Alternative RNA Processing of Topoisomerase IIα in Etoposide-Resistant Human Leukemia K562 Cells: Intron Retention Results in a Novel C-Terminal Truncated 90-kDa Isoform

Abstract

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.

The Novel C-terminal Truncated 90-kDa Isoform of Topoisomerase IIα (TOP2α/90) Is a Determinant of Etoposide Resistance in K562 Leukemia Cells via Heterodimerization with the TOP2α/170 Isoform

“…TOP2a/170 is decreased in K/VP.5 cells to approximately one-fifth the level found in parental K562 cells (Kanagasabai et al, 2017). In contrast, K/VP.5 cells express approximately threefold greater levels of TOP2a/90 compared with parental K562 cells (Kanagasabai et al, 2017).…”

“…Immunoassays. Cellular, cytoplasmic, nuclear, or tissue extracts were subjected to Western blot analysis as previously described (Kanagasabai et al, 2017). Unless otherwise noted, 12.5 mg of protein was loaded into each well.…”

“…no. ab74715; Abcam, Cambridge, MA; used at 1:1000 dilution) (Kanagasabai et al, 2017), the anti-rabbit TOP2a/170 C-terminal specific antibody (generated against a recombinant 70-kDa fragment corresponding to only the TOP2a/170 C-terminal sequence; used at 1:5000 dilution) (Ritke et al, 1994a), the anti-rabbit TOP2a/90 specific antibody (raised against a synthetic peptide corresponding to the C-terminal sequence of TOP2a/90 encoded by the exon 19/intron 19 "read-through"; used at 1:1000 dilution or 1:250) (Kanagasabai et al, 2017), a mouse monoclonal b-actin antibody (cat. no.…”

“…Recently, we demonstrated that both parental human leukemia K562 cells and a cloned K562 cell line (K/VP.5 cells) with acquired resistance to etoposide (Ritke and Yalowich, 1993;Ritke et al, 1994b) express two TOP2a isoforms, TOP2a/170 and a novel 90 kDa protein (i.e., TOP2a/90) (Kanagasabai et al, 2017). TOP2a/170 is decreased in K/VP.5 cells to approximately one-fifth the level found in parental K562 cells (Kanagasabai et al, 2017). In contrast, K/VP.5 cells express approximately threefold greater levels of TOP2a/90 compared with parental K562 cells (Kanagasabai et al, 2017).…”

“…As a result of intron 19 retention, TOP2a/90 is missing the C-terminal 770 aa present in TOP2a/170 (1531 aa), which are replaced by 25 unique aa encoded by translation of the exon 19/intron 19 "read-through." Therefore, TOP2a/90 lacks active site Tyr805, canonical dimerization domains (DD) (aa 1053-1069 and 1121-1143), and the nuclear localization signals (NLS) (aa 1259-1296 and 1454-1497) found in the TOP2a/170 isoform (Kanagasabai et al, 2017) (Fig. 1).…”