Poster abstracts
Poster number 59 submitted by Xinyu Zhao
PCF11-Mediated Regulation of TOP2α Intron 19 Intronic Polyadenylation in Acquired Etoposide-Resistant K562 Leukemia Cells
Xinyu Zhao (Division of Pharmaceutics and Pharmaceutics, The Ohio State University College of Pharmacy), Xinyi Wang, Sydney R. Cassel, Sarah E. Eaton (Division of Pharmaceutics and Pharmaceutics, The Ohio State University College of Pharmacy), Junan Li (Division of Pharmacy Practice and Science, The Ohio State University College of Pharmacy), Sophie Harvey, Terry S. Elton (Division of Pharmacy Education and Innovation, The Ohio State University College of Pharmacy), Jack C. Yalowich (Division of Pharmaceutics and Pharmaceutics, The Ohio State University College of Pharmacy)
Abstract:
DNA topoisomerase IIα (TOP2α, 170 kDa; TOP2α/170) is an essential enzyme that ensures proper chromosome segregation by generating transient double-stranded DNA breaks. It is also a major target of DNA-damaging chemotherapeutic agents such as etoposide. However, the clinical efficacy of TOP2α-targeting drugs can be undermined by acquired resistance. Our previous studies identified reduced TOP2α/170 expression in an etoposide-resistant human leukemia K562 subline, designated K/VP.5 cells. We demonstrated that the observed decrease in TOP2α/170 results from a TOP2α intronic polyadenylation (IPA) event. As a consequence of IPA, a C-terminally truncated isoform, TOP2α/90 (90 kDa), is expressed at very high levels in K/VP.5 cells. Using 3′-Rapid Amplification of cDNA Ends (3′-RACE), we demonstrated that TOP2α/90 is translated from a transcript generated by IPA at a cryptic polyadenylation site #3 (PAS #3) located within intron 19 of the TOP2α gene. We hypothesized that aberrant expression of cleavage and polyadenylation/splicing factors may drive TOP2α/90 IPA in K/VP.5 cells. Importantly, proteomics, qPCR, and immunoblot/Westerns demonstrated that the PCF11 Cleavage and Polyadenylation Factor Subunit (PCF11) is overexpressed in K/VP.5 cells. Notably, siRNA-mediated knockdown of PCF11 in K/VP.5 cells resulted in decreased TOP2α/90 mRNA/protein with a concomitant increase in TOP2α/170 mRNA/protein, ultimately enhancing etoposide-induced DNA damage and growth inhibition. In contrast, transiently forced and stably expressed PCF11 in parental K562 cells increased TOP2α/90 mRNA/protein with a concomitant decrease in TOP2α/170 mRNA/protein which resulted in decreased DNA damage and growth inhibition. Together, these results strongly suggest that PCF11 is playing an important role in mediating TOP2α/90 IPA in K/VP.5 cells.
Keywords: DNA Topoisomerase II-targeted drugs, Intronic polyadenylation, PCF11 Cleavage and Polyadenylation Factor Subunit