Selection of non-P-glycoprotein mediated high-level etoposide resistant cell lines by adriamycin with P-gp inhibitors.
In murine erythroleukemia (MEL) A20 cells (grown in 20 ng/ml adriamycin), mutation(s) producing 10-fold adriamycin (doxorubicin) resistance emerged via an unknown mechanism. Exposure of A20 cells to further stepwise increasing concentrations of ADR in combination with MDR modulators (PSC833 and verapamil) aimed to amplify the undetermined A20 mechanism while controlling P-glycoprotein (P-gp) overexpression. The growth of the derived cell lines A30P, A40P and A60P (grown in 30, 40 and 60 ng/ml ADR with PSC833 and verapamil) was initially slow, but eventually reached near WT rates. The new cell lines A30P and A40P were only 1.3- and 1.6-fold more resistant to adriamycin than PC4 A20. Resistance to vincristine was unchanged, but resistance to etoposide (VP-16) was 3.7-fold higher in A40P than A20 (itself 97-fold higher than wild-type). Expression of mdr3 and mrp mRNA tested by RT-PCR showed no increase. Daunorubicin and etoposide accumulation was not different among the cell lines, and no changes were detected in the number of daunorubicin fluorescent lysosomes. In comparison to WT, reduced topoisomerase IIalpha (EC 5.99.1.3) activity (20%) and protein expression (80%) was similar to the parental A20 cells. No mutations in the coding sequence of topoisomerase IIalpha could be located to account for the high etoposide resistance levels. The inhibitor combination of verapamil and PSC833 prevented the emergence of transporter mediated MDR, but not ADR selection of cell lines highly resistant to etoposide.
Ades
Steven
S
Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA.
Maxfield
Lori F
LF
Gould
Christopher J
CJ
Jones
Graham K
GK
Levy
Stuart B
SB
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003524:Cyclosporins / Q000494:pharmacology; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003630:Daunorubicin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014700:Verapamil / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D003524:Cyclosporins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D005047:Etoposide; D004317:Doxorubicin; C095122:multidrug resistance protein 3; D014700:Verapamil; D004250:DNA Topoisomerases, Type II; C070272:valspodar; D003630:Daunorubicin