ALTERATIONS IN THE TOPOISOMERASE II-ALPHA GENE, MESSENGER-RNA, AND SUBCELLULAR PROTEIN DISTRIBUTION AS WELL AS REDUCED EXPRESSION OF THE DNA TOPOISOMERASE II-BETA ENZYME IN A MITOXANTRONE-RESISTANT HL-60 HUMANLEUKEMIA-CELL LINE

A human HL-60 leukemia cell line selected for resistance to mitoxantro ne, HL-60/MX2, displays cross-resistance only to agents whose cytotoxi cities result from interaction with the nuclear enzyme DNA topoisomera se II (topo II). The topo II catalytic activity is reduced 2-fold in t he 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 t opo II alpha-related immunoreactive protein in these cells by immunobl ot. The topo II alpha (M(r) 170,000) protein levels in nuclear extract s from the HL-60/MX2 cells were noted on average to be similar to 40% lower than in comparable HL-60 nuclei. Studies of the subcellular loca lization of topo II by immunohistochemical and fractional extraction t echniques demonstrated that the M(r) 160,000 topo II alpha-related pro tein is primarily localized in the cytoplasm. Levels of the 6.3-kiloba se topo II alpha 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 alpha-related mRNA transcript that was present in HL-60/MX2 but not H L-60 cells. The absence of topo II beta protein in nuclear and whole c ell extracts from the HL-60/MX2 cells was associated with the virtual absence of detectable topo II beta mRNA in those cells by Northern blo t analysis. Using a reverse transcription-PCR assay we were able to de monstrate the presence of very low levels of topo II beta mRNA in HL-6 0/MX2 cells, representing <1% of that found in the HL-60 cells. In con trast, the nuclear catalytic activity and cellular mRNA levels of the related nuclear enzyme DNA topoisomerase I were nearly identical in th e two cell types. Southern blot analysis of DNA extracted from the dru g-sensitive and drug-resistant cells revealed a structural alteration in one topo II alpha allele in the HL-60/MX2 cells, but there was no e vidence of rearrangement or hypermethylation of the topo II beta locus . These results indicate that the reduced levels of topo II alpha and beta 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 alpha 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 alpha-related mRNA t ranscript and the M(r) 160,000 protein discovered in those cells.