Repair Of DNA Strand Breaks Research Articles

Aprataxin localizes to mitochondria and preserves mitochondrial function

Ataxia with oculomotor apraxia 1 is caused by mutation in the APTX gene, which encodes the DNA strand-break repair protein aprataxin. Aprataxin exhibits homology to the histidine triad superfamily of nucleotide hydrolases and transferases and removes 5'-adenylate groups from DNA that arise from aborted ligation reactions. We report herein that aprataxin localizes to mitochondria in human cells and we identify an N-terminal amino acid sequence that targets certain isoforms of the protein to this intracellular compartment. We also show that transcripts encoding this unique N-terminal stretch are expressed in the human brain, with highest production in the cerebellum. Depletion of aprataxin in human SH-SY5Y neuroblastoma cells and primary skeletal muscle myoblasts results in mitochondrial dysfunction, which is revealed by reduced citrate synthase activity and mtDNA copy number. Moreover, mtDNA, not nuclear DNA, was found to have higher levels of background DNA damage on aprataxin knockdown, suggesting a direct role for the enzyme in mtDNA processing.

Online imaging of initial DNA damages at the PTB microbeam

In an inter-disciplinary collaboration of Physikalisch-Technische Bundesanstalt (PTB), German Collection of Microorganisms and Cell Cultures (DSMZ) and Heinrich-Heine University, live-cell imaging has been established at the charged-particle microbeam facility of PTB. Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MRE11, MSH2, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterise and use these highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions.

PDE4 Inhibitors Enhance DNA Damage-Induced Apoptosis In CLL

AbstractAbstract 2902Treatment of primary chronic lymphocytic leukemia (CLL) cells with PDE4 inhibitors induces apoptosis while comparable treatment of primary human T and B cells from healthy donors does not. PDE4 inhibitor treatment also augments glucocorticoid-induced apoptosis in CLL cells. We now report that PDE4 inhibitor treatment enhances CLL apoptosis induced by DNA damage. Using drug combination analysis, treatment with etoposide and rolipram resulted in a greater than additive apoptotic effect than predicted by a Loewe additivity model at many dose combinations. In order to directly address the role of DNA damage and rule out effects on drug uptake and efflux, we next examined the ability of PDE4 inhibitors to modulate gamma-irradiation-induced apoptosis in CLL. While most patients demonstrated in vitro sensitivity to rolipram and gamma irradiation, a small subset of patients, including those known to be p53 mutant, that lacked a substantial response to ionizing radiation also lacked sensitivity to rolipram (p < 0.01). Those patients with intact sensitivity to radiation, demonstrated at least an additive, or greater-than-additive at some doses, effect on apoptosis when PDE4 inhibitor treatment was combined with gamma irradiation treatment (p < 0.05 at 1 Gy and 20 mM rolipram). As judged by comet assays, gamma radiation-induced DNA double stranded breaks were repaired within one hour of induction regardless of exposure to rolipram. As judged by Western analysis and flow cytometry, the kinetics of gamma irradiation-induced H2AX phosphorylation, a marker for DNA damage, were not altered by addition of rolipram in a manner that correlated with apoptosis. Finally, rolipram treatment did not further augment levels of p53, p21cip, or phospho-Chk2. These results suggest that PDE4 inhibitors did not alter double strand DNA break repair nor alter upstream radiation-induced p53-mediated signaling events. As PDE4 inhibitors are known to induce CLL apoptosis through a mitochondrial pathway, our current studies are focused on identifying the mechanism by which DNA damage and PDE4 inhibitor signaling pathways converge at the mitochondrion to enhance apoptosis. Regardless of the precise mechanism by which these supra-additive apoptotic effects occur, our study adds credence to the concept that PDE4 inhibitors may prove to be important sensitizing adjuvants in the treatment of human B cell malignancies. Disclosures:No relevant conflicts of interest to declare.

The effect of acute dose charge particle radiation on expression of DNA repair genes in mice

The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the remaining four genes (XPC, ERCC2, XRCC1, and MLH1) in either the experimental or control animals.

Role for the Mammalian Swi5-Sfr1 Complex in DNA Strand Break Repair through Homologous Recombination

In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5 −/− and Sfr1 −/− embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5 −/− and Sfr1 −/− cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5 −/− and Sfr1 −/− cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.

