Multidrug-resistant human sarcoma cells with a mutant P-glycoprotein, altered phenotype, and resistance to cyclosporins.
A variant of the multidrug-resistant human sarcoma cell line Dx5 was derived by co-selection with doxorubicin and the cyclosporin D analogue PSC 833, a potent inhibitor of the multidrug transporter P-glycoprotein. The variant DxP cells manifest an altered phenotype compared with Dx5, with decreased cross-resistance to Vinca alkaloids and no resistance to dactinomycin. Resistance to doxorubicin and paclitaxel is retained. The multidrug resistance phenotype of DxP cells is not modulated by 2 microM PSC 833 or cyclosporine. DxP cells manifest a decreased ability to transport [3H]cyclosporine. DNA heteroduplex analysis and sequencing reveal a mutant mdr1 gene (deletion of a phenylalanine at amino acid residue 335) in the DxP cell line. The mutant P-glycoprotein has a decreased affinity for PSC 833 and vinblastine and a decreased ability to transport rhodamine 123. Transfection of the mutant mdr1 gene into drug-sensitive MES-SA sarcoma cells confers resistance to both doxorubicin and PSC 833. Our study demonstrates that survival of cells exposed to doxorubicin and PSC 833 in a multistep selection occurred as a result of a P-glycoprotein mutation in transmembrane region 6. These data suggest that Phe335 is an important binding site on P-glycoprotein for substrates such as dactinomycin and vinblastine and for inhibitors such as cyclosporine and PSC 833.
Chen
G
G
Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5306, USA.
Durán
G E
GE
Steger
K A
KA
Lacayo
N J
NJ
Jaffrézou
J P
JP
Dumontet
C
C
Sikic
B I
BI
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D015153:Blotting, Western; D002470:Cell Survival; D003524:Cyclosporins / Q000627:therapeutic use*; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004095:Dihydropyridines / Q000378:metabolism; D004317:Doxorubicin / Q000627:therapeutic use; D018432:Drug Resistance, Multiple; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011224:Prazosin / Q000031:analogs & derivatives / Q000378:metabolism; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism; D012509:Sarcoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D020394:Vault Ribonucleoprotein Particles; D014700:Verapamil / Q000627:therapeutic use; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D003524:Cyclosporins; D004250:DNA Topoisomerases, Type II; D004095:Dihydropyridines; D004317:Doxorubicin; D009363:Neoplasm Proteins; D011224:Prazosin; D020112:Rhodamine 123; D012235:Rhodamines; D020394:Vault Ribonucleoprotein Particles; D014700:Verapamil; D000099187:Major Vault Protein; C041130:azidopine; C041552:azidoprazosin