oncology R, Herlev Hospital, University of Copenhagen, Her- lev Ringvej, DK-2730 Herlev, Denmark. Tel: +4544884714; Fax: 4544883094; E-mail: dln@dadlnet.dk Supported by a grant from the Foundation of 1870 and from the Danish Cancer Society. Acknowledgments—The authors are grateful to Marianne Fregil, Marianne Knudsen, and Bente Raatz for excellent technical assis- tance. We greatly appreciate the expert technical assistance of Henning Hansen, M.Sc. Received Dec 21, 2000, and in revised form Apr 26, 2001. Accepted for publication Jun 21, 2001. Int. J. Radiation Oncology Biol. Phys., Vol. 51, No. 4, pp. 1050 –1057, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/01/$–see front matter 1050