Mitotic recombination: a genotoxic effect of the antidepressant citalopram in Aspergillus nidulans

This report evaluates the potential of the antidepressant drug citalopram to induce homozygotization of genes previously present in a heterozygous condition, by homologous recombination. In order to address this question, a heterozygous diploid strain of the filamentous fungus Aspergillus nidulans and the homozygotization assay were utilized. Non-cytotoxic concentrations of citalopram (50, 75 and 100 μmol/L) showed a strong recombinogenic effect in A. nidulans, inducing homozygosis of the diploid strain's nutritional markers. The genetic markers exhibited homozygotization index (HI) rates higher than 2.0 and significantly different from HI control ones. Since citalopram has been previously characterized as a DNA synthesis inhibitor, the recombinogenic potential of this antidepressant in A. nidulans may be associated with the recombinational repair of citalopram-induced DNA strand breaks.

Poly(ADP-ribose) polymerase signaling of topoisomerase 1-dependent DNA damage in carcinoma cells

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2 +/+ and p53 +/+. HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT + PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT + PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24–72 h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors.

Poly (ADP-Ribose) Polymerase as a Novel Therapeutic Target in Cancer

Cancer chemotherapy exploits limitations in repairing DNA damage in order to kill proliferating malignant cells. Recent evidence suggests that cancers within and across tissue types have specific defects in DNA repair pathways, and that these defects may predispose for sensitivity and resistance to various classes of cytotoxic agents. Poly (ADP-ribose) polymerase (PARP) and BRCA proteins are central to the repair of DNA strand breaks and, when defective, lead to the accumulation of mutations introduced by error-prone DNA repair. Breast, ovarian, and other cancers develop in the setting of BRCA deficiency, and these cancers may be more sensitive to cytotoxic agents that induce DNA strand breaks, as well as inhibitors of PARP activity. A series of recent clinical trials has tested whether PARP inhibitors can achieve synthetic lethality in BRCA-pathway-deficient tumors. Future studies must seek to identify sporadic cancers that harbor genomic instability, rendering susceptibility to agents that induce additional and lethal DNA damage.

Characterization of a DNA damage‐inducible membrane protein kinase from Deinococcus radiodurans and its role in bacterial radioresistance and DNA strand break repair

Deinococcus radiodurans mutant lacking pyrroloquinoline-quinone (PQQ) synthesis shows sensitivity to γ-rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Δdr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3-log cycle both at 6 kGy γ-rays and 1200 Jm(-2) UV (254 nm) radiation, and 2.5-log cycle upon 14 days desiccation at 5% humidity. The Δdr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Δdr2518pqqE:cat double mutant showed γ-ray sensitivity similar to Δdr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15-fold increase in dr2518 transcription and several-fold enhanced in vivo phosphorylation of DR2518 were observed in response to γ irradiation. These results suggest that DR2518 as a DNA damage-responsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans.

Fibroblast Growth Factor Type 2 Signaling Is Critical for DNA Repair in Human Keratinocyte Stem Cells

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair. Stem Cells 2010; 28:1639–1648.

Gamma-radiation sensitivity and polymorphisms in RAD51L1 modulate glioma risk

DNA strand breaks pose the greatest threat to genomic stability. Genetically determined mutagen sensitivity predisposes individuals to a variety of cancers, including glioma. However, polymorphisms in DNA strand break repair genes that may determine mutagen sensitivity are not well studied in cancer risk, especially in gliomas. We correlated genotype data for tag single-nucleotide polymorphisms (tSNPs) of DNA strand break repair genes with a gamma-radiation-induced mutagen sensitivity phenotype [expressed as mean breaks per cell (B/C)] in samples from 426 glioma patients. We also conducted analysis to assess joint and haplotype effects of single-nucleotide polymorphisms (SNPs) on mutagen sensitivity. We further validate our results in an independent external control group totaling 662 subjects. Of the 392 tSNPs examined, we found that mutagen sensitivity was modified by one tSNP in the EME2 gene and six tSNPs in the RAD51L1 gene (P < 0.01). Among the six RAD51L1 SNPs tested in the validation set, one (RAD51L1 rs2180611) was significantly associated with mutagen sensitivity (P = 0.025). Moreover, we found a significant dose-response relationship between the mutagen sensitivity and the number of adverse tSNP genotypes. Furthermore, haplotype analysis revealed that RAD51L1 haplotypes F-A (zero adverse allele) and F-E (six adverse alleles) exhibited the lowest (0.42) and highest (0.93) mean B/C values, respectively. A similar dose-response relationship also existed between the mutagen sensitivity and the number of adverse haplotypes. These results suggest that polymorphisms in and haplotypes of the RAD51L1 gene, which is involved in the double-strand break repair pathway, modulate gamma-radiation-induced mutagen sensitivity.

