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Cytoplasmic Localization of a Mutant M_r 160,000 Topoisomerase II Is Associated with the Loss of Putative Bipartite Nuclear Localization Signals in a Drug-resistant Human Lung Cancer Cell Line¹

Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada

Many clinically important antineoplastic agents exert their cytotoxicity through interaction with the M_r 170,000 topoisomerase II, an essential nuclear enzyme. Resistance to these agents has been associated frequently with either a decrease in the levels of topoisomerase II or a qualitative change that alters the interaction of this enzyme with a drug or DNA. Using a VP-16-selected lung cancer cell line, H209/V6, we have identified a third resistance mechanism which involves an aberrant subcellular location of the topoisomerase II isoenzyme. We have shown previously that H209/V6 cells express two topoisomerase II mRNAs (6.1 and 4.8 kilobases) but only a single catalytically active protein which has a M_r of 160,000 and is located primarily in the cytoplasm (Mirski et al., Cancer Res., 53: 4866–4873, 1993; Feldhoff et al., Cancer Res., 54: 756–762, 1994). In the present study we have determined that this mutant M_r 160,000 topoisomerase II is encoded by the shorter 4.8-kilobase mRNA. The sequencing of reverse transcriptase-PCR products from H209/V6 cells and subsequent Northern blot analyses showed that a sequence of 988 nucleotides from the 3'-coding and 3'-noncoding region of the normal topoisomerase II is absent from the 4.8-kilobase mRNA. This shorter mRNA is predicted to encode a topoisomerase II protein that no longer contains the 109 COOH-terminal amino acids of the normal enzyme but instead contains 34 new amino acids encoded by a sequence that was previously in the 3'-noncoding region of the mRNA. Confirmation that the COOH terminus of topoisomerase II is no longer present in the M_r 160,000 protein in H209/V6 cells was obtained by immunoblot analysis. Sequence analyses indicate that 3 putative bipartite nuclear localization signals in the M_r 160,000 protein are disrupted or lost. Our results suggest that sequences within the COOH-proximal domain of human topoisomerase II serve an important nuclear localization function.

¹ Supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (S. P. C. C.), and from the Clare Nelson Bequest of Kingston General Hospital (S. P. C. C., S. E. L. M.). S. P. C. C. is a Career Scientist of the Ontario Cancer Foundation.

² To whom requests for reprints should be addressed.

Received 12/27/94. Accepted 3/15/95.

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