Characterization of WIF-B9/R cells as an in vitro model with hepatocyte-like polarity and enhanced expression of canalicular ABC transporters involved in phase III of hepatic detoxification.

摘要

The rat hepatoma/human fibroblast hybrid cell line WIF-B9 was developed to be used in studies requiring maintained hepatocyte-like polarity. To enhance their usefulness in order to investigate hepatic phase III detoxification process, we have characterized a subline of WIF-B9 cells (WIF-B9/R) obtained by exposure to progressively increasing cisplatin concentrations (up to 10 microM) and double sub-clonal selection. As compared to WIF-B9 cells, the cytostatic effect of cisplatin and doxorubicin on WIF-B9/R cells was lower (>10-fold), whereas the ability to reduce cell loading of cisplatin, doxorubicin, rhodamine 123 and calcein was higher. As their parent cells, WIF-B9/R cells express hepatocyte-like polarity. However, they have enhanced stable expression of Mdr1, Mrp1, Mrp2, Mrp3 and BCRP, but not Bsep/BSEP, as determined by real-time quantitative RT-PCR and western blot. Differentiation to hepatocyte-like phenotype was characterized by the formation of canalicular-like structures, containing in their membranes immunocytochemically detectable Mdr1, Mrp2 and BCRP. Functionality of these ABC transporters was evaluated by using specific substrates and inhibitors. Thus, canalicular-like structures were able to concentrate calcein, rhodamine 123 and doxorubicin. Moreover, verapamil, probenecid and Hoechst-33342 inhibited doxorubicin efflux and enhanced its content in WIF-B9/R cells. Probenecid inhibited calcein efflux and increased calcein cell load, but had no effect on cell loading of rhodamine 123, which was increased by verapamil and Hoechst-33342. In conclusion, WIF-B9/R cells are a useful model of polarized cells to study, in the absence of Bsep/BSEP, hepatic phase III of the detoxification process of several compounds whose canalicular transport is mediated by ABC proteins.