DNA Damage-inducing Agents Research Articles

Abstract 736: Combining PD1- and CTLA4-inhibiting antibodies with cisplatin or PARP inhibition in an attempt to eradicate BRCA1-deficient mouse mammary tumors

Abstract Solid tumors are usually not eradicated by conventional chemotherapy resulting in disease relapse and mortality. An intriguing example is BRCA-associated breast cancer. A genetically engineered mouse model for BRCA1-associated breast cancer (K14Cre;Brca1F/F;p53F/F), which highly resembles disease in human patients, can be used to study therapy escape and residual disease in BRCA-deficient tumors. Due to the BRCA inactivation, the tumors that arise in this model lack homologous recombination-directed DNA repair, an Achilles heel that has provided a therapeutic opportunity to eradicate tumors with DNA damage-inducing agents. Despite repeated sensitivity some residual cancer cells escape the deadly effect of anticancer therapy and lead to disease relapse. A special histopathological feature of BRCA1-mutated tumors in both humans and mice is the presence of infiltrating lymphocytes. This may be explained by the high frequency of genomic alterations of BRCA1-mutated tumors resulting in the generation of many neo-antigens. Our hypothesis is that blocking inhibitory T-cell signaling, using antibodies directed against CTLA4 and PD1, may increase the activity of the T-cell compartment towards a diverse pool of antigens, and successfully eliminate residual tumor cells in combination with DNA damage-inducing drugs. In particular, we will present data of combining CTLA4- and PD1- targeting antibodies with PARP inhibition or cisplatin to eliminate residual tumor cells in this mouse model for BRCA1-mutated breast cancer. Citation Format: Sohvi Blatter, Charlotte Guyader, Aslı Küçükosmanoğlu, Stephan Freriks, Karin de Visser, Piet Borst, Sven Rottenberg. Combining PD1- and CTLA4-inhibiting antibodies with cisplatin or PARP inhibition in an attempt to eradicate BRCA1-deficient mouse mammary tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 736. doi:10.1158/1538-7445.AM2015-736

Identification and characterization of a novel gene encoding the NBS1 protein in Pyricularia oryzae.

The ascomycete Pyricularia oryzae (teleomorph: Magnaporthe oryzae) causes one of the most serious diseases known as rice blast. The Nijmegen breakage syndrome protein (NBS1) is essential for DNA repair; thus, we studied the P. oryzae NBS1 homolog (PoNBS1). A PoNBS1 null mutant exhibited high sensitivity to DNA damage-inducing agents. The mutant also exhibited the retarded hyphal growth, and induced abnormal conidial germination and shape, but showed normal appressorium formation. The phenotypes of the null mutant were complemented by introducing the cDNA of PoNBS1 driven by a TrpC promoter of Aspergillus nidulans. In addition, the null mutant similarly complemented with the PoNBS1 cDNA lacking the FHA domain that had a normal phenotype except for hyphal growth. These results suggest that PoNBS1 is involved in DNA repair and normal development in P. oryzae. Moreover, the FHA domain of PoNBS1 participates in normal hyphal growth.

Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents.

The Fanconi anemia (FA) pathway plays a key role in interstrand crosslink (ICL) repair and maintenance of the genomic stability, while inhibition of this pathway may sensitize cancer cells to DNA ICL agents and ionizing radiation (IR). The active FA core complex acts as an E3 ligase to monoubiquitinate FANCD2, which is a functional readout of an activated FA pathway. In the present study, we aimed to identify FANCD2-targeting agents, and found that the natural compound celastrol induced degradation of FANCD2 through the ubiquitin–proteasome pathway. We demonstrated that celastrol downregulated the basal and DNA damaging agent-induced monoubiquitination of FANCD2, followed by proteolytic degradation of the substrate. Furthermore, celastrol treatment abrogated the G2 checkpoint induced by IR, and enhanced the ICL agent-induced DNA damage and inhibitory effects on lung cancer cells through depletion of FANCD2. These results indicate that celastrol is a FANCD2 inhibitor that could interfere with the monoubiquitination and protein stability of FANCD2, providing a novel opportunity to develop FA pathway inhibitor and combinational therapy for malignant neoplasms.

