Two COOH-Terminal Truncated Cytoplasmic Forms of Topoisomerase IIα in a VP-16-Selected Lung Cancer Cell Line Result from Partial Gene Deletion and Alternative Splicing

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Abstract
Topoisomerase IIα is a nuclear enzyme involved in chromosome segregation and other essential cellular processes. It is also the target of several clinically important antineoplastic agents such as the epipodophyllotoxin, VP-16 (etoposide). We have previously described a VP-16-selected lung cancer cell line, H209/V6, that expresses reduced levels of two species of topoisomerase IIα-related mRNAs and a catalytically active, predominantly cytoplasmic topoisomerase IIα-related protein that is 10 kDa smaller than the wild-type protein [Mirski, S. E. L., et al. (1993) Cancer Res. 53, 4866−4873; Feldhoff, P. W. et al. (1994) Cancer Res. 54, 756−762]. The smaller H209/V6 4.8 kb mRNA is missing 988 nucleotides of contiguous coding and non-coding sequence at its 3‘ end resulting in an mRNA predicted to encode a truncated polypeptide missing three previously unrecognized potential COOH-proximal bipartite nuclear localization signals [Mirski, S. E. L., & Cole, S. P. C. (1995) Cancer Res. 55, 2129−2134]. We have now determined the structure of the larger 6.2 kb topoisomerase IIα-related mRNA and show that it is missing 684 nucleotides of contiguous 3‘ coding and non-coding sequence between nucleotide positions 4267 and 4951. This sequence is replaced by 847 nucleotides of new sequence, containing an in-frame stop codon after 41 nucleotides. The translation product of the 6.2 kb mRNA is predicted to contain 13 new amino acids replacing the COOH-terminal 109 residues of wild-type topoisomerase IIα, producing a truncated polypeptide of approximately 160 kDa. Immunoblot analyses using antisera against the unique COOH-terminal 13 and 34 amino acids encoded by H209/V6 6.2 kb and 4.8 kb mRNAs, respectively, confirmed that both mRNAs are translated. Restriction enzyme analysis and sequencing of the 3‘-proximal region of the TOP2A gene in the H209 and H209/V6 DNA revealed that a partial deletion has occurred in H209/V6 and the novel sequence identified in the H209/V6 6.2 kb mRNA is derived from the adjacent 3‘ intron as a consequence of read-through at a concensus splice donor site. These observations suggest a mechanism for the generation of the two mutant topoisomerase IIα mRNAs in H209/V6 cells and provide the first reported example of a drug resistant cell line containing two different cytoplasmic forms of topoisomerase IIα.

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