Co-expression of MDR-associated markers, including P-170, MRP and LRP and cytoskeletal proteins, in three resistant variants of the human ovarian carcinoma cell line, OAW42.
Variants of the human ovarian carcinoma cell line, OAW42, exhibiting low-level intrinsic resistance (OAW42-SR) and drug-induced higher-level resistance (OAW42-A1 & OAW42-A), were studied along with a sensitive clonal population (OAW42-S) which was isolated from OAW42-SR. Expression of the MDR-associated protein P-170, the more recently discovered LRP (lung resistance-related protein) and MRP (multidrug resistance-associated protein), topoisomerase II alpha and beta, GST pi and the cytoskeletal proteins, cytokeratin 8 and vimentin, were studied (using immunocytochemistry and Western blotting techniques) in conjunction with drug (doxorubicin) accumulation and subcellular distribution. Expression of mRNA for P-170, MRP, topoisomerase 11 alpha and beta and GST pi was studied using RT-PCR (reverse transcriptase polymerase chain reaction). Results indicate differential co-expression of four MDR-associated parameters (P-170, MRP, LRP and reduced topoisomerase II alpha and beta) in the OAW42-SR and OAW42-A1 variants, whereas resistance in the OAW42-A variant appeared to be mainly P-170 mediated. Comparable amounts of MRP and greater amounts of LRP were detected in the OAW42-S cells compared to the OAW42-SR variant (which showed increased resistance compared to the OAW42-S cells), but all cell lines expressed similar low-level amounts of MRP mRNA (by RT-PCR). GST pi levels did not differ markedly between variants. Increased levels of the cytoskeletal proteins were observed with increasing levels of resistance. The relative resistance of the variants, OAW42-SR and OAW42-A1, compared with OAW42-S was seen to change during increased serial passaging of the cells. There was greater drug accumulation by the sensitive OAW42-S cell line compared with that of the resistant variants, particularly the most highly resistant OAW42-A cells. Both verapamil and cyclosporin A effectively restored the accumulation defects seen in the resistant variants, cyclosporin A being the more effective of the two. Sub-cellular location of drug was predominantly in the nucleus with maximum levels seen in the sensitive OAW42-S variant and minimum levels in the most resistant OAW42-A clone.
Moran
E
E
National Cell and Tissue Culture Centre, BioResearch Ireland, Dublin City University, Ireland.
Cleary
I
I
Larkin
A M
AM
Amhlaoibh
R N
RN
Masterson
A
A
Scheper
R J
RJ
Izquierdo
M A
MA
Center
M
M
O'Sullivan
F
F
Clynes
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western; D004317:Doxorubicin / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000032:analysis*; D010051:Ovarian Neoplasms / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000903:Antibiotics, Antineoplastic; D014408:Biomarkers, Tumor; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein