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Loss of amino acids 1490Lys-Ser-Lys1492 in the COOH-terminal region of topoisomerase IIalpha in human small cell lung cancer cells selected for resistance to etoposide results in an extranuclear enzyme localization

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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 IIalpha, the target of etoposide. Immunoblots showed a decrease in Mr 170,000 topoisomerase IIalpha 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 IIalpha 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 IIalpha mRNA. Subsequent sequencing of H69/VP genomic DNA revealed a G-->T 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 IIalpha and extranuclear localization point to a pivotal role for the basic cluster 1490Lys-Ser-Lys1492 in the nuclear import of this enzyme.

Original languageEnglish
JournalCancer Research
Volume57
Issue number20
Pages (from-to)4451-4
Number of pages4
ISSN0008-5472
Publication statusPublished - 15 Oct 1997

    Research areas

  • ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis, ATP-Binding Cassette Transporters/biosynthesis, Amino Acid Sequence, Amino Acid Substitution, Antigens, Neoplasm, Carcinoma, Small Cell/enzymology, Cell Nucleus/enzymology, Consensus Sequence, DNA Topoisomerases, Type II/chemistry, DNA-Binding Proteins, Drug Resistance, Multiple/genetics, Drug Resistance, Neoplasm, Etoposide/toxicity, Humans, Isoenzymes/chemistry, Lung Neoplasms/enzymology, Lysine, Molecular Sequence Data, Molecular Weight, Multidrug Resistance-Associated Proteins, Peptide Fragments/chemistry, Point Mutation, Polymerase Chain Reaction, RNA, Messenger/biosynthesis, Sequence Deletion, Serine, Tumor Cells, Cultured

ID: 59178485