Effect of 2.45 mT sinusoidal 50 Hz magnetic field on Saccharomyces cerevisiae strains deficient in DNA strand breaks repair

Purpose: To investigate whether extremely-low frequency magnetic field (MF) exposure produce alterations in the growth, cell cycle, survival and DNA damage of wild type (wt) and mutant yeast strains.Materials and methods: wt and high affinity DNA binding factor 1 (hdf1), radiation sensitive 52 (rad52), rad52 hdf1 mutant Saccharomyces cerevisiae strains were exposed to 2.45 mT, sinusoidal 50 Hz MF for 96 h. MF was generated by a pair of Helmholtz coils. During this time the growth was monitored by measuring the optical density at 600 nm and cell cycle evolution were analysed by microscopic morphological analysis. Then, yeast survival was assayed by the drop test and DNA was extracted and electrophoresed.Results: A significant increase in the growth was observed for rad52 strain (P = 0.005, Analysis of Variance [ANOVA]) and close to significance for rad52 hdf1 strain (P = 0.069, ANOVA). In addition, the surviving fraction values obtained for MF-exposed samples were in all cases less than for the controls, being the P value obtained for the whole set of MF-treated strains close to significance (P = 0.066, Student's t-test). In contrast, the cell cycle evolution and the DNA pattern obtained for wt and the mutant strains were not altered after exposure to MF.Conclusions: The data presented in the current report show that the applied MF (2.45 mT, sinusoidal 50 Hz, 96 h) induces alterations in the growth and survival of S. cerevisiae strains deficient in DNA strand breaks repair. In contrast, the MF treatment does not induce alterations in the cell cycle and does not cause DNA damage.

DNA End-Processing Enzyme Polynucleotide Kinase As a Potential Target in the Treatment of Cancer

Pharmacological inhibition of DNA-repair pathways as an approach for the potentiation of chemo- and radio-therapeutic cancer treatments has attracted increasing levels of interest in recent years. Inhibitors of several enzymes involved in the repair of DNA strand breaks are currently at various stages of the drug development process. Polynucleotide kinase (PNK), a bifunctional DNA-repair enzyme that possesses both 3'-phosphatase and 5'-kinase activities, plays an important role in the repair of both single strand and double strand breaks and as a result, RNAi-mediated knockdown of PNK sensitizes cells to a range of DNA-damaging agents. Recently, a small molecule inhibitor of PNK has been developed that is able to sensitize cells to ionizing radiation and the topoisomerase I poison, camptothecin. Although still in the early stages of development, PNK inhibition represents a promising means of enhancing the efficacy of existing cancer treatments.

Automated method to quantify formation and repair of DNA strand breaks

Automated method to quantify formation and repair of DNA strand breaks

Abstract 689: EGFR nuclear translocation modulates DNA repair following cisplatin or ionizing radiation treatment

