Overexpression of P-glycoprotein and alterations in topoisomerase II in P388 mouse leukemia cells selected in vivo for resistance to mitoxantrone.
The overexpression of P-glycoprotein (PGP) and alterations in DNA topoisomerase II (TOPO II) were evaluated in mouse leukemia P388 cells selected in vivo for mitoxantrone (MTT) resistance (P388/MTT) and compared to doxorubicin (DOX) resistant (P388/DOX) or vincristine (VCR) resistant (P388/VCR) models. Among a panel of TOPO II inhibitors which included etoposide (VP-16), DOX, MTT and 4'-[(9-acridinyl)-amino]methanesulfon-m-anisidide (m-AMSA), the relative resistance compared to parental sensitive P388/S cells was: P388/DOX greater than P388/MTT greater than P388/VCR. All the resistant sublines exhibited minimal cell kill (less than 20%) at vincristine concentrations greater than 100-fold the IC50 for P388/S cells. In a soft-agar colony-forming assay, the modulation of cytotoxicity in P388/MTT cells by the calmodulin inhibitor trifluoperazine following a 3-hr drug treatment demonstrated a marked potentiation in cell kill with MTT, VP-16, DOX and m-AMSA but not VCR. Immunoblotting data revealed that while PGP was not detectable in P388/S cells, the overexpression of PGP was apparent in P388/MTT cells and the relative expression between the resistant sublines was: P388/DOX greater than P388/MTT greater than P388/VCR. Although the amount and DNA cleavage activity of TOPO II in nuclear extracts from P388/VCR cells were comparable to those in P388/S cells, they were markedly lower in both P388/DOX and P388/MTT cells. However, decatenation activity of TOPO II in nuclear extracts was comparable between the sensitive (P388/S) and resistant sublines (P388/MTT, P388/DOX, and P388/VCR). Results from the present study demonstrated that P388 cells selected for resistance to mitoxantrone exhibit changes in TOPO II and overexpression of PGP similar to P388/DOX cells, while vincristine resistant cells only overexpress PGP. Since therapeutic strategies are primarily designed to interfere with PGP-mediated drug efflux, the choice of agents for modulating resistance in tumors which overexpress PGP versus tumors which overexpress PGP with altered TOPO II could be different.
Research Institute, Cleveland Clinic Foundation, OH 44195.
N
N
Kamath
D
D
Grabowski
J
J
Ford
D
D
Kerrigan
Y
Y
Pommier
R
R
Ganapathi
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D016923:Cell Death; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000627:therapeutic use; D004351:Drug Resistance; D007941:Leukemia P388 / Q000188:drug therapy* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008942:Mitoxantrone / Q000627:therapeutic use*; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000627:therapeutic use
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D014750:Vincristine; D004317:Doxorubicin; D008942:Mitoxantrone; D004250:DNA Topoisomerases, Type II