Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform.

Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and ß), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIß revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.

The analysis of protein recruitment to laser microirradiation-induced DNA damage in live cells: Best practices for data analysis.

Laser microirradiation coupled with live-cell fluorescence microscopy is a powerful technique that has been used widely in studying the recruitment and retention of proteins at sites of DNA damage. Results obtained from this technique can be found in published works by both seasoned and infrequent users of microscopy. However, like many other microscopy-based techniques, the presentation of data from laser microirradiation experiments is inconsistent; papers report a wide assortment of analytic techniques, not all of which result in accurate and/or appropriate representation of the data. In addition to the varied methods of analysis, experimental and analytical details are commonly under-reported. Consequently, publications reporting data from laser microirradiation coupled with fluorescence microscopy experiments need to be carefully and critically assessed by readers. Here, we undertake a systematic investigation of commonly reported corrections used in the analysis of laser microirradiation data. We validate the critical need to correct data for photobleaching and we identify key experimental parameters that must be accounted for when presenting data from laser microirradiation experiments. Furthermore, we propose a straightforward, four-step analytical protocol that can readily be applied across platforms and that aims to improve the quality of data reporting in the DNA damage field.

M011L-deficient oncolytic myxoma virus induces apoptosis in brain tumor-initiating cells and enhances survival in a novel immunocompetent mouse model of glioblastoma.

BACKGROUND: Myxoma virus (MYXV) is a promising oncolytic agent and is highly effective against immortalized glioma cells but less effective against brain tumor initiating cells (BTICs), which are believed to mediate glioma development/recurrence. MYXV encodes various proteins to attenuate host cell apoptosis, including an antiapoptotic Bcl-2 homologue known as M011L. Such proteins may limit the ability of MYXV to kill BTICs, which have heightened resistance to apoptosis. We hypothesized that infecting BTICs with an M011L-deficient MYXV construct would overcome BTIC resistance to MYXV. METHODS: We used patient-derived BTICs to evaluate the efficacy of M011L knockout virus (vMyx-M011L-KO) versus wild-type MYXV (vMyx-WT) and characterized the mechanism of virus-induced cell death in vitro. To extend our findings in a novel immunocompetent animal model, we derived, cultured, and characterized a C57Bl/6J murine BTIC (mBTIC0309) from a spontaneous murine glioma and evaluated vMyx-M011L-KO efficacy with and without temozolomide (TMZ) in mBTIC0309-bearing mice. RESULTS: We demonstrated that vMyx-M011L-KO induces apoptosis in BTICs, dramatically increasing sensitivity to the virus. vMyx-WT failed to induce apoptosis as M011L protein prevented Bax activation and cytochrome c release. In vivo, intracranial implantation of mBTIC0309 generated tumors that closely recapitulated the pathological and molecular profile of human gliomas. Treatment of tumor-bearing mice with vMyx-M011L-KO significantly prolonged survival in immunocompetent-but not immunodeficient-mouse models, an effect that is significantly enhanced in combination with TMZ. CONCLUSIONS: Our data suggest that vMyx-M011L-KO is an effective, well-tolerated, proapoptotic oncolytic virus and a strong candidate for clinical translation.

DNA damage-induced activation of ATM and ATM-dependent signaling pathways.

Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to ionizing radiation. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage response pathways, most notably cell cycle checkpoints. In this review, we describe recent developments in our understanding of the mechanism of activation of ATM and its downstream signaling pathways, and explore whether DNA double-strand breaks are the sole activators of ATM and ATM-dependent signaling pathways.

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX.

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here, we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20, 46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 1981, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets.

In vitro screen of a small molecule inhibitor drug library identifies multiple compounds that synergize with oncolytic myxoma virus against human brain tumor-initiating cells.

BACKGROUND: Brain tumor-initiating cells (BTICs) are stem-like cells hypothesized to form a disease reservoir that mediates tumor recurrence in high-grade gliomas. Oncolytic virotherapy uses replication-competent viruses to target and kill malignant cells and has been evaluated in clinic for glioma therapy with limited results. Myxoma virus (MyxV) is a safe and highly effective oncolytic virus (OV) in conventional glioma models but, as seen with other OVs, is only modestly effective for patient-derived BTICs. The objective of this study was to determine whether MyxV treatment against human BTICs could be improved by combining chemotherapeutics and virotherapy. METHODS: A 73-compound library of drug candidates in clinical use or preclinical development was screened to identify compounds that sensitize human BTICs to MyxV treatment in vitro, and synergy was evaluated mathematically in lead compounds using Chou-Talalay analyses. The effects of combination therapy on viral gene expression and viral replication were also assessed. RESULTS: Eleven compounds that enhance MyxV efficacy were identified, and 6 were shown to synergize with the virus using Chou-Talalay analyses. Four of the synergistic compounds were shown to significantly increase viral gene expression, indicating a potential mechanism for synergy. Three highly synergistic compounds (axitinib, a VEGFR inhibitor; rofecoxib, a cyclooxygenase-2 inhibitor; and pemetrexed, a folate anti-metabolite) belong to classes of compounds that have not been previously shown to synergize with oncolytic viruses in vitro. CONCLUSIONS: This study has identified multiple novel drug candidates that synergistically improve MyxV efficacy in a preclinical BTIC glioma model.

