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Alterations in the Topoisomerase II Gene, Messenger RNA, and Subcellular Protein Distribution as well as Reduced Expression of the DNA Topoisomerase IIß Enzyme in a Mitoxantrone-resistant HL-60 Human Leukemia Cell Line¹

Medicine Service, Veterans Affairs Medical Center and Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah 84148 [W. G. H., D. L. S., R. L. P., M. H. H.] and Departments of Medicine and Biochemistry, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky 40292 [P. W. F., D. M. S.]

A human HL-60 leukemia cell line selected for resistance to mitoxantrone, HL-60/MX2, displays cross-resistance only to agents whose cytotoxicities result from interaction with the nuclear enzyme DNA topoisomerase II (topo II). The topo II catalytic activity is reduced 2-fold in the drug-resistant cell line in association with the absence of the M_r 180,000 isoform of topo II and the finding of a novel M_r 160,000 topo II -related immunoreactive protein in these cells by immunoblot. The topo II (M_r 170,000) protein levels in nuclear extracts from the HL-60/MX2 cells were noted on average to be 40% lower than in comparable HL-60 nuclei. Studies of the subcellular localization of topo II by immunohistochemical and fractional extraction techniques demonstrated that the M_r 160,000 topo II-related protein is primarily localized in the cytoplasm. Levels of the 6.3-kilobase topo II mRNA were noted to be reduced 2-fold in the HL-60/MX2 cells in association with the finding of a novel 4.8-kilobase topo II-related mRNA transcript that was present in HL-60/MX2 but not HL-60 cells. The absence of topo IIß protein in nuclear and whole cell extracts from the HL-60/MX2 cells was associated with the virtual absence of detectable topo IIß mRNA in those cells by Northern blot analysis. Using a reverse transcription-PCR assay we were able to demonstrate the presence of very low levels of topo IIß mRNA in HL-60/MX2 cells, representing <1% of that found in the HL-60 cells. In contrast, the nuclear catalytic activity and cellular mRNA levels of the related nuclear enzyme DNA topoisomerase I were nearly identical in the two cell types. Southern blot analysis of DNA extracted from the drug-sensitive and drug-resistant cells revealed a structural alteration in one topo II allele in the HL-60/MX2 cells, but there was no evidence of rearrangement or hypermethylation of the topo IIß locus. These results indicate that the reduced levels of topo II and ß isoenzymes observed in mitoxantrone-resistant HL-60/MX2 cells are related to changes in the levels of their respective mRNA transcripts. The identification of structural changes in one topo II allele in the HL-60/MX2 cell line suggests that the altered allele may serve as the source of the unique 4.8-kilobase topo II-related mRNA transcript and the M_r 160,000 protein discovered in those cells.

¹ Supported in part by the Department of Veterans Affairs Medical Research (W. G. H., D. L. S., R. L. P., M. H. H.), by NIH Grants R29-33044 (M. H. H.) and CA59747 (D. M. S.), and by National Cancer Institute CCSG Grant 5P30-CA42014-08 (W. G. H., D. L. S., R. L. P., M. H. H.).

² To whom requests for reprints should be addressed, at Medical Service 111C, Veterans Affairs Medical Center, 500 Foothill Drive, Salt Lake City, UT 84148.

Received 11/15/94. Accepted 2/15/95.

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