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.

DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), 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. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2/90 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 TOP2/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2/90 expression decreases drug-induced TOP2-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2/170. Alternative processing of TOP2 pre-mRNA, and subsequent synthesis of TOP2/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2/170-DNA covalent complexes in response to TOP2-targeting agents.

Two new (1 and 2) and four known arylnaphthalene lignan lactones (3–6) were isolated from different plant parts of Phyllanthus poilanei collected in Vietnam, with two further known analogues (7 and 8) being prepared from phyllanthusmin C (4). The structures of the new compounds were determined by interpretation of their spectroscopic data and by chemical methods, and the structure of phyllanthusmin D (1) was confirmed by single-crystal X-ray diffraction analysis. Several of these arylnaphthalene lignan lactones were cytotoxic toward HT-29 human colon cancer cells, with compounds 1 and 7-O-[(2,3,4-tri-O-acetyl)-α-l-arabinopyranosyl)]diphyllin (7) found to be the most potent, exhibiting IC50 values of 170 and 110 nM, respectively. Compound 1 showed activity when tested in an in vivo hollow fiber assay using HT-29 cells implanted in immunodeficient NCr nu/nu mice. Mechanistic studies showed that this compound mediated its cytotoxic effects by inducing tumor cell apoptosis through activation of caspase-3, but it did not inhibit DNA topoisomerase IIα activity.

Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity. Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity. Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity. Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding. Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9. These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction. Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments. Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIa as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phosphohistone gH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzymeto-DNA assay; and to display cross-resistance in etoposideresistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10-to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the b isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIa in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIa over topoisomerase IIb.

Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.

Transcriptional activation of p53 target genes, due to DNA damage, causes either apoptosis or survival by cell cycle arrest and DNA repair. However, the regulators of the choice between cell death and survival signaling have not been completely elucidated. Here, we report that human adenocarcinoma cells (MCF-7) survive UV-induced DNA damage by heat shock protein 27 (Hsp27)-assisted Akt/p21 phosphorylation/translocation. Protein levels of the p53 target genes, such as p21, Bcl-2, p38MAPK, and Akt, showed a positive correlation to Hsp27 level during 48 hours postirradiation, whereas p53 expression increased initially but started decreasing after 12 hours. Hsp27 prevented the G 1 -S phase cell cycle arrest, observed after 8 hours of post-UV irradiation, and PARP-1 cleavage was inhibited. Conversely, silencing Hsp27 enhanced G 1 -S arrest and cell death. Moreover, use of either Hsp27 or Akt small interference RNA reduced p21 phosphorylation and enhanced its retention in nuclei even after 48 hours postirradiation, resulting in enhanced cell death. Our results showed that Hsp27 expression and its direct chaperoning interaction increases Akt stability, and p21 phosphorylation and nuclear-to-cytoplasm translocation, both essential effects for the survival of UV-induced DNA-damaged cells. We conclude that the role of Hsp27 in cancer is not only for enhanced p53 proteolysis per se, rather it is also a critical determinant in p21 phosphorylation and translocation. Mol Cancer Res; 8(10); 1399-412. ©2010 AACR.

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor B (NF-B) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-B (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/ adr cells, and induced cell death by increased G 2 /M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-B-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-B level will be a potential approach to circumvent drug resistance in breast cancer.Development of a multidrug-resistant phenotype is a major obstacle to the successful treatment of breast cancer (1, 2). There are two major pathways by which cancer cells acquire drug resistance, drug efflux and direct suppression of apoptosis. Drug efflux is due to increased plasma membrane accumulation of various ATP-binding cassette (ABC) 2 transporters, including ABCB1, also known as P-glycoprotein (P-gp), which extrude the internalized drugs from the cancer cells (3-5). Various approaches have been reported to overcome the drug efflux, including pharmacological inhibition of ABCs and modulation of endogenous regulators of MDR1 (6). Drug resistance is also acquired via direct suppression of apoptotic pathways due to accumulation of mutant p53 (mutp53) with "gain of function" (7, 8) and increased expression of antiapoptotic proteins, such as BCl-2 (4). In addition to abrogating the proapoptotic function of wild type p53 (wtp53), these p53 missense mutations have been shown to have unusual gain of function properties both in vitro and in vivo (9 -11). Recent studies have established a link between these two pathways of drug resistance (12).Mutant p53 has been found to be the prominent common mediator of both pathways (11). Wild type p53 is generally known to repress the expression of the MDR1 gene, which codes for the ABC protein P-gp through interaction with basal transcription facto...