Accelerated DNA Repair Research Articles

Palmitoyl ascorbic acid glucoside enhanced cell survival with post irradiation treatment

Radiation exposure poses a significant threat to cellular integrity by inducing DNA damage through the generation of free radicals and reactive oxygen species. Ascorbic acid, particularly its derivative Palmitoyl Ascorbic Acid 2-Glucoside (PA2G), has demonstrated remarkable radioprotective properties. While previous research focused on its pre-irradiation application, this study explores the post-irradiation radiomitigation potential of PA2G. Our findings reveal that post-irradiation treatment with PA2G enhances cell survival and accelerates DNA repair processes, particularly the non-homologous end-joining (NHEJ) repair pathway. Notably, PA2G treatment reduces the frequency of lethal chromosomal aberrations and micronuclei formation, indicating its ability to enhance the repair of complex DNA lesions. Furthermore, PA2G is shown to play a role in potentially lethal damage repair (PLDR). These radioprotective effects are specific to NHEJ and ATM pathways, as cells deficient in these mechanisms do not benefit from PA2G treatment. This study highlights PA2G as a versatile radioprotector, both pre- and post-irradiation, with significant potential for applications in radiation therapy and protection, offering new insights into its mechanism of action. Further research is required to elucidate the precise molecular mechanisms underlying PA2G's radiomitigation effects and its potential clinical applications.

Sphingosine kinase inhibitor, SKI-II confers protection against the ionizing radiation by maintaining redox homeostasis most likely through Nrf2 signaling

Exposure to ionizing radiation (IR) set a series of deleterious events causing acute radiation syndrome and mortality, posing the need for a potent and safe radio-protective drug. IR induces cell death predominantly by causing oxidative stress and macromolecular damage. The pre-existing antioxidant defence machinery of the cellular system plays a crucial role in protecting the cells against oxidative stress by activation of Nrf2. The current study was undertaken to investigate the radio-protective potential of sphingosine kinase inhibitor (SKI-II), which was demonstrated to activate Nrf2 signaling. The safety and efficacy of SKI-II were evaluated with cell cytotoxicity, proliferation index, and clonogenic survival assays in different cell lines, namely Raw 264.7, INT-407, IEC-6 and NIH/3T3 cell lines. A safe dose of SKI-II was found radio-protective in all the cell lines linked with the activated antioxidant defence system, thereby resulting in the amelioration of IR induced oxidative stress. SKI-II pretreatment also significantly reduced DNA damage, micronuclei expression, and accelerated DNA repair kinetics as compared to IR exposed cells. Reduced oxidative stress and enhanced DNA repair significantly reduced apoptosis and suppressed the pro-death signaling associated with IR exposure. Furthermore, the in-vitro observation was verified in the in-vivo model (C57 BL/6). The Intra-peritoneal (IP) administration of SKI-II, 2 h before a lethal dose of IR exposure (7.5 Gy) resulted in 75% survival. These results imply that SKI-II ameliorates IR-induced oxidative stress and cell death by inducing anti-oxidant defence system and DNA repair pathways, thus strengthening its potential to be used as radiation countermeasure.

Bach2 Deficiency Promotes Intestinal Epithelial Regeneration by Accelerating DNA Repair in Intestinal Stem Cells.

SummaryEpithelial regeneration is critical for barrier maintenance and organ function after intestinal injury, although the repair mechanisms are unclear. Here, we found that Bach2 deficiency promotes intestinal epithelial cell proliferation during homeostasis. Moreover, genetic inactivation of Bach2 in mouse intestinal epithelium facilitated crypt regeneration after irradiation, resulting in a reduction in mortality. RNA-sequencing analysis of isolated crypts revealed that Bach2 deficiency altered the expression of numerous genes, including those regulating double-strand break repair. Mechanistic characterizations indicated that Bach2 deletion facilitated DNA repair in intestinal crypt cells, as evidenced by faster resolution of γ-H2AX and 53BP1 foci in Bach2−/− crypt cells, compared with Bach2+/+ control. Together, our studies highlight that Bach2 deficiency promotes intestinal regeneration by accelerating DNA repair in intestinal stem cells after radiation damage.