Atomic Insight into the Altered O6-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer.

O6-methylguanine-DNA methyltransferase (MGMT) is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O6-methylguanine (mutagenic lesion) back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon151 resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS). The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the “protein stabilizing hing” mapped on C3-C4 cluster, preceding the active site.

Association between Polymorphisms of Antioxidant Gene (Mnsod, CAT and Gpx1) and Risk of Coronary Artery Disease

Background and Objective: Reactive oxygen species (ROS) been cited as one of the major causes of atherosclerosis and coronary artery disease (CAD) which are possible agents inducing DNA damage. Manganese superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase-1 (GPX1) have evolved to address primary defense against free radical mediated damage in mitochondria. The aim of this study was to delineate the association of MnSOD, CAT, and GPX1 genetic polymorphisms and risk of CAD in Taiwan. Methods: We conducted a case-control study with 657 participants who received quantitative coronary angiography to assess CAD at a medical center. Individuals with at least one coronary artery diameter stenosis = 50% were defined as cases (n = 447) and the others as controls (n = 210). Polymorphisms of MnSOD (Ala-9Val), CAT (C-262T), and GPX1 (Pro198Leu) genes were determined by PCR-RFLP methods. Multivariate logistic regression model was used to calculate the odds ratios (ORs) and 95% confidence intervals ...

Abstract B5: Identification of a role for REST in promoting preneoplastic events in a novel genetically engineered mouse model

Abstract Medulloblastoma is a pediatric cerebellar tumor that arises from undifferentiated neuronal stem/progenitor cells (NSCs). The Repressor Element-1 (RE1) Silencing Transcription Factor (REST) negatively regulates neurogenesis by repressing numerous genes involved in terminal neuronal differentiation. We previously showed that REST levels were abnormally elevated in medulloblastoma samples and that this was associated with poor prognosis for patients. Previous studies from two groups including ours showed that REST knockdown in human tumor cells blocked their tumorigenic potential in mouse orthotopic models, suggesting that REST was required for tumor maintenance. In addition, constitutively elevated expression of REST in neural progenitors in the context of Myc overexpression promoted tumorigenesis in the murine brain, implicating REST in tumor progression. However, a role for REST in tumor initiation has not been demonstrated. To examine if elevated REST expression in neuronal progenitors was sufficient to promote pre-neoplastic events such as proliferation, delayed differentiation and genetic instability, we generated a novel transgenic (Tg) mouse model in which human REST Tg expression could be conditionally elevated in the post-natal cerebellar progenitor cells. These studies revealed that cerebellar progenitors harvested from REST Tg mice, had sustained neurosphere formation and proliferation potential compared to progenitors from age-matched wild type littermates. Progenitors from REST Tg mice also exhibited substantially decreased expression of REST target neuronal differentiation genes such as Syn1 and SCG10 and displayed delayed onset of neurogenesis compared to wild type controls. Interestingly, progenitors with elevated REST expression had significantly perturbed telomere homeostasis associated with altered telomerase levels and increased genomic instability compared to wild type controls. The above findings were recapitulated in vivo where cerebella from REST Tg mice exhibited prolonged Ki-67 staining accompanied by lack of differentiation and accumulation of genetic instability. Furthermore, REST mediated genetic instability was associated with increased sensitivity to DNA damaging reagents such as etoposide compared to normal wild type progenitors. Thus, we provide the first demonstration of a role for elevated REST expression in neural progenitors in promoting tumor initiating events such as hyperproliferation, blockade of differentiation and telomere defects. We also show that the genetic instability contributes to the accelerated response of these cells to clinically relevant DNA damage-inducing agents. These observations may have therapeutic implications for high REST medulloblastomas. Citation Format: Tara Dobson, Ellen Busschers, Pete Taylor, Julianne Humphries, Sadhan Majumder, Vidya Gopalakrishnan. Identification of a role for REST in promoting preneoplastic events in a novel genetically engineered mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B5.