Structural determinants of the catalytic inhibition of human topoisomerase IIα by salicylate analogs and salicylate-based drugs.

We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.3) that preferentially targets the alpha isoform by interfering with topo II-mediated DNA cleavage. Many pharmaceuticals and compounds found in foods are salicylate-based. We have now investigated whether these are also catalytic inhibitors of topo II and the structural determinants modulating these effects. We have determined that a number of hydroxylated benzoic acids attenuate doxorubicin-induced DNA damage signaling mediated by the ATM protein kinase and inhibit topo II decatenation activity in vitro with varying potencies. Based on the chemical structures of these and other derivatives, we identified unique properties influencing topo II inhibition, including the importance of substitutions at the 2'- and 5'-positions. We extended our findings to a number of salicylate-based pharmaceuticals including sulfasalazine and diflunisal and found that both were effective at attenuating doxorubicin-induced DNA damage signaling, topo II DNA decatenation and they blocked stabilization of doxorubicin-induced topo II cleavable complexes in cells. In a manner similar to salicylate, we determined that these agents inhibit topo II-mediated DNA cleavage. This was accompanied by a concomitant decrease in topo II-mediated ATP-hydrolysis. Taken together, these findings reveal a novel function for the broader class of salicylate-related compounds and highlight the need for additional studies into whether they may impact the efficacy of chemotherapy regimens that include topo II poisons.

Sodium salicylate is a novel catalytic inhibitor of human DNA topoisomerase II alpha.

We have previously reported that pretreatment of human lymphoblastoid cells with the hydroxyl radical scavenger, N-acetyl cysteine, attenuates doxorubicin-induced DNA damage signalling through the ATM protein kinase. We sought to extend these studies to examine the effects of other hydroxyl radical scavengers in human breast cancer cells. Using MCF-7 cells, we observed that doxorubicin treatment triggered autophosphorylation of ATM on serine 1981 and the ATM-dependent activation of its downstream effectors p53, Chk2, and SMC1. Furthermore, we demonstrate that this effect was attenuated by pretreatment of cells with the hydroxyl radical scavengers sodium benzoate, sodium salicylate and, to a lesser extent, N-acetyl cysteine, but not Trolox™. Intriguingly, these effects were independent of doxorubicin's ability to redox cycle, were observed with multiple classes of topoisomerase II poisons, but did not represent a general damage-attenuating response. In addition, the observed effects were independent of the ability of sodium salicylate to inhibit cyclooxygenase-2 or NFκB. We demonstrate that sodium salicylate prevented doxorubicin-induced DNA double-strand break generation, which was attributable to inhibition of doxorubicin-stabilized topoisomerase IIα-DNA cleavable complex formation in vivo. Using topoisomerase IIα-DNA cleavage and decatenation assays, we determined that sodium salicylate is a catalytic inhibitor of topoisomerase IIα. Consistent with the observed inhibition of double-strand break formation, pretreatment of cells with sodium salicylate attenuated doxorubicin and etoposide cytotoxicity. These results demonstrate a novel mechanism of action for sodium salicylate and suggest that further study on the mechanism of topoisomerase II inhibition and the effects of related therapeutics on doxorubicin and etoposide cytotoxicity are warranted.

Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis.

In mammals, stress elicits a stereotyped endocrine response that requires an increase in the activity of hypothalamic parvocellular neuroendocrine neurons. The output of these cells is normally constrained by powerful GABA-mediated synaptic inhibition. We found that acute restraint stress in rats released the system from inhibitory synaptic drive in vivo by down-regulating the transmembrane anion transporter KCC2. This manifested as a depolarizing shift in the reversal potential of GABA(A)-mediated synaptic currents that rendered GABA inputs largely ineffective. Notably, repetitive activation of GABA synapses after stress resulted in a more rapid collapse of the anion gradient and was sufficient to increase the activity of neuroendocrine cells. Our data indicate that hypothalamic neurons integrate psychological cues to mount the endocrine response to stress by regulating anion gradients.

Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species.

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser(343)), SMC1 (Ser(957)), Chk1 (Ser(317) and Ser(345)), and Chk2 (Ser(33/35) and Thr(68)). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.

Cloning and Characterization of the Murine and Rat mrp1 Promoter Regions.

The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents. The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression. As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene. All three promoters are GC-rich, TATA-less, and CAAT-less. Conservation of sequence between rodent and human promoters is limited to a proximal region of 100 nucleotides containing binding sites for members of the Sp1 family and a putative activator protein-1 element. The 5'-untranslated region (UTR) of human MRP1 contains an insertion of approximately 160 nucleotides comprising a GCC-triplet repeat and a GC-rich tandem repeat that is absent from the rodent sequences. Transient transfection analyses demonstrated that the conserved GC-boxes of all three genes are the major determinants of basal activity. Based on electrophoretic mobility shift assays, each GC-box can be bound by Sp1 or Sp3. Unlike the rodent genes, the human MRP1 5'UTR also binds Sp1 but not Sp3, and the human promoter retains substantial activity even in the absence of the conserved GC-boxes. Finally, we show that the tumor suppressor protein p53 can repress the human and rodent promoters by a mechanism that is independent of the Sp1 elements.