Abstract Overexpression of the epidermal growth factor receptor (EGFR) is associated with resistance to chemotherapy and radiotherapy. Nuclear translocation of EGFR occurs through a tripartite nuclear localisation sequence (NLS). This process has been shown to enhance DNA repair in cells treated with ionising radiation (IR). The association of EGFR with the catalytic subunit of DNA protein kinase (DNAPKcs) contributes to this effect. The L858R mutation, found in tyrosine kinase inhibitor-sensitive non-small cell lung cancer, which shows reduced nuclear translocation, has been shown to sensitise cells to radiation. We investigated the role of EGFR nuclear import, and its association with DNAPKcs on DNA repair, following treatment with cisplatin or IR. NIH3T3 cells, which do not express EGFR, were transfected with plasmids expressing wild type EGFR (wtEGFR), L858R, EGFR variant III (EGFRvIII), wtEGFR with mutation in the NLS (NLS123) and an L858R construct with the same NLS mutation (LNLS123). Immunoprecipitation, western blot and confocal microscopy demonstrated that mutation of the NLS impairs EGFR nuclear translocation and activation following treatment with IR or cisplatin. Confocal microscopy confirmed the role of nuclear translocation in mediating association of EGFR with DNAPKcs following these treatments. Immunoprecipitation experiments showed association of EGFR with DNAPKcs only when EGFR is expressed in the nucleus. This association was shown to increase 2-fold by 18 hours following cisplatin treatment, and resulted in stimulation of DNA-PKcs activity as shown by a DNAPKcs kinase assay. The DNA interstrand crosslink (ICL) is a critical determinant of platinum cytotoxicity. Effects of nuclear translocation of EGFR on repair (unhooking) of cisplatin-induced DNA ICL and IR-induced DNA strand breaks were determined by the single cell agarose gel electrophoresis (comet assay). Unhooking of cisplatin-induced ICL was 100% at 36 hours in cells expressing wt EGFR and EGFRvIII. In contrast, in cells expressing L858R, unhooking was 46 ±3.8% as compared with peak levels of ICL. NLS123 showed 19.5 ±3.7% and LNLS123 16.5 ±5.2% unhooking at 36 hours. Additionally, following IR, repair of DNA strand breaks was 100% at 4 hours in cells expressing wtEGFR and EGFRvIII. In cells expressing the L858R, repair of DNA strand breaks was 88 ± 6.3% at four hours whereas NLS123 showed 75 ± 5.7% and LNLS123 73 ±3.9%. These results demonstrate that EGFR nuclear translocation and activation are involved in the modulation of DNA repair following treatment with cisplatin or IR. EGFR-DNAPKcs association and EGFR nuclear expression may be a critical determinant of response to chemotherapy or IR. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 689.

Abstract 682: Dual inhibition of BER by TRC102 and PARP inhibitor (ABT 888) synergistically enhances cytotoxicity of TMZ in human melanoma

Abstract Melanoma, the most fatal skin cancer, has increased in incidence by 15-fold in the past 40 years, the fastest rate increase of any human malignancy. This disease metastasizes rapidly and is highly resistant to chemotherapy. Currently, temozolomide (TMZ) is an important part of treatment regimens for advanced metastatic melanoma. However, drug resistance often results in treatment failure. A major resistance factor is the presence of elaborate mechanisms of DNA repair. We have previously studied the role of base excision repair (BER) in conferring TMZ resistance and explored a combined therapy by targeting BER with methoxyamine (TRC102), an inhibitor of BER, which binds to abasic sites and disrupts the BER pathway. The potentiation of TMZ by TRC102 has been validated in different tumor types in vitro and in xenograft models. Results showed TRC102 potentiation of TMZ was accompanied by a remarkable induction of DNA strand breaks. In this study, we examined the therapeutic efficacy of the combination of TMZ with TRC102 and a PARP inhibitor (ABT888) in three melanoma cell lines (A375, WM9, and WM164) and in melanoma xenografts. PARP, an enzyme critical in BER pathway, is actively involved in the repair of DNA strand breaks. We hypothesized the inhibition of PARP would inhibit BER with the accumulation of large numbers of unrepaired DNA strand breaks and the combination of TRC102 and ABT888 would synergistically enhance the anti-tumor effect of TMZ through dual inhibition of BER. Results showed that compared to TMZ alone, the combination with either ABT888 or TRC102 more effectively inhibited cell viability and induced apoptosis in A375 and WM9, but not in WM164 melanoma cell lines. The resistance in WM164 cells is probably related to a deficiency in methylpurine-DNA glycosylase, which is responsible for removing TMZ-induced methylated DNA adducts (N7mG and N3mA) and producing toxic AP sites. When combined with TMZ and TRC102, ABT888 at a concentration of 5 µM efficiently potentiated cytotoxicity by 8-10 fold in A375 and WM9 melanoma cell lines, respectively, and 4 fold in WM164 cells. Cytotoxicity was correlated with the induction of DNA single and double stranded breaks as assayed by comet assay and induction of γ-H2AX. For in vivo studies, the therapeutic regimen was initiated when tumor xenografts (WM9) in nude mice reached ∼100 mm3 in volume and treatment was continued for 5 days. Tumor volume was measured for assessment of therapeutic effect. At termination, we found 30-40% reduction in tumor volume following treatment with the combination of TMZ with ABT888 or with TRC2, and 80% reduction in tumor volume by combining the three drugs relative to the TMZ alone group, which had no significant differences in tumor growth in comparison to untreated group. Data indicate that combining a PARP inhibitor with TMZ and TRC102 results in greater inhibition of BER and induces a synergistic cytotoxic effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 682.

