High cell density-dependent resistance and P-glycoprotein-mediated multidrug resistance in mitoxantrone-selected Chinese hamster cells.
Mitoxantrone (MIT) resistance has been studied in a colony selected from the CHO AA8 parental line in one step under a low degree of selective pressure (9 nM). The cells of the clonal isolate AA8/MIT C1(0) were sensitive to 9 nM MIT at low cell density but able to grow at high density. Parental AA8 cells were not able to grow under the latter condition. Decreased MIT accumulation (-20%) was observed at this step (step 0) in the absence of overexpression of mdr RNA coding for the drug efflux pump P-glycoprotein. Furthermore, AA8/MIT C1(0) did not exhibit cross resistance to vincristine, Adriamycin and etoposide at low cell density. During subsequent controlled growth for 2 months at high cell density in the presence of 9 nM drug, an additional selection occurred leading to a 4-fold MIT-resistant subline AA8/MIT C1(+). This subline was characterized at this step (step I) and after an additional 4 months of culture in the presence of 9 nM MIT (step II). Analysis of mdr gene expression and gene copy number showed an increase in mdr RNA and a pattern of mdr gene amplification which changed between step I and II. AA8/MIT C1(+)II exhibited a classical multidrug resistance phenotype with decreased accumulation of [14C]MIT and cross-resistance to vincristine, Adriamycin and etoposide. The ability to form the cleavable complex in the presence of etoposide in DNA topoisomerase II-containing nuclear extracts was identical in AA8/MIT C1(+)II and AA8 cell lines. These results demonstrate a new sequence of events in MIT resistance: low level of drug resistance at high cell density followed by mdr gene amplification.
Muller
C
C
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, Toulouse, France.
Laval
F
F
Soues
S
S
Birck
C
C
Charcosset
J Y
JY
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology; D005784:Gene Amplification; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008942:Mitoxantrone / Q000494:pharmacology*; D012313:RNA / Q000302:isolation & purification; D014750:Vincristine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D014750:Vincristine; D012313:RNA; D005047:Etoposide; D004317:Doxorubicin; D004247:DNA; D008942:Mitoxantrone