YY1-mediated PTEN dephosphorylation antagonizes IR-induced DNA repair contributing to tongue squamous cell carcinoma radiosensitization

Ionizing radiation (IR) confers a survival advantage in tongue squamous cell carcinoma (TSCC), however, IR resistance limits its efficacy. Although Yin Yang 1 (YY1) has been reported to play a role in genotoxic drug resistance by accelerating DNA repair, its role in TSCC radioresistance remains unclear. In this study, we examined YY1 mRNA and protein expression in human tongue cancer samples using qRT-PCR and western blotting, respectively. DNA array data identified YY1 mRNA expression in IR sensitivity or resistance cell lines and tissues. Tongue carcinoma primary cells and CAL27 cells with YY1 stably overexpressed or knocked-down were exposed to IR and evaluated for cell proliferation and apoptosis by CCK8-assay and caspase-3 assay, respectively. We also examined DNA damage- or repair-related indicators, such as YY1, p-H2AX, nuclear PTEN, p-PTEN, and Rad51 through Western blot analysis. Additionally, we explored the mechanism of IR-induced PTEN nuclear translocation by introducing a series of PTEN phosphorylation site mutations and co-IP assay. We observed that YY1 mRNA and protein are highly expressed in TSCC tissues, which was correlated with worse overall survival. Moreover, higher expression of YY1 and Rad51 was observed in radioresistant cells and tissues, overexpression of YY1 led to IR resistance in TSCC cells, whereas YY1 knockdown sensitized TSCC cells to IR. The underlying mechanism showed that the overexpression of YY1 upregulated nuclear PTEN and Rad51 expression, which is essential for DNA repair. IR upregulated YY1, nuclear PTEN, and Rad51; thus, knockdown of YY1 completely blocked IR-induced upregulation of nuclear PTEN/Rad51. IR upregulated PTEN phosphorylation, and mutation of the phosphorylation site of Ser380 nearly completely blocked IR-induced PTEN nuclear translocation. Furthermore, the phosphatase PP2A negatively regulated pS380-PTEN, and knockdown of YY1 completely blocked IR-induced pS380-PTEN through PP2A. In conclusion, knockdown of YY1 enhanced TSCC radiosensitivity through PP2A-mediated dephosphorylation of PTEN Ser380; thus, antagonizing the IR-induced nuclear PTEN/Rad51 axis and targeting YY1 may reverse IR resistance in TSCC.

Nono Recruits Ribosomal Protein RPLP0 to DNA Damage Sites to Enhance Non-Homologous End Joining and Tumor Radio-Resistance

Colorectal cancer (CRC) is one of the most common and deadly cancers in the world. Currently, radio-therapy is one of the major treatments for CRC. NONO, an RNA/DNA binding protein, has been known to enhance radio-resistance by accelerating DNA repair, whereas its underlying mechanism remains largely unknown. In this study, we aimed to explore its molecular mechanism and clinical significance in CRC. The radiation-induced NONO-binding protein was identified by Co-immunoprecipitation (CoIP) and mass spectrum (MS). The association between proteins was verified by CoIP, immunofluorescence and proximal ligation assay. The expression of RNAs and proteins were evaluated by quantitative real-time PCR and western blotting, respectively. The DNA damage level was evaluated by the analyses of γ-H2A.X level and counting of γ-H2A.X foci. The double strand break repair efficiency was examined by in vitro non-homologous end joining (NHEJ) assay. NONO knockout enhanced radio-sensitivity, reduced the repair of radiation-induced DNA damage and NHEJ efficiency. Through CoIP and MS, RPLP0 was identified as a novel NONO-binding protein, and their association was further enhanced upon irradiation. NONO bound to free RPLP0, but not that within ribosome, in nucleus. The RPLP0-binding domain of NONO was mapped to RRM1/2 domain. Furthermore, RPLP0 silencing reduced the repair of radiation-induced DNA damage and the efficiency of NHEJ. As translation blockage with Cycloheximide had no influence on DNA damage repair, RPLP0 silencing-induced DNA repair deficiency was not a result of translation inhibition. Mechanically, RPLP0 was recruited to DNA damage site by NONO and enhanced DNA repair. The analyses of clinical samples showed that both the RNA and protein level of NONO and RPLP0 were upregulated in colorectal cancer tissues, and high expression of NONO was associated with poor response of radio-therapy. Moreover, compared with adjacent normal tissues, the interaction between NONO and RPLP0 was stranger in CRC tissues. NONO enhanced radio-resistance and DNA damage repair by recruiting RPLP0 to DNA damage site. High expression of NONO was a biomarker for poor treatment response of radio-therapy.

