DNA topoisomerase IIα (TOP2α) is a prominent target for anticancer drugs whose clinical efficacy is often limited by chemoresistance. Using antibody specific for the N-terminal of TOP2α, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, present in K562 leukemia cells and in an acquired etoposide (VP-16)-resistant clone (K/VP.5). TOP2α/90 expression was dramatically increased in etoposide-resistant K/VP.5 compared with parental K562 cells. We hypothesized that TOP2α/90 was the translation product of novel alternatively processed pre-mRNA, confirmed by 3'-rapid amplification of cDNA ends, polymerase chain reaction, and sequencing. TOP2α/90 mRNA includes retained intron 19, which harbors an in-frame stop codon, and two consensus poly(A) sites. The processed transcript is polyadenylated. TOP2α/90 mRNA encodes a 90,076-Da translation product missing the C-terminal 770 amino acids of TOP2α/170, replaced by 25 unique amino acids through translation of the exon 19/intron 19 read-through. Immunoassays, utilizing antisera raised against these unique amino acids, confirmed that TOP2α/90 is expressed in both cell types, with overexpression in K/VP.5 cells. Immunodetection of complex of enzyme-to-DNA and single-cell gel electrophoresis (Comet) assays demonstrated that K562 cells transfected with a TOP2α/90 expression plasmid exhibited reduced etoposide-mediated TOP2α-DNA covalent complexes and decreased etoposide-induced DNA damage, respectively, compared with similarly treated K562 cells transfected with empty vector. Because TOP2α/90 lacks the active site tyrosine (Tyr) of full-length TOP2α, these results strongly suggest that TOP2α/90 exhibits dominant-negative properties. Further studies are underway to characterize the mechanism(s) by which TOP2α/90 plays a role in acquired resistance to etoposide and other TOP2α targeting agents.

Nonsense-mediated mRNA decay (NMD) represents a eukaryotic quality control pathway that recognizes and rapidly degrades transcripts harbouring nonsense mutations to limit accumulation of non-functional and potentially toxic truncated polypeptides. A critical component of the NMD machinery is UPF1, an RNA helicase whose ATPase activity is essential for NMD, but for which the precise function and site of action remain unclear. We provide evidence that ATP hydrolysis by UPF1 is required for efficient translation termination and ribosome release at a premature termination codon. UPF1 ATPase mutants accumulate 3′ RNA decay fragments harbouring a ribosome stalled during premature termination that impedes complete degradation of the mRNA. The ability of UPF1 to impinge on premature termination, moreover, requires ATP-binding, RNA-binding and NMD cofactors UPF2 and UPF3. Our results reveal that ATP hydrolysis by UPF1 modulates a functional interaction between the NMD machinery and terminating ribosomes necessary for targeting substrates to accelerated degradation.