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.
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.
Wessel
I
I
Department of Pathology, Laboratory Center, Rigshospitalet 5444, Copenhagen, Denmark.
Jensen
P B
PB
Falck
J
J
Mirski
S E
SE
Cole
S P
SP
Sehested
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000951:Antigens, Neoplasm; D018288:Carcinoma, Small Cell / Q000201:enzymology* / Q000235:genetics; D002467:Cell Nucleus / Q000201:enzymology; D016384:Consensus Sequence; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000633:toxicity*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics; D008239:Lysine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D017384:Sequence Deletion; D012694:Serine; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000951:Antigens, Neoplasm; D004250:DNA Topoisomerases, Type II; D004268:DNA-Binding Proteins; D005047:Etoposide; D007527:Isoenzymes; D008239:Lysine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D012694:Serine