The Human SRCAP Chromatin Remodeling Complex Promotes DNA-End Resection

Repair of DNA double-strand breaks (DSBs) byhomologous recombination requires 5'-3' resection of the DSB ends. In vertebrates, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. However, how this process occurs within the context of chromatin is still not well understood. Here we identify the human SRCAP chromatin remodeling complex as a factor that promotes CtIP-dependent DNA-end resection. We show that SRCAP, which is mutated in Floating-Harbor syndrome, confers resistance to DNA damage-inducing agents and is recruited to DSBs. Moreover, we demonstrate that SRCAP is required for DNA-end resection, and thereby for recruitment of RPA and RAD51 to DSBs, and for the ensuing homologous recombination. Finally, we reveal that SRCAP forms a complex with CtIP and promotes accumulation of CtIP at DSBs through a mechanism involving its ATPase activity. Our study implicates the human SRCAP chromatin remodeling complex as a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs in the context of chromatin.

Genotoxic effect of doxorubicin–transferrin conjugate on human leukemia cells

Doxorubicin (DOX) is an effective anthracycline antibiotic against a wide spectrum of tumors and hematological malignancies. It mainly interacts with DNA, but can also generate reactive oxygen species (ROS), which damage cell components. Unfortunately, numerous side effects, such as severe cardiotoxicity and bone-marrow suppression, limit its use. To reduce this obstacle and improve its pharmacokinetics, we conjugated DOX to transferrin (TRF), a human plasma protein. In our study, we compared the effect of DOX and the doxorubicin–transferrin conjugate (DOX-TRF) on human leukemic lymphoblasts (CCRF-CEM), and on normal peripheral blood mononuclear cells (PBMC). In parallel, experiments were carried out on two human chronic myeloid leukemia (CML) cell lines derived from K562 cells, of which one was sensitive and the other resistant to doxorubicin (K562/DOX). By use of the alkaline comet assay, the effect of the agents on the induction of DNA damage in normal human cells and human leukemia cells was determined. Oxidative and alkylating DNA damage were assayed by a slightly modified comet assay that included the use of the DNA-repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). To investigate whether DNA breaks are the result of apoptosis, we examined the induction of DNA fragmentation visualized as oligosomal ladders after simple agarose electrophoresis under neutral conditions. Modifications of the genome induced by the different drugs were analyzed following assessment of the cell-cycle phase.The DOX-TRF conjugate caused more DNA damage than the free drug, the degree of DNA fragmentation being dependent on the duration of treatment and the cell type analyzed. With neutral agarose electrophoresis we showed that the test compounds caused the formation of a characteristic DNA-ladder pattern. Furthermore, the DOX-TRF conjugate generated a higher percentage of apoptotic cells in the subG1 fraction and blocked more cells in the G2/M phase of the cell cycle than did free DOX. In summary, both agents induced DNA damage in cancer cells, but the DOX-TRF conjugate generated more genotoxic effects and apoptosis than the unconjugated drug.

Methodological considerations for mutagen exposure in C. elegans

Maintenance of the genome requires the continual repair of DNA lesions. Exposure of nematodes to DNA damage-inducing agents is a powerful method to rapidly ascribe a role for specific genes in DNA repair and to define epistatic relationships to other repair genes which allows for the construction of repair pathways. Despite the extensive use of these agents, however, differences in dosing, timing, and handling makes it difficult to compare results across laboratories. We provide herein a consideration of the parameters that influence the results of these exposures and detailed protocols for the exposure to mutagenic inducing agents.

H3K9 histone methyltransferase G9a-mediated transcriptional activation of p21

We report that H3K9 HMTase G9a activates transcription of the cell cycle regulatory gene, p21, in p53-null H1299 cells. Positive regulation of p21 by G9a is independent of its HMTase activity. We demonstrate that G9a upregulates p21 via interaction with PCAF, and provide evidence that the activating complex is recruited to the p21 promoter upon DNA damage-inducing agent etoposide treatment. Our study suggests that G9a decreases proliferation and cell viability by increasing the level of p21-mediated apoptosis. Our results suggest that G9a functions as a coactivator for p21 transcription, and directs cells to undergo apoptosis.

