Cytoplasmic localization of a mutant M(r) 160,000 topoisomerase II alpha is associated with the loss of putative bipartite nuclear localization signals in a drug-resistant human lung cancer cell line.

Many clinically important antineoplastic agents exert their cytotoxicity through interaction with the M(r) 170,000 topoisomerase II alpha, an essential nuclear enzyme. Resistance to these agents has been associated frequently with either a decrease in the levels of topoisomerase II alpha 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 alpha isoenzyme. We have shown previously that H209/V6 cells express two topoisomerase II alpha 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 alpha 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 alpha is absent from the 4.8-kilobase mRNA. This shorter mRNA is predicted to encode a topoisomerase II alpha 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 alpha 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 alpha serve an important nuclear localization function.