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    Molecular biology of P-glycoprotein mediated multidrug resistance

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    Author
    Ma, Jian Feng
    Advisor
    Melera, Peter W.
    Date
    1995
    Type
    dissertation
    
    Metadata
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    Abstract
    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.
    Description
    University of Maryland, Baltimore. Molecular and Cell Biology. Ph.D. 1995
    Keyword
    Biology, Molecular
    Biology, Genetics
    Biology, Cell
    Health Sciences, Oncology
    p-glycoprotein
    Drug resistance in cancer cells
    ATP-Binding Cassette, Sub-Family B, Member 1--adverse effects
    Drug Resistance, Multiple
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1523
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