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Loss of Amino Acids ¹⁴⁹⁰Lys-Ser-Lys¹⁴⁹² in the COOH-Terminal Region of Topoisomerase II in Human Small Cell Lung Cancer Cells Selected for Resistance to Etoposide Results in an Extranuclear Enzyme Localization¹

Department of Pathology, Laboratory Center, Rigshospitalet 5444, DK-2100 Copenhagen, Denmark [I. W., J. F., M. S.]; Department of Oncology, Finsen Center, Rigshospitalet 5074, DK-2100 Copenhagen, Denmark [I. W., J. F., P. B. J.]; and Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada [S. E. L. M., S. P. C. C.]

The human small cell lung cancer NCI-H69 cell line selected for resistance to etoposide (H69/VP) has been reported previously to sequentially overexpress both the MRP and MDR1 multidrug resistance-conferring genes. In addition, immunocytochemistry of H69/VP cells demonstrated a distinct extranuclear localization of the nuclear enzyme topoisomerase II, the target of etoposide. Immunoblots showed a decrease in M_r 170,000 toposiomerase II in nuclear extracts in H69/VP but equal amounts of the enzyme in whole-cell extracts. Topoisomerase II catalytic activities in H69 and H69/VP whole-cell extracts were equal, as were their inhibition by etoposide. Sequencing of the entire H69/VP topoisomerase II cDNA showed a homozygous 9-nucleotide deletion encompassing nucleotides 4468-76, coding for Lys-Ser-Lys, overlapping two potential bipartite nuclear localization signals. The deletion occurred at the initial nine nucleotides of an exon, suggesting alternative splicing of topoisomerase II mRNA. Subsequent sequencing of H69/VP genomic DNA revealed a GT point mutation in the 3' acceptor splice site consensus sequence, resulting in the use of an alternate splice site. Comparison with previous reports on three drug-resistant cell lines with large truncations/deletions in the COOH-terminal region of topoisomerase II and extranuclear localization point to a pivotal role for the basic cluster ¹⁴⁹⁰Lys-Ser-Lys¹⁴⁹² in the nuclear import of this enzyme.

¹ This work was supported by the Danish Cancer Society, the Novo Nordisk Foundation, Danish Medical Research Council, Director E. Danielsen's Foundation, Fonden til Laegevidenskabens Fremme, The Hojmosegaard Legacy, and the Nationla Cancer Institute of Canada with funds from the Canadian Cancer Society. S. P. C. C. is a Senior Scientist of the Ontario Cancer Foundation.

² To whom requests for reprints should be addressed. Phone: (45) 3545 5432; Fax: (45) 3545 5414; E-mail: maxwell@rh.dk.

Received 6/26/97. Accepted 8/22/97.

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