• Diversity of multidrug resistance in mammalian cells

      Devine, Scott E.; Melera, Peter W. (1993)
      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.
    • Molecular biology of P-glycoprotein mediated multidrug resistance

      Ma, Jian Feng; Melera, Peter W. (1995)
      The emergence of drug-resistance in cancer cells during chemotherapy remains a major obstacle in the treatment of neoplasia. Multidrug resistance (MDR) to a group of unrelated cytotoxic compounds can be conferred to eucaryotic cells by the expression of P-glycoprotein (Pgp), a putative plasma membrane transporter believed to mediate the efflux of these agents out of cells. A variety of agents are able to reverse this MDR phenotype by inhibiting the Pgp transporter. Blocking the action of this protein increases the effectiveness of cancer chemotherapeutic agents and, hence, has significant clinical implications. A mutant Pgp1 cDNA containing the substitution (Gly{dollar}\sp{lcub}338{rcub}{dollar}Ala{dollar}\sp{lcub}339{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}Pro{dollar}\sp{lcub}339{rcub}{dollar}) within the sixth transmembrane domain (tm6) has been cloned. The expression of this mutant confers an MDR phenotype preferentially resistant to actinomycin D. In this thesis we report that this MDR phenotype also has a decreased sensitivity toward reversal by cyclosporin A, while the sensitivity toward verapamil is unaltered. The accumulation of {dollar}\rm\lbrack\sp3H\rbrack{dollar} vincristine in cells expressing the wild-type Pgp1, not the mutant, increases dramatically in the presence of cyclosporin A, which correlates well with the reversal profile. We have altered only one amino acid residue at this location (Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar} or Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar}). The transfectants expressing the Pgp1 containing the proline substitution, rather than the alanine, demonstrate an MDR phenotype which is preferentially resistant to actinomycin D, and insensitive to reversal by cyclosporin A. Modeling the whole tm6 domain (with the Quanta modeling program and energy minimization by the CHARMm program) reveals that the proline substitution at position 339 rather than the alanine at 338 drastically changes the local {dollar}\alpha{dollar}-helice conformation, especially the polar side chain alignment along the hydrophilic side of this amphipathic {dollar}\alpha{dollar}-helice. We hypothesize that the Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar} substitution, rather than the Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}, is the primary contributor to the aforementioned altered phenotype. We have also attempted to determine the functions of two spliced variants of Pgp1, previously cloned in this laboratory, by expressing them in an in vitro system. The biogenesis of one of the variants, ADX124, has also been investigated. We conclude that it is derived from a splicing event that involves the internal splicing signals that are maintained in the mature full-length Pgp1 transcript.