MMSET/WHSC1 Enhances DNA Damage Repair Leading To An Increase In Resistance To Chemotherapeutic Agents

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, and is believed to be the driving factor in the pathogenesis of this subtype of MM. Overexpression of MMSET also occurs in solid cancers, including neuroblastoma, colon and prostate. MMSET overexpression in MM and prostate cells leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3). This altered epigenetic landscape is accompanied by changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones, including H3K36, to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET might play a role in DNA damage repair and response.To investigate the role of MMSET in DNA damage repair, we transfected U2OS cells with a linearized vector expressing a neomycin-resistant gene. In the presence of G418, only cells that are able to integrate this plasmid through non-homologous end joining (NHEJ) can survive. siRNA knockdown of MMSET led to a decrease in cell survival, suggesting that MMSET is necessary for efficient DNA repair. We also used U2OS cells engineered to express the AsiSI enzyme fused to an estrogen receptor hormone-binding domain. Upon tamoxifen treatment, double strand breaks (DSBs) are induced at multiple AsiSI recognition sites, accompanied by an increase in γH2AX foci. The extent of repair after AsiSI-induced damage was ascertained by the ability of a DNA fragment that spans a specific cut site to be PCR amplified. With MMSET knockdown, there was a >10 fold increase in unrepaired DNA. ChIP analysis showed that with the depletion of MMSET, γH2AX persisted at the cut site. ChIP for specific effectors of DNA damage showed a marked decrease of recruitment of CtIP and RAD51 to the DSB. However, immunoblot analysis showed that CtIP and RAD51 levels were drastically decreased with MMSET depletion, thus explaining the loss of their recruitment to DSBs. In contrast, XRCC4 levels were maintained with MMSET siRNA, but its recruitment to the DSB decreased. CtIP is important for both NHEJ and homologous recombination (HR), RAD51 is critical for HR, and XRCC4 is necessary for NHEJ, suggesting that MMSET is important in multiple pathways of DNA repair.To study the effect of MMSET in MM, we used the t(4;14)+ KMS11 cell line, NTKO, and genetically matched TKO cells in which the overexpressed MMSET allele was knocked out. NTKO cells have elevated levels of DNA damage at baseline, as measured by a comet assay and by the presence of elevated numbers of 53BP1-positive foci. Upon addition of the DNA damaging agent melphalan, NTKO cells showed increased damage as measured by an increase in the tail moment by the comet assay. Paradoxically, upon treatment of these cells with the DNA damaging agents, NTKO cells survived better than TKO cells. NTKO repaired DNA damage at an enhanced rate and continued to proliferate after a significant DNA damage insult, whereas TKO cells accumulated DNA damage and entered cell cycle arrest. We repleted TKO cells with constructs expressing either wild-type MMSET or an HMT-dead (Y1118A) isoform. Upon treatment, cells expressing the wild-type MMSET have showed enhanced DNA repair and continued proliferation after DNA damage, whereas cells expressing the HMT-dead protein repaired DNA damage more slowly and entered cell cycle arrest. The HMT activity of MMSET was critical for the induction of expression of genes required for multiple DNA repair pathways including CHEK2, DDB2, DDIT3, RAD51, and MRE11, again suggesting that MMSET modulates DNA repair by affecting expression of critical components of the repair machinery.The clinical relevance of these finds becomes more apparent in vivo. Luciferase-tagged KMS11 cells harboring doxycycline-inducible MMSET shRNA were injected into nude mice. After one week, mice were treated with doxycycline and injected with melphalan or saline. Knockdown of MMSET or melphalan treatment alone decreased tumor growth but eventually all mice had progressive disease. Only when MMSET was knocked down and chemotherapy given were the mice rendered tumor free. These findings indicate a new mechanism for the ability of MMSET to enhance DNA repair and identify the protein as a potential therapeutic target in MM and other cancers. Disclosures:No relevant conflicts of interest to declare.

