Functional expression and characterization in Xenopus laevis oocytes of the ABCG2 transporter derived from A549 human lung adenocarcinoma cells

We cloned the ATP-binding cassette sub-family G member 2 (ABCG2) transporter,the most recently identified among several major human multidrug-resistance pumps,from A549 human lung adenocarcinoma cells in order to characterize its functionand substrate specificity. In a previous report, we confirmed that a stem cell-likeside population of A549 cells highly expressed the ABCG2 gene and had a uniqueability to resist the anticancer drug methotrexate (MTX). In this study, ABCG2cDNA was cloned by RT-PCR and converted into cRNA by an in vitro transcriptionsystem for expression in Xenopus laevis (X. laevis) oocytes. The transcribed cRNAof the ABCG2 gene was injected into the oocytes under the absence of cofactorsor heterologous partner proteins or some lipids from the media. A high expressionof ABCG2 was observed on the oocyte surface by immunofluorescence and confocallaser microscopy. We tested the functional effect of ABCG2 expression on drugefflux by directly injecting MTX into X. laevis oocytes. The drug concentrationwithin the oocytes was quantified with LC-MS/MS; the analysis showed that theaccumulation of MTX was significantly decreased in the X. laevis oocytes expressingABCG2 compared with the control oocytes not expressing ABCG2. These findings showthat the ABCG2 protein has an important role in the efflux of MTX through thecell membrane of X. laevis oocytes. Therefore, it might be that ABCG2, abundantlyexpressed in the stem cell population of A549 cells, can modulate resistance toMTX in lung cancer therapy.