Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux.
P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.
Tainton
K M
KM
Cancer Immunology Program, The Peter MacCallum Cancer Centre, Trescowthick Research Laboratories, East Melbourne, Victoria, Australia.
Smyth
M J
MJ
Jackson
J T
JT
Tanner
J E
JE
Cerruti
L
L
Jane
S M
SM
Darcy
P K
PK
Johnstone
R W
RW
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D045304:Cytochromes c / Q000378:metabolism; D004252:DNA Mutational Analysis; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000494:pharmacology; D015255:Idarubicin / Q000494:pharmacology; D008223:Lymphoma / Q000188:drug therapy; D008938:Mitosis; D009154:Mutation; D012190:Retroviridae / Q000235:genetics; D013329:Structure-Activity Relationship; D013997:Time Factors; D014750:Vincristine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D006877:Hydroxamic Acids; C100692:oxamflatin; D049452:Green Fluorescent Proteins; D014750:Vincristine; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D020169:Caspases; D000251:Adenosine Triphosphatases; D015255:Idarubicin