INDUCTION OF CMRP CMOAT GENE-EXPRESSION BY CISPLATIN, 2-ACETYLAMINOFLUORENE, OR CYCLOHEXIMIDE IN RAT HEPATOCYTES/

The human multidrug-resistance-associated protein (MRP), a member of t he adenosine triphosphate (ATP)-binding cassette transporter superfami ly, is frequently overexpressed in tumor cells resistant to antineopla stic drugs, In the rat, two Mrp isoforms have been identified, Mrp and cMrp. cMrp, also called Mrp2 or cMoat (canalicular multispecific orga nic anion transporter), is expressed in the canalicular membrane of ra t hepatocytes and mediates the excretion of glucuronate, sulfate, and glutathione conjugates into bile, We investigated the expression of cM rp and Mrp in rat hepatocytes in primary culture. Treatment with the c hemical carcinogen 2-acetylaminofluorene (2-AAF), the antineoplastic d rug cisplatin, and the protein-synthesis inhibitor cycloheximide led t o a dose-dependent and time-dependent increase in cmrp gene expression . A 347-base pair cmrp complementary DNA (cDNA) probe served to demons trate the induction of cmrp messenger RNA (mRNA) with 40 mu mol/L 2-AA F, 5 mu mol/L cisplatin, or 5 mu mol/L cycloheximide, An analogous res ponse was obtained for the increase in cMrp protein, Mrp mRNA was belo w the detection limit in Northern blots of RNA from liver and hepatocy te cultures, in contrast to rat testis mRNA which served as a positive control, Immunofluorescence microscopy of cultured hepatocytes was us ed to visualize cMrp in the plasma membrane. Treatment with 2-AAF led to a marked increase in the immunofluorescence signal confirming the c Mrp-inducing potency of 2-AAF. In conclusion, the inducing effect of t he compounds studied may reflect a general inducibility of hepatic cMr p by a variety of cytotoxic, carcinogenic, and chemotherapeutic agents which is likely to be of relevance for the acquisition of multidrug r esistance during chemotherapy and in the process of chemical carcinoge nesis in the liver.