Diversity of multidrug resistance in mammalian cells

Abstract

The emergence of drug-resistant tumor cells during chemotherapy remains a major problem in the treatment of cancer. Cells that become refractory to the lethal effects of certain natural product drugs may also simultaneously acquire cross resistance to a large number of structurally and functionally unrelated compounds; this phenotype is known as multidrug resistance (mdr). P-glycoprotein (pgp) is a plasma-membrane transporter that is thought to confer mdr to otherwise drug-sensitive cells by acting as an ATP-dependent drug-efflux pump with broad substrate specificity. Interestingly, the cross-resistance profiles of drug-resistant cell lines developed by growth in chemotherapeutic agents can be extremely variable. Hence, to study this diversity in mdr, we first cloned and analyzed a full-length hamster pgp1 cDNA from Chinese hamster lung DC-3F/ADX cells, which overexpress pgp and display mdr. The pgp encoded by this cDNA was very similar to mouse and human pgps, both structurally and functionally. Upon comparison with a pgp1 cDNA cloned from normal hamster liver, the pgp1 cDNA cloned from drug-resistant DC-3F/ADX cells was found to contain two point mutations. These point mutations were located at nucleotides 1123 and 1125 of the pgp1 transcript, and each led to an amino acid substitution within the predicted sixth transmembrane (tm6) domain of the encoded pgp: the glycine at codon 338 was changed to alanine, and the adjacent alanine at 339 was changed to proline. Transfection experiments showed that the introduction and overexpression of the normal hamster pgp1 cDNA conferred mdr to otherwise drug-sensitive hamster DC-3F cells. Furthermore, the tm6-mutant pgp1 conferred a very different cross-resistance profile from that conferred by the normal protein, indicating that the tm6 domain plays a role in the mechanisms of drug recognition and efflux. Finally, a number of drug-resistant clones of the Chinese hamster lung cell line, DC-3F, were isolated by growth in actinomycin D for short periods of time, and these clones were compared to pgp1 transfectants, as well as other DC-3F sublines (including DC-3F/ADX). These studies indicated that somatically-acquired mutations in pgp genes play a role in shaping multidrug resistance phenotypes in mammalian cells.