Glutamine Synthetase Promotes Radiation Resistance via Facilitating Nucleotide Metabolism and Subsequent DNA Damage Repair

Radiation resistance is a critical problem in radiotherapy for cancer. Radiation kills tumor cells mainly through causing DNA damage. Thus, efficiency of DNA damage repair is one of the most important factors that limits radiotherapy efficacy. Glutamine physiologically functions to generate protein and nucleotides. Here, we study the impact of glutamine metabolism on cancer therapeutic responses, in particular under irradiation-induced stress. We show that radiation-resistant cells possessed low glycolysis, mitochondrial respiration, and TCA cycle but high glutamine anabolism. Transcriptome analyses revealed that glutamine synthetase (GS), an enzyme catalyzing glutamate and ammonia to glutamine, was responsible for the metabolic alteration. ChIP and luciferase reporter assays revealed that GS could be transcriptionally regulated by STAT5. Knockdown of GS delayed DNA repair, weakened nucleotide metabolism, and enhanced radiosensitivity both invitro and invivo. Our data show that GS links glutamine metabolism to radiotherapy response through fueling nucleotide synthesis and accelerating DNA repair.

Inhibition of novel GCN5-ATM axis restricts the onset of acquired drug resistance in leukemia.

Leukemia is majorly treated by topoisomerase inhibitors that induce DNA double strand breaks (DSB) resulting in cell death. Consequently, modulation of DSB repair pathway renders leukemic cells resistant to therapy. As we do not fully understand the regulation of DSB repair acquired by resistant cells, targeting these cells has been a challenge. Here we investigated the regulation of DSB repair pathway in early drug resistant population (EDRP) and late drug resistant population (LDRP). We found that doxorubicin induced equal DSBs in parent and EDRP cells; however, cell death is induced only in the parent cells. Further analysis revealed that EDRP cells acquire relaxed chromatin via upregulation of lysine acetyl transferase KAT2A (GCN5). Drug treatment induces GCN5 interaction with ATM facilitating its recruitment to DSB sites. Hyperactivated ATM maximize H2AX, NBS1, BRCA1, Chk2, and Mcl-1 activation, accelerating DNA repair and survival of EDRP cells. Consequently, inhibition of GCN5 significantly reduces ATM activation and survival of EDRP cells. Contrary to EDRP, doxorubicin failed to induce DSBs in LDRP because of reduced drug uptake and downregulation of TOP2β. Accordingly, ATM inhibition prior to doxorubicin treatment completely eliminated EDRP but not LDRP. Furthermore, baseline AML samples (n = 44) showed significantly higher GCN5 at mRNA and protein levels in MRD positive compared to MRD negative samples. Additionally, meta-analysis (n = 221) showed high GCN5 expression correlates with poor overall survival. Together, these results provide important insights into the molecular mechanism specific to EDRP and will have implications for the development of novel therapeutics for AML.

Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

Bone atrophy and its related fragility fractures are frequent, late side effects of radiotherapy in cancer survivors and have a detrimental impact on their quality of life. In another study, we showed that parathyroid hormone 1-34 and anti-sclerostin antibody attenuates radiation-induced bone damage by accelerating DNA repair in osteoblasts. DNA damage responses are partially regulated by the ubiquitin proteasome pathway. In the current study, we examined whether proteasome inhibitors have similar bone-protective effects against radiation damage. MG132 treatment greatly reduced radiation-induced apoptosis in cultured osteoblastic cells. This survival effect was owing to accelerated DNA repair as revealed by γH2AX foci and comet assays and to the up-regulation of Ku70 and DNA-dependent protein kinase, catalytic subunit, essential DNA repair proteins in the nonhomologous end-joining pathway. Administration of bortezomib (Bzb) reversed the loss of trabecular bone structure and strength in mice at 4 wk after focal radiation. Histomorphometry revealed that Bzb significantly increased the number of osteoblasts and activity in the irradiated area and suppressed the number and activity of osteoclasts, regardless of irradiation. Two weeks of Bzb treatment accelerated DNA repair in bone-lining osteoblasts and thus promoted their survival. Meanwhile, it also inhibited bone marrow adiposity. Taken together, we demonstrate a novel role of proteasome inhibitors in treating radiation-induced osteoporosis.-Chandra, A., Wang, L., Young, T., Zhong, L., Tseng, W.-J., Levine, M. A., Cengel, K., Liu, X. S., Zhang, Y., Pignolo, R. J., Qin, L. Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

Abstract 3174: GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia

Abstract Abstract Introduction- Resistance to therapeutics targeting topoisomerase 2 is a major problem in the treatment of leukemia. Cells which survive and give rise to relapsed leukemia are known to modulate different pathways like downregulation of drug target, reduced drug accumulation, and improved DNA repair; ultimately leading to survival of drug resistant cells. Here, we wanted to identify the earliest detectable changes occurring when cells become resistant to topoisomerase 2 inhibitors. Methodology- We generated early and late stage drug (doxorubicin) resistant leukemic sub cell line from K562 and THP-1 parent cells, HL-60/MX2 resistant sub cell line of HL60 was also used for this study. Molecular changes were analyzed by electron microscopy, quantitative PCR, western blotting, immunofluorescence, flow cytometry, molecular inhibitors and MTT assay. Results were further confirmed in blast cells of AML (n=44) patient samples collected for this study. Furthermore, meta-analysis for survival was done from microarray expression data of 221 patient samples using PrognoScan. Results- We mapped molecular changes acquired by leukemic cells during evolution from Early Drug Resistant Population (EDRP) to Late Drug Resistant Population (LDRP). We found unlike LDRP, EDRP cells do not possess known bona fide drug-resistance mechanisms namely limited drug accumulation, reduced DNA damage or expression of drug target. Instead they survive by acquiring ‘poised epigenetic state’ that enhanced their DNA repair. Mechanistically, GCN5, a histone acetyl transferase get selectively upregulated in EDRP cells mediating higher H4K16 acetylation levels and consequent euchromatin state of EDRP. Upon Doxorubicin treatment, H4K16ac facilitate higher ATM recruitment and activation causing efficient activation of H2AX, NBS1, BRCA1, Chk2 and Mcl-1, accelerating DNA repair and survival of EDRP cells. Inhibition of GCN5 with Doxorubicin treatment significantly reduces H4K16ac levels, ATM recruitment and cell survival of EDRP cells. Similarly, ATM inhibition along with Doxorubicin completely ablates EDRP but not LDRP. Furthermore, baseline AML patient samples (n=44) showed significantly higher GCN5 expression in MRD positive compared to MRD negative samples. Additionally, meta-analysis of 221 AML patients showed increased GCN5 expression associates with poor survival of AML patients. Conclusion- We identify GCN5 expression as marker that defines onset of resistance in leukemia and GCN5 mediated ATM activation via H4K16ac as a novel non-genetic route facilitating EDRP cell survival with enhanced DNA repair. These data also highlight the clinical relevance of targeting GCN5 and ATM during early resistance to prevent emergence of difficult to treat stable diverse resistance in leukemia. Citation Format: Sameer J. Salunkhe, Jyothi Nair, Ekjot Kaur, Neha Chaoudhary, Sanket Shah, Ketaki Patkar, Dev Anand, Navin Khattry, Syed K. Hasan, Shilpee Dutt. GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2017-3174

Special AT-rich Sequence-binding Protein 1 (SATB1) Functions as an Accessory Factor in Base Excision Repair

