Two COOH-terminal truncated cytoplasmic forms of topoisomerase II alpha in a VP-16-selected lung cancer cell line result from partial gene deletion and alternative splicing.
Topoisomerase II alpha 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 II alpha-related mRNAs and a catalytically active, predominantly cytoplasmic topoisomerase II alpha-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 II alpha-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 II alpha, 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 II alpha mRNAs in H209/V6 cells and provide the first reported example of a drug resistant cell line containing two different cytoplasmic forms of topoisomerase II alpha.
Yu
Q
Q
Department of Pharmacology & Toxicology, Queen's University, Kingston, Ontario, Canada.
Mirski
S E
SE
Sparks
K E
KE
Cole
S P
SP
D000595:Amino Acid Sequence / Q000187:drug effects; D001482:Base Composition; D001483:Base Sequence / Q000187:drug effects; D015152:Blotting, Northern; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D005047:Etoposide / Q000494:pharmacology*; D017353:Gene Deletion; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D005047:Etoposide; D012194:RNA-Directed DNA Polymerase; D004250:DNA Topoisomerases, Type II