Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation.
OBJECTIVE
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
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 semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-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 3H-VCR and 3H-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.


CONCLUSIONS
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.
Nielsen
D
D
Department of Oncology, Herlev Hospital, University of Copenhagen, Herlev, Denmark. dln@dadlnet.dk
Maare
C
C
Eriksen
J
J
Litman
T
T
Skovsgaard
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism / Q000528:radiation effects*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000378:metabolism* / Q000627:therapeutic use; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism* / Q000532:radiotherapy; D003630:Daunorubicin / Q000378:metabolism / Q000627:therapeutic use; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000378:metabolism / Q000627:therapeutic use; D005978:Glutathione / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism / Q000528:radiation effects*; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000528:radiation effects; D014410:Tumor Stem Cell Assay; D014700:Verapamil / Q000494:pharmacology; D014750:Vincristine / Q000378:metabolism / Q000627:therapeutic use
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014750:Vincristine; D005047:Etoposide; D014700:Verapamil; D005978:Glutathione; D003630:Daunorubicin