Base excision repair is initiated by DNA glycosylases that recognize specific altered bases. DNA glycosylases for oxidized bases carry both a glycosylase activity that removes the faulty base and an apyrimidinic/apurinic lyase activity that introduces a single-strand DNA incision. In particular, the CUT domains within the CUX1 and CUX2 proteins were recently shown to interact with the 8-oxoguanine (8-oxoG) DNA glycosylase and stimulate its enzymatic activities. SATB1, which contains two CUT domains, was originally characterized as a T cell-specific genome organizer whose aberrant overexpression in breast cancer can promote tumor progression. Here we investigated the involvement of SATB1 in DNA repair. SATB1 knockdown caused a delay in DNA repair following exposure to H2O2, an increase in OGG1-sensitive oxidized bases within genomic DNA, and a decrease in 8-oxoG cleavage activity in cell extracts. In parallel, we observed an increase in phospho-CHK1 and γ-H2AX levels and a decrease in DNA synthesis. Conversely, ectopic expression of SATB1 accelerated DNA repair and reduced the levels of oxidized bases in genomic DNA. Moreover, an enhanced GFP-SATB1 fusion protein was rapidly recruited to laser microirradiation-induced DNA damage. Using purified proteins, we showed that SATB1 interacts directly with OGG1, increases its binding to 8-oxoG-containing DNA, promotes Schiff base formation, and stimulates its glycosylase and apyrimidinic/apurinic lyase enzymatic activities. Structure/function analysis demonstrated that CUT domains, but not the homeodomain, are responsible for the stimulation of OGG1. Together, these results identify another CUT domain protein that functions both as a transcription factor and an accessory factor in base excision repair.

Suppression of Sclerostin Alleviates Radiation-Induced Bone Loss by Protecting Bone-Forming Cells and Their Progenitors Through Distinct Mechanisms.

Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/β-catenin pathway can overcome radiation-induced DNA damage and apoptosis of osteoblastic cells. Neutralization of circulating sclerostin with a monoclonal antibody (Scl-Ab) is an innovative approach for treating osteoporosis by enhancing Wnt/β-catenin signaling in bone. Together with the fact that focal radiation increases sclerostin amount in bone, we sought to determine whether weekly treatment with Scl-Ab would prevent focal radiotherapy-induced osteoporosis in mice. Micro-CT and histomorphometric analyses demonstrated that Scl-Ab blocked trabecular bone structural deterioration after radiation by partially preserving osteoblast number and activity. Consistently, trabecular bone in sclerostin null mice was resistant to radiation via the same mechanism. Scl-Ab accelerated DNA repair in osteoblasts after radiation by reducing the number of γ-H2AX foci, a DNA double-strand break marker, and increasing the amount of Ku70, a DNA repair protein, thus protecting osteoblasts from radiation-induced apoptosis. In osteocytes, apart from using similar DNA repair mechanism to rescue osteocyte apoptosis, Scl-Ab restored the osteocyte canaliculi structure that was otherwise damaged by radiation. Using a lineage tracing approach that labels all mesenchymal lineage cells in the endosteal bone marrow, we demonstrated that radiation damage to mesenchymal progenitors mainly involves shifting their fate to adipocytes and arresting their proliferation ability but not inducing apoptosis, which are different mechanisms from radiation damage to mature bone forming cells. Scl-Ab treatment partially blocked the lineage shift but had no effect on the loss of proliferation potential. Taken together, our studies provide proof-of-principle evidence for a novel use of Scl-Ab as a therapeutic treatment for radiation-induced osteoporosis and establish molecular and cellular mechanisms that support such treatment. © 2016 American Society for Bone and Mineral Research.