Abstract PR11: Use of a DNA damage multiplex immunofluorescence assay to monitor pharmacodynamic markers following in vitro exposure to therapeutic agents.

Abstract Most current therapeutic approaches to cancer include agents which induce genotoxic damage, either through direct damaging effects on DNA or indirectly, by altering cellular checkpoint controls or by causing a synthetic lethal response in patients with genetic abnormalities in DNA damage repair pathways. As candidate agents or novel combination therapies come into the clinic, the development of multiplexed immunoassays that quantitatively measure several pharmacodynamic biomarkers of DNA repair could improve our understanding of the relationship between target engagement and therapeutic efficacy. We previously reported the initial development of a quantitative, multiplex immunofluorescence assay comprised of a nuclear marker and four DNA damage repair proteins (γH2AX, pNbs1S343, Rad51, and ERCC1) as a method for monitoring drug action in vitro and in vivo. Here, we report the use of this multiplex assay to measure the effect of a PARP inhibitor (BMN-673) on an isogenic pair of HR competent and incompetent cell lines. The multiplex immunofluorescence assay utilizes a cassette approach, with all validated primary antibodies conjugated directly to a fluorophore or hapten such that multiple antibodies raised in the same host species can be used without the requirement of an anti-species reporter. The cassette approach allows for rearrangement of the composition of markers in the multiplex assay based on the investigational agent or clinical trial in question. We will also report on the development and utility of additional DNA damage markers for future multiplex assays, including markers specific for ATR-activated repair pathways. We have developed an antibody to the pATR T1989 site and will demonstrate its use in immunofluorescence applications as a pharmacodyamic marker following in vitro exposure to DNA damage-inducing agents and an ATR inhibitor. Additionally, we show in vitro data which supports the use of phosphorylated checkpoint proteins (pChk1S345, pChk2T68) as downstream indicators of DNA damage in the multiplex format. These biomarkers, in conjunction with our previously-reported multiplex panel, will provide an effective technique to monitor DNA repair responses and checkpoint controls in the clinical evaluation of combination therapy approaches targeting DNA damage repair deficiencies. Funded by NCI Contract No. HHSN261200800001E. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR11. Citation Format: Allison M. Marrero, Deborah Wilsker, Scott M. Lawrence, Thomas D. Pfister, John Hirt, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Use of a DNA damage multiplex immunofluorescence assay to monitor pharmacodynamic markers following in vitro exposure to therapeutic agents. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR11.

Proteomic analysis of mismatch repair-mediated alkylating agent-induced DNA damage response

BackgroundMediating DNA damage-induced apoptosis is an important genome-maintenance function of the mismatch repair (MMR) system. Defects in MMR not only cause carcinogenesis, but also render cancer cells highly resistant to chemotherapeutics, including alkylating agents. To understand the mechanisms of MMR-mediated apoptosis and MMR-deficiency-caused drug resistance, we analyze a model alkylating agent (N-methyl-N’-nitro-N-nitrosoguanidine, MNNG)-induced changes in protein phosphorylation and abundance in two cell lines, the MMR-proficient TK6 and its derivative MMR-deficient MT1.ResultsUnder an experimental condition that MNNG-induced apoptosis was only observed in MutSα-proficient (TK6), but not in MutSα-deficient (MT1) cells, quantitative analysis of the proteomic data revealed differential expression and phosphorylation of numerous individual proteins and clusters of protein kinase substrates, as well differential activation of response pathways/networks in MNNG-treated TK6 and MT1 cells. Many alterations in TK6 cells are in favor of turning on the apoptotic machinery, while many of those in MT1 cells are to promote cell proliferation and anti-apoptosis.ConclusionsOur work provides novel molecular insights into the mechanism of MMR-mediated DNA damage-induced apoptosis.

Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

Abstract 4614: β-lapachone suppresses glucose metabolism in a NQO1-dependent, tumor-selective manner.

Abstract β-Lapachone (β-lap) is a very unique quinone that can be reduced by two-electron oxidoreductase, NQO1, but unlike most other quinones, its hydroquinone form is unstable and spontaneously oxidizes back to β-lap in two steps. This redox cycle occurs in a futile manner in which one mole of β-lap generates ∼120 moles of superoxide in two minutes, causing predominately DNA base and single strand break damage. This results in PARP1 hyperactivation and programmed necrosis in an NQO1-dependent manner. Most solid cancers, such as nearly 90% of pancreatic and non-small cell lung cancers have 10- to 40- fold of elevated levels of NQO1. Since β-lap causes DNA damage that is dependent on NQO1 expression, and therefore tumor-selective for most solid cancers, use of this ‘NQO1 bioactivatable’ drug is perfect for improving efficacy of cancer therapy. Our recent work also showed that β-lap efficaciously kills pancreatic cancer cells in an NQO1-dependent manner (Li et al., Clin. Cancer Res., 2011). In mechanism of action studies of β-lap, we found that PARP1 hyperactivation led to dramatic depletion of NAD+ and ATP pools in NQO1+ cancer cells (Bentle et al., JBC, 2006). Since NAD+ is a key cofactor for many enzymes in cellular metabolism, we theorized that NAD+ loss may significantly affect cellular metabolism and energy generation. To elucidate how β-lap affects metabolism, we examined metabolites using 13C-labeled glucose. After two hours exposure of β-lap, glucose utilization in MiaPaca-2 cells was completely repressed, lasting 12 h after removal of β-lap. β-Lap treatment also inhibited lactate generation, indicating a suppression of glycolysis after β-lap treatment. In examining metabolic recovery, however, we found that the TCA cycle was not, or only moderately affected by β-lap, indicating that this metabolic process was essential for recovery from this agent. A proportion of 13C-labeled metabolites (i.e., citrate, fumarate) generated by TCA cycle increased vs untreated control cells. Consistently, ATP levels in β-lap-treated NQO1+ pancreatic cancer cells gradually recovered from <10% of untreated cells to ∼50% in 12 h. These data indicate that β-lap predominately represses glycolysis, while the TCA cycle remains functional and possibly the only source of ATP production in the cell. Our results indicated that β-lap a very unique DNA damage-inducing NQO1-dependent agent that simultaneously suppresses glucose metabolism and energy generation. This may explain why β-lap is so potent in cell death induction in comparison with other DNA damage-inducing agents. Further studies will be performed to elucidate the exact mechanism how β-lap represses glycolysis while apparently stimulating the TCA cycle. Understanding how cells recover from β-lap exposures should shed light on improved therapies for NQO1 bioactivatable drugs. This work was supported by an AACR Innovator Award from the George and June Block Foundation to DAB. Citation Format: Xiuquan Luo, Malina Patel, Longshan Li, Ralph Deberardinis, David A. Boothman. β-lapachone suppresses glucose metabolism in a NQO1-dependent, tumor-selective manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4614. doi:10.1158/1538-7445.AM2013-4614

BH3-Mimetics- and Cisplatin-Induced Cell Death Proceeds through Different Pathways Depending on the Availability of Death-Related Cellular Components

