A truncated cytoplasmic topoisomerase II alpha in a drug-resistant lung cancer cell line is encoded by a TOP2A allele with a partial deletion of exon34

To study the problem of acquired resistance to widely used anti-cancer drug s that target the 170 kDa topoisomerase II alpha (topo II alpha), a drug-re sistant 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 II alpha that is localized normally in the nucleus and also express lower levels of a 16 0 kDa topo II alpha-related protein that is located predominantly in the cy toplasm. Band depletion immunoblotting experiments suggest that the H209/VP nuclear 170 kDa topo II alpha is able to form ternary complexes with DNA a nd VP-16 in intact cells, but the ability of the cytoplasmic 160 kDa protei n to do so is greatly diminished. Sequence analysis of the 3' end of the H2 09/VP mutant topo II alpha mRNA and the TOP2A gene indicates that the mRNA is missing 200 nt that corresponds to exon 34 because the partial loss of t he 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 t his mutant mRNA does not contain the COOH-terminal 109 amino acids of the w ild-type enzyme that we have demonstrated contain a strongly functional nuc lear localization signal sequence. Consequently, our data explain both the size and the cytoplasmic localization of the H209/VP mutant topo II alpha. The mutant TOP2A allele in H209/VP cells differs from those in previously c haracterized cell lines with cytoplasmic topo II alpha and extends the numb er of types of resistance-associated deletions in this region to 4. These f indings indicate that this region of the TOP2A gene may be a hot spot for m utations. Int. J, Cancer 85:534-539, 2000. (C) 2000 Wiley-Liss, Inc.