Abstract A15: The integrin α6β4 promotes pancreatic cancer invasion by altering DNA repair-mediated epigenetics

Abstract The integrin α6β4 is upregulated in pancreatic carcinoma and signaling from this integrin promotes metastatic properties, including cancer cell invasion. We have previously shown that the integrin α6β4 promotes such properties by dramatically altering the transcriptome toward an invasive phenotype. Furthermore, we have found that transcriptome alterations can be accomplished through targeted DNA demethylation of specific promoters, as we have shown with the pro-metastatic gene S100A4. In this study, we find that signaling from the integrin α6β4 dramatically upregulates expression of amphiregulin (Areg) and epiregulin (Ereg), ligands for the epidermal growth factor receptor (EGFR), and that these ligands promote pancreatic carcinoma invasion. To determine if Areg and Ereg are regulated by DNA methylation, pancreatic cancer cells with low expression of the integrin α6β4 were treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC), which resulted in stable over expression of Areg and Ereg, as measured by QPCR. Furthermore, expression of a dominant negative form of integrin β4 or blockade of β4 expression by RNAi suppressed induction of Areg and Ereg by DAC. Similarly, treatment of cells with high integrin α6β4 with the methyl donor S-adenosylmethionine inhibited gene expression of Areg and Ereg. In order to confirm these results, genomic DNA from pancreatic cancer cells with either high or low expression of integrin α6β4 was isolated and whole genome bisulfite sequencing was performed. We find that cells with high integrin α6β4 have lower overall methylation levels compared to cells with low integrin α6β4. As expected, we also find lower methylation levels within the first exon of both Areg and Ereg when the integrin α6β4 is high. These regions of hypomethylation correspond to known H3K27 acetylation marks found in ENCODE databases and reported in the USCS genome browser. Thus, these data implicate promoter demethylation as the driver of Areg and Ereg gene expression mediated by integrin α6β4. It is currently accepted that active DNA demethylation can occur via DNA repair. To test our hypothesis that DNA repair is responsible for DNA demethylation of Areg and Ereg in pancreatic cancer cells, we modulated several components of the DNA repair pathway including GADD45A, TDG and Parp-1. The chemotherapeutic agent Gemcitabine has been shown to inhibit multiple components of DNA repair, as well as DNA demethylation mediated by Gadd45A. Treatment of pancreatic cancer cells with Gemcitabine resulted in a marked reduction in Areg and Ereg expression when the integrin α6β4 is high, indicating DNA repair is required for gene expression. To further test the involvement of Gadd45A, we used either RNAi-mediated knockdown or cDNA overexpression to alter Gadd45A levels and examined their effect on Areg and Ereg. In both instances, Areg and Ereg expression positively correlated with Gadd45A, particularly when the integrin α6β4 is high, indicating that Gadd45A is a rate-limiting step in Areg and Ereg overexpression. Thymine DNA glycosylase (TDG) has likewise been shown to mediate active DNA demethylation. Using stable shRNA, we show that TDG is required for Areg and Ereg expression in integrin α6β4 high cells, and nuclear localization of TDG is much higher in cells with high integrin α6β4. We further used a specific inhibitor for Parp-1 and show that Areg and Ereg expression is dependent on Parp-1. Finally, we determine that integrin α6β4 signaling accelerates DNA repair rates by 2.5-fold. Taken together, these data indicated that DNA repair is required to maintain overexpression of Areg and Ereg in response to signaling from the integrin α6β4 and that integrin α6β4 promotes this overexpression by enhancing DNA repair and DNA repair-mediated DNA demethylation at specific sites within the promoter regions of Areg and Ereg. Citation Format: Brittany L. Carpenter, Jinpeng Liu, Chi Wang, Kathleen O'Connor. The integrin α6β4 promotes pancreatic cancer invasion by altering DNA repair-mediated epigenetics. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A15.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Silibinin enhances the repair of ultraviolet B-induced DNA damage by activating p53-dependent nucleotide excision repair mechanism in human dermal fibroblasts.

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 μM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention.

Vascular Smooth Muscle Cell Senescence Promotes Atherosclerosis and Features of Plaque Vulnerability.