BackgroundOwing to their important function in regulating cell death, pharmacological inhibition of Bcl-2 proteins by dubbed BH3-mimetics is a promising strategy for apoptosis induction or sensitization to chemotherapy. However, the role of Apaf-1, the main protein constituent of the apoptosome, in the process has yet not been analyzed. Furthermore as new chemotherapeutics develop, the possible chemotherapy-induced toxicity to rapidly dividing normal cells, especially sensitive differentiated cells, has to be considered. Such undesirable effects would probably be ameliorated by selectively and locally inhibiting apoptosis in defined sensitive cells.Methodology and Principal FindingsMouse embryonic fibroblasts (MEFS) from Apaf-1 knock out mouse (MEFS KO Apaf-1) and Bax/Bak double KO (MEFS KO Bax/Bak), MEFS from wild-type mouse (MEFS wt) and human cervix adenocarcinoma (HeLa) cells were used to comparatively investigate the signaling cell death-induced pathways of BH3-mimetics, like ABT737 and GX15-070, with DNA damage-inducing agent cisplatin (cis-diammineplatinum(II) dichloride, CDDP). The study was performed in the absence or presence of apoptosis inhibitors namely, caspase inhibitors or apoptosome inhibitors. BH3-mimetic ABT737 required of Apaf-1 to exert its apoptosis-inducing effect. In contrast, BH3-mimetic GX15-070 and DNA damage-inducing CDDP induced cell death in the absence of both Bax/Bak and Apaf-1. GX15-070 induced autophagy-based cell death in all the cell lines analyzed. MEFS wt cells were protected from the cytotoxic effects of ABT737 and CDDP by chemical inhibition of the apoptosome through QM31, but not by using general caspase inhibitors.ConclusionsBH3-mimetic ABT737 not only requires Bax/Bak to exert its apoptosis-inducing effect, but also Apaf-1, while GX15-070 and CDDP induce different modalities of cell death in the absence of Bax/Bak or Apaf-1. Inclusion of specific Apaf-1 inhibitors in topical and well-localized administrations, but not in systemic ones, to avoid interferences with chemotherapeutics would be of interest to prevent chemotherapeutic-induced unwanted cell death which could improve cancer patient care.

Mutagenicity and antimutagenicity of (−)-hinokinin a trypanosomicidal compound measured by Salmonella microsome and comet assays

BackgroundThe dibenzylbutyrolactone lignan (−)-hinokinin (HK) was derived by partial synthesis from (−)-cubebin, isolated from the dry seeds of the pepper, Piper cubeba. Considering the good trypanosomicidal activity of HK and recalling that natural products are promising starting points for the discovery of novel potentially therapeutic agents, the aim of the present study was to investigate the (anti) mutagenic∕ genotoxic activities of HK.MethodsThe mutagenic∕ genotoxic activities were evaluated by the Ames test on Salmonella typhimurium strains TA98, TA97a, TA100 and TA102, and the comet assay, so as to assess the safe use of HK in the treatment of Chagas’ disease. The antimutagenic ∕antigenotoxic potential of HK were also tested against the mutagenicity of a variety of direct and indirect acting mutagens, such as 4- nitro-o-phenylenediamine (NOPD), sodium azide (SA), mitomycin C (MMC), benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1), 2-aminoanthracene (2-AA) and 2-aminofluorene (2-AF), by the Ames test, and doxorubicin (DXR) by the comet assay.ResultsThe mutagenicity∕genotoxicity tests showed that HK did not induce any increase in the number of revertants or extent of DNA damage, demonstrating the absence of mutagenic and genotoxic activities. On the other hand, the results on the antimutagenic potential of HK showed a strong inhibitory effect against some direct and indirect-acting mutagens.ConclusionsRegarding the use of HK as an antichagasic drug, the absence of mutagenic effects in animal cell and bacterial systems is encouraging. In addition, HK may be a new potential antigenotoxic ∕ antimutagenic agent from natural sources. However, the protective activity of HK is not general and varies with the type of DNA damage-inducing agent used.

Evaluation of cytotoxic, genotoxic and antigenotoxic potential of Solanum lycocarpum fruits glicoalkaloid extract in V79 cells

