A truncated cytoplasmic topoisomerase IIα in a drug-resistant lung cancer cell line is encoded by a TOP2A allele with a partial deletion of exon 34

Abstract

To study the problem of acquired resistance to widely used anti-cancer drugs that target the 170 kDa topoisomerase IIα (topo IIα), a drug- resistant human small-cell lung cancer cell line, H209/VP, was selected in VP-16. H209/VP cells express reduced levels of the 170 kDa topo IIα that is localized normally in the nucleus and also express lower levels of a 160 kDa topo IIα-related protein that is located predominantly in the cytoplasm. Band depletion immunoblotting experiments suggest that the H209/VP nuclear 170 kDa topo IIα is able to form ternary complexes with DNA and VP-16 in intact cells, but the ability of the cytoplasmic 160 kDa protein to do so is greatly diminished. Sequence analysis of the 3' end of the H209/VP mutant topo IIα mRNA and the TOP2A gene indicates that the mRNA is missing 200 nt that corresponds to exon 34 because the partial loss of the minimal 3' splice-acceptor sequence at the beginning of exon 34 results in splicing of exon 33 to exon 35. The protein predicted to be encoded by this mutant mRNA does not contain the COOH-terminal 109 amino acids of the wild-type enzyme that we have demonstrated contain a strongly functional nuclear localization signal sequence. Consequently, our data explain both the size and the cytoplasmic localization of the H209/VP mutant topo IIα. The mutant TOP2A allele in H209/VP cells differs from those in previously characterized cell lines with cytoplasmic topo IIα and extends the number of types of resistance-associated deletions in this region to 4. These findings indicate that this region of the TOP2A gene may be a hot spot for mutations.