Differential recognition of mdr1a and mdr1b gene products in multidrug resistant mouse tumour cell lines by different monoclonal antibodies

Abstract

An immunocytochemical method was used to test the reactivity of the anti-P-glycoprotein antibodies, C219, MRK 16, JSB-1 and 265/F4 against multidrug resistant (MDR) variants derived from the human small cell lung carcinoma line, NCI-H69, the mouse fibrosarcoma line, RIF-1 and the mouse mammary tumour cell line, EMT6. C219 produced positive staining in MDR variants of both human and mouse tumour cell lines. MRK 16 and JSB-1 however recognised P-glycoprotein only in the human MDR cell lines and not in the mouse MDR cells. 265/F4 appeared the most selective of the monoclonal antibodies used, producing positive staining of MDR variants derived from the RIF-1 line, but not of MDR variants derived from the EMT6 line. Total RNA was prepared from the mouse cell lines and, following reverse transcription, cDNA sequences were amplified by the polymerase chain reaction with primers specific for either the murine mdr1a or the mdr1b genes. From this it was possible to show that only the mdr1a gene is overexpressed in the resistant EMT6 lines that do not stain with 265/F4 whereas both mdr1a and mdr1b are overexpressed in the positively staining resistant fibrosarcoma line, RIF/1.0. Low level expression of mdr1b was detected in the sensitive parent RIF-1 cells and increasing levels of expression correlated with increasing resistance in the lines, RIF/0.1, 0.2, 0.4 and 1.0. Expression of mdr1a was found only in the more resistant fibrosarcoma cell lines. It seems that 265/F4 recognises only the mdr1b P-glycoprotein. Western blotting confirmed that this antibody detects a 170 kDa protein only in membranes derived from the resistant fibrosarcoma cells. 265/F4 may thus be used to distinguish between the murine P-glycoprotein isoforms so revealing differences between tumour cell lines in cellular localisation and in the time of appearance of mdr1a and mdr1b P-glycoprotein following drug exposure.