Solanum lycocarpum St.-Hil (Solanaceae) is a hairy shrub or small much-branched tree of the Brazilian Cerrado, popularly known as “fruit-of-wolf”. Considering that the induction of chromosomal mutations is involved in the process of carcinogenesis, and that S. lycocarpum is often used in folk medicine, it becomes relevant to study its effect on genetic material. In this sense, the aim of present study was to determine the possible cytotoxic, genotoxic and antigenotoxic potentials of S. lycocarpum fruits glycoalkaloid extract (SL) in Chinese hamster lung fibroblasts (V79 cells). The cytotoxicity was evaluated by the colony forming assay, apoptosis and necrosis assay, Trypan blue exclusion dye method and mitotic index. Genotoxic and antigenotoxic potential were evaluated by comet and chromosomal aberrations assays. Four concentrations of SL (4, 8, 16 and 32μg/mL) were used for the evaluation of its genotoxic potential. The DNA damage-inducing agent methyl methanesulfonate (MMS, 22μg/mL) was utilized in combination with extract to evaluate a possible protective effect. The results showed that SL was cytotoxic at concentrations above 32μg/mL by the colony forming assay. For apoptosis and necrosis assay, the concentration of 64μg/mL of SL showed statistically significant increase in cell death by apoptosis and necrosis, while the concentrations of 128 and 256μg/mL of SL demonstrated statistically significant increase in cell death by necrosis, compared with the control group. Analysis of cell viability by Trypan blue exclusion indicated >96% viability for treatments with concentrations up to 32μg/mL of SL. No significant differences in MI were observed between cultures treated with different concentrations of SL (4, 8, 16 and 32μg/mL) alone or in combination with MMS and the negative control, indicating that these treatments were not cytotoxic. The comet and chromosomal aberrations assays revealed that SL does not display genotoxic activity. Moreover, the different concentrations of SL showed protective effect against both genomic and chromosomal damages induced by MMS.

Telomere dysfunction as a tool to identify novel DNA damage factors

The DNA damage response (DDR) pathway plays an essential tumor suppressive role ensuring the integrity of genomic information. Two major limitations have hindered the research in this field: i) the unpredictable location of DNA damage sites induced by conventional DNA damage‐inducing agents, ii) the lack of a technique for the isolation of proteins associated with a given DNA substrate. I have overcome these limitations by taking advantage of: i) telomere dysfunction as a tool to induce DNA damage to specific genomic loci, ii) a novel technique developed to isolate proteins associated with specific chromatin loci termed Proteomics of Isolated Chromatin segments (PICh). I performed the PICh purification on Terf2F/F Rosa26‐Cre‐ERT2 mouse embryonic fibroblasts (MEFs), in which deletion of both alleles of Terf2 gives rise to a DDR at chromosome ends. The purification identified 95 proteins uniquely associated with dysfunctional telomeres, among which I selected a subset of candidates for confirmation of their localization to damaged telomeres and for their characterization as potential novel DDR factors.Research Support Source ADI

DNA damage-induced apoptosis and genetic background of the host: host-specific signaling enhancers of apoptosis.

Radiation therapy, inducing DNA damage, is one of the most effective tools for treatments of human cancers, but the effectiveness of the therapy is largely depending on the host specific conditions. Recently genetic constitution has proven to be important for apoptosis-induction responding to DNA damage. Regarding the host-specific manner of responses against DNA-damage in animal model, we have reported that infection with Friend leukemia virus (FLV) enhances the DNA damage-induced apoptosis in hematopoietic cells derived from C3H but DBA/2 mice. Furthermore, p53 or ATM knockout mice of C3H background and DNA-PK-deficient C3H SCID mice did not show the enhanced apoptosis by FLV. Recently, we could show that this host-specific apoptosis was mediated by the kinase activity of DNA-PK in association with FLV env-coding protein, gp70. Interestingly, two host proteins, acinus and MCM2, were also associated with DNA-PK and gp70 and were host-specifically overexpressed in C3H-derived cells. Our data suggest that gp70 enhances cellular DNA damage-induced signaling in association with host-specific cellular proteins, including acinus and MCM2, resulting in the activation of DNA-PK to phosphorylate P53. By introducing gp70/acinus/MCM2-associated pathways into tumor cells, cancer therapy with DNA damage-inducing agents might become much more effective. Our aim is to develop a novel form of targeted therapy that can be combined with other treatment modalities, such as radiotherapy and chemotherapy, using the host-specific regulatory mechanisms of apoptotic enhancement.