Although vascular smooth muscle cell (VSMC) proliferation is implicated in atherogenesis, VSMCs in advanced plaques and cultured from plaques show evidence of VSMC senescence and DNA damage. In particular, plaque VSMCs show shortening of telomeres, which can directly induce senescence. Senescence can have multiple effects on plaque development and morphology; however, the consequences of VSMC senescence or the mechanisms underlying VSMC senescence in atherosclerosis are mostly unknown. We examined the expression of proteins that protect telomeres in VSMCs derived from human plaques and normal vessels. Plaque VSMCs showed reduced expression and telomere binding of telomeric repeat-binding factor-2 (TRF2), associated with increased DNA damage. TRF2 expression was regulated by p53-dependent degradation of the TRF2 protein. To examine the functional consequences of loss of TRF2, we expressed TRF2 or a TRF2 functional mutant (T188A) as either gain- or loss-of-function studies in vitro and in apolipoprotein E(-/-) mice. TRF2 overexpression bypassed senescence, reduced DNA damage, and accelerated DNA repair, whereas TRF2(188A) showed opposite effects. Transgenic mice expressing VSMC-specific TRF2(T188A) showed increased atherosclerosis and necrotic core formation in vivo, whereas VSMC-specific TRF2 increased the relative fibrous cap and decreased necrotic core areas. TRF2 protected against atherosclerosis independent of secretion of senescence-associated cytokines. We conclude that plaque VSMC senescence in atherosclerosis is associated with loss of TRF2. VSMC senes cence promotes both atherosclerosis and features of plaque vulnerability, identifying prevention of senescence as a potential target for intervention.

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

IntroductionChemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].MethodsStudies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.ResultsTN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.ConclusionThese studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

NKX3.1 Suppresses TMPRSS2-ERG Gene Rearrangement and Mediates Repair of Androgen Receptor-Induced DNA Damage.

TMPRSS2 gene rearrangements occur at DNA breaks formed during androgen receptor-mediated transcription and activate expression of ETS transcription factors at the early stages of more than half of prostate cancers. NKX3.1, a prostate tumor suppressor that accelerates the DNA repair response, binds to androgen receptor at the ERG gene breakpoint and inhibits both the juxtaposition of the TMPRSS2 and ERG gene loci and also their recombination. NKX3.1 acts by accelerating DNA repair after androgen-induced transcriptional activation. NKX3.1 influences the recruitment of proteins that promote homology-directed DNA repair. Loss of NKX3.1 favors recruitment to the ERG gene breakpoint of proteins that promote error-prone nonhomologous end-joining. Analysis of prostate cancer tissues showed that the presence of a TMPRSS2-ERG rearrangement was highly correlated with lower levels of NKX3.1 expression consistent with the role of NKX3.1 as a suppressor of the pathogenic gene rearrangement.

PTH1–34 Blocks Radiation-induced Osteoblast Apoptosis by Enhancing DNA Repair through Canonical Wnt Pathway

Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1-34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1-34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/β-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the β-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1-34 or Wnt agonist as possible therapy for radiation-induced osteoporosis.

Abstract 1774: Emerging role of Smad7 in DNA damage response pathway as a positive regulator.

Abstract The cellular response to DNA damage is essential in maintaining genomic stability. As DNA damage accumulates, the occurrence rate of cancer is significantly enhanced. Smad7 is known as an inhibitory Smad which negatively regulates TGF-β-induced signaling pathway. Smad7 is predominantly localized in the nucleus even without TGF-β stimulation. However, TGF-β-independent Smad7 function in the nucleus is still not clear. Here, we report Smad7 regulates DNA damage signaling in response to double-strand break induced by radiomimetic drug, neocarzinostatin (NCS). Smad7 overexpression accelerates DNA repair by enhancing phosphorylation of ATM and γH2AX, major DNA damage response signaling, after DNA damage. In Smad7−/− MEF cell, loss of Smad7 leads to failure of ATM phosphorylation and nuclear foci formation at the site of DNA damage upon NCS treatment. We show that Smad7 interacts with activated ATM/MRN complex and this interaction is significantly increased after DNA damage. Additionally, Smad7 co-localizes with pATM, MRN complex and γH2AX at the sites of DNA damage. Taken together, our data suggests that Smad7 plays a critical role in DNA damage response pathway through interaction with activated ATM. In this study, we reveal the de novo function of Smad7 in the nucleus. Citation Format: Sujin Park, Jin Muk Kang, Staci Jakyong Kim, Hyojung Kim, Seong-Jin Kim. Emerging role of Smad7 in DNA damage response pathway as a positive regulator. [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 1774. doi:10.1158/1538-7445.AM2013-1774