Abstract

DNA topoisomerase IIα (TOP2α) is a prominent target for clinically utilized anticancer drugs. However, the efficacy of these agents is limited by chemoresistance. We have previously characterized acquired resistance to etoposide (VP-16) in a cloned human K562 leukemia cell line, K/VP.5, containing reduced TOP2α. In the present study, using an antibody specific for the amino-terminus of TOP2α protein, immunoassays indicated the existence of two TOP2α isoforms, 170 and 90 kDa, in both parental K562 and K/VP.5 cell lysates. TOP2α/90 was dramatically increased in VP-16-resistant K/VP.5 compared to parental K562 cells. An antibody specific for the carboxy-terminus revealed only the TOP2α/170 protein which was reduced in K/VP.5 compared to K562 cells. We hypothesized that TOP2α/90 was the translation product of a novel alternatively processed pre-mRNA, subsequently confirmed by 3′-RACE, PCR, and sequencing studies. This novel mRNA harbors an in-frame stop codon, consensus poly(A) sites within retained intron 19, and encodes a predicted 90,076 Da translation product. TOP2α/90 is missing the carboxyl-terminal 770 amino acids present in TOP2α/170 which are replaced by 25 unique amino acids encoded by 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. Since TOP2α/90 does not harbor the active site tyrosine (Tyr805) of full length TOP2α, this protein may exhibit dominant-negative properties and/or impact the nuclear levels/accumulation of active TOP2α/170. Further studies are underway to characterize the role of this truncated form of TOP2α.