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.

DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2α/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2α/90 (786 aa) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2α/170 isoform (1531 aa). Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2α/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents.

Capsicodendrin from Cinnamosma fragrans Exhibits Antiproliferative and Cytotoxic Activity in Human Leukemia Cells: Modulation by Glutathione.

Capsicodendrin (CPCD, 1), an epimeric mixture of a dimeric drimane-type sesquiterpene, is one of the major compounds present in the three endemic species of Madagascan traditional chemopreventive plants: Cinnamosma species ( C. fragrans, C. macrocarpa, and C. madagascariensis). Despite the popular use of Cinnamosma in Madagascan traditional medicine and the reported antiproliferative properties of CPCD, elucidation of its mechanism(s) of action is still to be accomplished. In the present study, CPCD at low micromolar concentrations was cytotoxic and induced apoptosis in human myeloid leukemia cells in a time- and concentration-dependent manner. The activity of CPCD in HL-60 and K562 cells was modulated by glutathione (GSH), since depletion of this intracellular thiol-based antioxidant with buthionine sulfoximine resulted in significantly ( p < 0.05) greater potency in antiproliferation assays. GSH depletion also significantly potentiated the cytotoxic activity in CPCD-treated human HL-60 cells. Single-cell gel electrophoresis (Comet) assays revealed that GSH depletion in HL-60 cells enhanced the formation of DNA strand breaks in the presence of CPCD. Although CPCD does not contain an obvious Michael acceptor in its structure, 1H NMR analyses indicated that cinnamodial (2), a monomer of CPCD, was formed within a few hours when dissolved in DMSO- d6 and interacts with GSH to form a covalent bond via Michael addition at the C-7 carbon. Together the results strongly suggest that 2 is responsible for the DNA-damaging, pro-apoptotic, and cytotoxic effects of CPCD and that depletion of GSH enhances overall activity by diminishing covalent interaction between GSH and this 2-alkenal decomposition product of CPCD.

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.

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 (Tyr805) 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.