Acquired resistance to the DNA topoisomerase II (topo II)-targeting agent, etoposide (VP-16) has been shown previously in this lab to be associated with decreased levels of topo II mRNA and protein in a K562 clone, K/VP.5. Utilizing exon junctional Taqman hydrolysis probes, real-time quantitative PCR (qPCR) revealed ∼2-fold decrease in topo IIα mRNA expression in K/VP.5 compared to parental K562 cells when probes were downstream of exon 19. However, use of probes upstream of exon 19 revealed a small but statistically significant increase in topo IIα mRNA in K/VP.5 compared to K562 cells. In contrast, expression of topo IIß mRNA was reduced in K/VP.5 compared to K562 cells in all 5’ and 3’ regions of the gene queried. RNA stability studies using the RNA polymerase II inhibitor DRB and 4 qPCR probes, regionally targeted to 3’ and 5’ domains, revealed similar topo IIα mRNA half-lives in K562 cells (6.55 hr [extreme 3’ probe] to 7.96 hr [extreme 5’ probe]) but a statistically significant increase in topo IIα mRNA half-lives in K/VP.5 cells (7.21 hr [extreme 3’ probe] to 11.86 hr [extreme 5’ probe (p=0.034)]). The differential RNA decay rate in K/VP.5 cells suggested alterations in exo- or endo-nuclease expression/function in resistant cells and/or alternative splicing events generating transcripts of different size and stability characteristics. Experiments using Rapid Amplification of cDNA Ends (3’-RACE) were consistent with loss of ∼3 kb of the total 5.8 kb topo IIα in K/VP.5 cells. PCR products derived from this truncated transcript were sequenced, revealing that the 5’ splice site of intron 19 is suppressed, resulting in the retention of ∼300 nt of intronic sequence in the mature mRNA, and the use of an alternative polyadenylation site. The retained portion of intron 19 contains an in-frame stop codon, as well as the consensus AAUAAA hexamer of the poly(A) signal. As a result of alternative 3’ end processing, exons 20-35 of the topo IIα gene are not included in the truncated transcript. Since the codon corresponding to the topo IIα active site tyrosine is contained in Exon 20 (Tyr 705), translation of the alternatively spliced transcript likely produces a non-functional protein. Surprisingly, the truncated transcript is also present in K562 cells, albeit at a greatly reduced level compared to K/VP.5, suggesting that alternative polyadenylation is a normal mechanism to regulate topo IIα expression. Taken together results indicate that posttranscriptional alterations in topo IIα may account, in part, for acquired resistance to VP-16, and potentially for regulation of topo II expression that may dictate intrinsic chemosensitivity as well.

Citation Format: Lucas D. Serdar, Ragu Kanagasabai, Jack C. Yalowich. Alternative RNA processing leads to decreased DNA topoisomerase IIα in etoposide (VP-16) resistant human leukemia K562 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 786. doi:10.1158/1538-7445.AM2014-786