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.

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Execution of apoptotic program in mitochondria is associated with accumulation of cardiolipin peroxidation products required for the release of proapoptotic factors into the cytosol. This suggests that lipid antioxidants capable of inhibiting cardiolipin peroxidation may act as antiapoptotic agents. Etoposide, a widely used antitumor drug and a topoisomerase II inhibitor, is a prototypical inducer of apoptosis and, at the same time, an effective lipid radical scavenger and lipid antioxidant. Here, we demonstrate that cardiolipin oxidation during apoptosis is realized not via a random cardiolipin peroxidation mechanism but rather proceeds as a result of peroxidase reaction in a tight cytochrome c/cardiolipin complex that restrains interactions of etoposide with radical intermediates generated in the course of the reaction. Using low-temperature and ambient-temperature electron paramagnetic resonance spectroscopy of H 2 O 2 -induced protein-derived (tyrosyl) radicals and etoposide phenoxyl radicals, respectively, we established that cardiolipin peroxidation and etoposide oxidation by cytochrome c/cardiolipin complex takes place predominantly on protein-derived radicals of cytochrome c. We further show that etoposide can inhibit cytochrome c-catalyzed oxidation of cardiolipin competing with it as a peroxidase substrate. Peroxidase reaction of cytochrome c/cardiolipin complexes causes cross-linking and oligomerization of cytochrome c. With nonoxidizable tetraoleoylcardiolipin, the cross-linking occurs via dityrosine formation, whereas bifunctional lipid oxidation products generated from tetralinoleoyl-cardiolipin participate in the production of high molecular weight protein aggregates. Protein aggregation is effectively inhibited by etoposide. The inhibition of cardiolipin peroxidation by etoposide, however, is realized at far higher concentrations than those at which it induces apoptotic cell death. Thus, oxidation of cardiolipin by the cytochrome c/cardiolipin peroxidase complex, which is essential for apoptosis, is not inhibited by proapoptotic concentrations of the drug.Mitochondria play a central role in the execution of apoptotic program realized through intrinsic mechanisms and extrinsic pathways in type II cells (Scaffidi et al., 1998). It is well-documented that one of the early mitochondrial responses to proapoptotic stimuli is the generation of reactive oxygen species (ROS) (Raha and Robinson, 2001). Whereas the overall significance of ROS production in apoptosis has been established by its inhibition by different antioxidant enzymes and free radical scavengers (Nomura et al., 1999;Genova et al., 2003), specific ROS-dependent mechanisms of apoptosis are still elusive. We have demonstrated recently that cytochrome c acts as a redox catalyst in oxidizing a mitochondria-specific phospholipid, cardiolipin, thus facilitating the accumulation of cardiolipin hydroperoxides (CL-OOH) required for the release of proapoptotic factors from mitochondria into the cytosol . This suggests that free radical scave...