Abstract 16: Thermal enhancement with optically activated gold nanoshells sensitizes breast cancer stem cells to radiation therapy

Abstract Background Metastatic disease and recurrence are hypothesized to result, at least in part, from the residual cancer stem cells (CSCs), also referred to as tumor-initiating cells (TICs), which evade initial treatment. In several genetically engineered mouse (GEM) models that resemble human breast cancer, the tumor subpopulation CD29+/CD24+/lin− has been shown to be more tumorigenic as compared to other subpopulations by both in vitro and in vivo assays. We and others have demonstrated that the tumorigenic CD29+/CD24+/lin− subpopulation repairs radiation-induced DNA strand breaks more efficiently than the other subpopulations. Therefore, we hypothesized that hyperthermia, known to inhibit DNA repair, may radiosensitize CSCs to radiation therapy (RT) by delaying and inhibiting the repair of DNA strand breaks. Methods To test this hypothesis, we assayed for tumorigenic CD29+/CD24+/lin− cells in a p53null mouse mammary tumor model with and without radiation, and in combination with hyperthermia (42°C) administered using optically-activated gold nanoshells. We then determined the percentage of functional CSCs by FACS analysis and limiting dilution transplantation assays. Results These studies confirmed that CD29+/CD24+/lin− cells were more radiation resistant and that their relative proportion was increased after RT, as compared to the other cell populations. In contrast, post-irradiation treatment with 20 minutes of local hyperthermia, using intravenously administered optically activated gold nanoshells, a larger reduction in tumor size was observed, without a concomitant increase in the percentage of CD29+/CD24+/lin− tumorigenic cells. Using functional limiting dilution transplantation assays to assay for CSCs, we demonstrated that 48 hours after treatment cells derived from tumors treated with RT and hyperthermia exhibited a decreased frequency of TICs compared to untreated controls, while in contrast tumors treated with RT alone exhibited an increase in TIC frequency. Thus, there was a 20-fold difference in TIC frequency between the RT only, and RT and hyperthermia treated tumors. Importantly some of the tumors that grew out after radiation and hyperthermia treatment were less aggressive histologically as compared to those treated with radiation alone, which actually displayed a more aggressive histology than mock treated tumors. These results obtained with the GEM model were validated using two different primary triple-negative breast cancer xenografts. Conclusion These results suggest that localized hyperthermia administered using optically-active gold nanoshells serves as a simple strategy to selectively sensitize radioresistant CSCs to enhance their response to radiation therapy. Supported by grants NCI R01 CA112305 and CA16303. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 16.

Abstract 693: Histone deacetylase inhibitors attenuate epirubicin induced p53 expression

