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Evidence for a Mutant Allele of the Gene for DNA Topoisomerase II in Adriamycin-resistant P388 Murine Leukemia Cells¹

Manitoba Institute of Cell Biology [A. M. D., D. J. B., G. J. G.] and Department of Medicine, University of Manitoba [G. J. G.], Winnipeg, Manitoba R3E 0V9, Canada

Previous studies have shown that DNA topoisomerase II enzyme activity and protein levels are reduced in cloned lines of Adriamycin-resistant P388 leukemia cells relative to drug-sensitive cells (Deffie et al., Cancer Res., 49: 58–62, 1989). The molecular basis of the reduced topoisomerase II levels in these resistant cells has been investigated. Northern blot analysis of total cellular RNA from drug-sensitive and -resistant cells using a 1.8-kilobase human topoisomerase II complementary DNA revealed the presence of two mRNA species: a 6.6-kilobase transcript that was strongly expressed in drug-sensitive cells but reduced 7- to 8-fold in resistant cells; and a 5.5-kilobase transcript detected only in drug-resistant cells. Southern blot analysis of genomic DNA digested with BamHI, StuI, or PvuII and probed with the 1.8-kilobase complementary DNA for human topoisomerase II showed that, in Adriamycin-resistant cells, there were two different alleles for topoisomerase II, one identical to the native allele but with a lower gene copy number than that found in sensitive cells, and a second allele containing a mutation present only in resistant cells. These findings suggest that the reduced levels of topo II protein in drug-resistant cells may be due to reduced amounts of the native 6.6-kilobase mRNA. The unique 5.5-kilobase mRNA in resistant cells may represent a shortened transcript of the mutated topoisomerase II allele.

¹ Supported by a grant from the National Cancer Institute of Canada.

² To whom requests for reprints should be addressed.

Received 2/24/89. Revised 8/ 1/89. Accepted 9/21/89.