Abstract Preclinical and clinical studies have demonstrated that Histone Deacetylase Inhibitors (HDACIs) can act synergistically with topoisomerase II inhibitors (e.g. doxorubicin, epirubicin) to increase the induction of apoptosis in breast cancer cells. We have previously shown that HDACIs (i.e. valproic acid and suberoylanilide hydroxamic acid, SAHA/vorinostat) caused a reduction in the expression of specific chromatin maintenance genes including HP1-, DNMT1 and SMC1-5 with subsequent chromatin decondensation. Chromatin decondensation facilitates access for DNA damaging agents such, as topoisomerase inhibitors, to their DNA substrate resulting in an increase in the topoisomerase induced DNA strand breaks and cell death. Using siRNA mediated depletion of HDAC2, similar synergistic effects were observed compared to valproic acid or SAHA. This was not observed with depletion of HDAC1 or HDAC6. This suggested the specific role of HDAC2 in mediating the synergy in increased DNA damage and apoptosis with epirubicin. However, efficient DNA strand break repair may be an alternate mechanism of resistance. To determine whether the HDAC inhibitors play a role in the inhibition of repair enzymes in addition to the increased induction of epirubicin induced strand breaks, we sought to determine the effects of HDACIs on epirubicin mediated DNA damage repair. Previous studies have shown that various HDACis down-regulate DNA repair associated genes including BRCA1, Ku70, and Rad51. Micro-array analysis performed by our group of cultured breast cancer cells treated with valproic acid or siRNA targeting HDAC2 revealed the down-regulation of the dsDNA repair genes Rad51b and Rad51c. These Rad51 paralogs have distinct functions in homologous recombination. Rad51b has been shown to associate with, and phosphorylate p53. Rad51c is able to exchange DNA strands vital for homologous recombination, and has been suggested to recruit Rad51 to sites of DNA damage. It is also suggested to be a Trp53-dependent tumor suppressor. We show that treatment of breast cancer cells with vorinostat caused a decrease in p53 protein expression. Furthermore, pre-treatment of these cells with valproic acid or SAHA significantly inhibited the induction of p53 in the presence of epirubicin. At the time points tested (0-120 minutes), this corresponded to an initial decrease in DNA repair kinetics as demonstrated by γ-H2AX expression. We hypothesize therefore that the treatment of breast cancer cells with HDACis in the presence of epirubicin results in a decreased expression of the homologous recombination Rad51 paralog proteins and downstream p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 693.

Non‐Homologous End‐Joining Gene Profiling Reveals Distinct Expression Patterns Associated with Lymphoma and Multiple Myeloma

Repair of DNA strand breaks induced during lymphoid antigen receptor rearrangement involves non-homologous end-joining (NHEJ). We investigated NHEJ in the aetiology of lymphoproliferative disorders (LPDs) and the disease subtypes therein through real-time quantitative RT-PCR gene expression analysis. Lower expression of XRCC6 and MRE11A was observed in all tumours, with higher expression of both XRCC4 and RAD50 observed only in multiple myeloma (MM). Hierarchical clustering enabled tumours to be clearly distinguished from controls, and by morphological sub-type. We postulate this identifies targets worthy of investigation in the genetic predisposition, pathogenesis and prognosis of lymphoid malignancies.

The SbcCD complex of Deinococcus radiodurans contributes to radioresistance and DNA strand break repair in vivo and exhibits Mre11–Rad50 type activity in vitro

Deinococcus radiodurans lacks a homologue of the recB and recC genes, and the sbcA/B genes, of Escherichia coli. Thus, DNA strand break repair in Deinococcus proceeds by pathways that do not utilize these proteins. Unlike E. coli, the absence of recBC and sbcA/sbcB, and presence of only sbcC and sbcD in Deinococcus, indicates an enigmatic role of SbcCD in this bacterium. Studies on sbcCD mutation in Deinococcus showed nearly a 100-fold increase in gamma radiation sensitivity as compared to wild type. The mutant showed a higher rate of in vivo DNA degradation during the post-irradiation recovery period that corresponds to the RecA-dependent DSB repair phase. These cells showed a typical NotI pattern of DNA reassembly during the early phase of DSB repair, but were defective for the subsequent RecA-dependent phase II of DSB repair. Hydrogen peroxide had no effect on cell survival of the mutant. While its tolerance to higher doses of UVC and mitomycin C was significantly decreased as compared to wild type. Purified recombinant SbcCD proteins showed single-stranded endonuclease and 3′ → 5′ double-stranded DNA exonuclease activities similar to that of the Mre11–Rad50 complex, which is required for DNA strand break repair in higher organisms. These results suggested that the Mre11–Rad50 type nuclease activity of SbcCD proteins contributes to the radiation resistance of D. radiodurans perhaps by promoting the RecA-dependent DSB repair required for polyploid genome maturation.