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PDF file, 185K, Supplementary Table S1. Sequences of the primers used in plasmid instruction or in real-time PCR. Supplementary Figure S1. The ROS levels in miR-21 over-expressed cells. Supplementary Figure S2 The ROS levels in 5 Gy-irradiated cells. Supplementary Figure S3 IR could not further increase miR-21 levels in the cells over-expressed with miR-21. Supplementary Figure S4 The SOD levels in miR-21 over-expressed cells at different times after exposure to 5 Gy. Supplementary Figure S5 The sequence of SOD33'UTR contains a potential miR-21 binding site. Supplementary Figure S6 SOD3 activity in the cells over-expressed with SOD3. Supplementary Figure S7 The sequence of TNFa3'UTR contains a potential miR-21 binding site. Supplementary Figure S8 The SOD2 mRNA levels in the cells over-expressed with TNFa. Supplementary Figure S9 SOD activity. Supplementary Figure S10 miR-21 does not affect the levels and activities of catalase and glutathione peroxidase.

It is well-known that Radiation-induced fibrosis (RIF) is a late event occurring months to years after the initial radiation exposure. Fibrotic lesions have been shown to manifest in many tissues including the skin, heart, lung, liver and kidney. Fibrosis occurs due to abnormal accumulation of extracellular matrix (ECM) proteins that result in loss of normal tissue and organ function. The cell type involved in RIF is myofibroblasts, which do not undergo apoptosis after healing but instead continue to accumulate, producing excessive amounts of ECM proteins, thereby damaging the tissues and organs. Reactive oxygen species, generated in response to radiation, is one signal that helps maintain the myofibroblast phenotype. In this review, we discuss molecular mechanisms leading to this late radiation event, known biomarkers for prediction, preclinical animal models of radiation-induced toxicity and current clinical trials designed for mitigation and treatment of radiation-induced fibrosis. We also discuss other physical properties such as linear energy transfer (LET) than the ones used in the clinics today which may have the potential to change our understanding on this inevitable pathway from radiation treatment to organ fibrosis.

Simple Summary We applied the genomic fabric principles for personalized gene therapy to a case of clear cell renal cell carcinoma (ccRCC). Despite decades of research, the process of finding the molecular mechanisms responsible for the disease and, more importantly, the therapeutic solution is still a work in progress. We analyzed the transcriptomes of the chest wall metastasis, two distinct cancer nodules, and the cancer-free surrounding tissue in the surgically removed right kidney of a Fuhrman grade 3 metastatic ccRCC patient. The studies revealed that even histopathologically equally classified cancer nodules from the same kidney have different transcriptomic topologies, requiring tailored therapeutic solutions not only for each patient but even for each cancer nodule. We identified death-associated protein kinase 3 (DAPK3); transcription activation suppressor (TASOR); family with sequence similarity 27, member C, long non-coding RNA (FAM27C); and UDP-N-acetylglucosaminyltransferase subunit (ALG13) as the gene master regulators of the four profiled regions and proposed molecular mechanisms by which expression manipulation of TASOR and ALG13 may selectively destroy the cancer cells without affecting many of the normal cells. Abstract Published transcriptomic data from surgically removed metastatic clear cell renal cell carcinoma samples were analyzed from the genomic fabric paradigm (GFP) perspective to identify the best targets for gene therapy. GFP considers the transcriptome as a multi-dimensional mathematical object constrained by a dynamic set of expression controls and correlations among genes. Every gene in the chest wall metastasis, two distinct cancer nodules, and the surrounding normal tissue of the right kidney was characterized by three independent measures: average expression level, relative expression variation, and expression correlation with each other gene. The analyses determined the cancer-induced regulation, control, and remodeling of the chemokine and vascular endothelial growth factor (VEGF) signaling, apoptosis, basal transcription factors, cell cycle, oxidative phosphorylation, renal cell carcinoma, and RNA polymerase pathways. Interestingly, the three cancer regions exhibited different transcriptomic organization, suggesting that the gene therapy should not be personalized only for every patient but also for each major cancer nodule. The gene hierarchy was established on the basis of gene commanding height, and the gene master regulators DAPK3, TASOR, FAM27C and ALG13 were identified in each profiled region. We delineated the molecular mechanisms by which TASOR overexpression and ALG13 silencing would selectively affect the cancer cells with little consequences for the normal cells.

Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed.

The relationship between mass and mass accretion rate in young objects is a powerful tool for interrogating formation and evolution. We analyze a comprehensive database of published accretion diagnostics for ∼400 objects (0.005–2 M ⊙), including 64 brown dwarfs. To eliminate systematics between studies as the source of the four orders of magnitude of observed scatter, we uniformly re-estimate masses and accretion rates from direct observables (e.g., spectral types, line luminosities). In particular, we adopt a single grid of evolutionary models to estimate masses, a single family of scaling relations to estimate accretion luminosity, and update line luminosities using Gaia DR2 distances. We find that this unification produces minimal effects on the observed relation, suggesting systematic and methodological variation is not responsible for the scatter. This paves the way for investigation of physical sources of scatter, including: age variation, intrinsic variability, formation mechanism, and accretion paradigm.

Radiation Therapy (RT) has been critical in cancer treatment regimens to date. However, it has been shown that ionizing radiation is also associated with increased risk of damage to healthy tissues. At high radiation doses, varied effects including inactivation of cells in treated tissue and associated functional impairment are seen. These range from direct damage to the heart; particularly, diffuse fibrosis of the pericardium and myocardium, adhesion of the pericardium, injury to the blood vessels and stenosis. Cardiac damage is mostly a late responding end-point, occurring anywhere between 1 and 10 years after radiation procedures. Cardiovascular disease following radiotherapy was more common with radiation treatments used before the late 1980s. Modern RT regimens with more focused radiation beams, allow tumors to be targeted more precisely and shield the heart and other healthy tissues for minimizing the radiation damage to normal cells. In this review, we discuss radiation therapeutic doses used and post-radiation damage to the heart muscle from published studies. We also emphasize the need for early detection of cardiotoxicity and the need for more cardio-protection approaches where feasible.

Psychological stress can have devastating and lasting effects on a variety of behaviors, especially those associated with mental illnesses such as anxiety and depression. Animal models of chronic stress are frequently used to elucidate the mechanisms underlying the relationship between stress and mental health disorders and to develop improved treatment options. The current study expands upon a novel chronic stress paradigm for mice: predatory stress. The predatory stress model incorporates the natural predator-prey relationship that exists among rats and mice and allows for greater interaction between the animals, in turn increasing the extent of the stressful experience. In this study, we evaluated the behavioral effects of exposure to 15 days of predatory stress on an array of behavioral indices. Up to 2 weeks after the end of stress, adult male mice showed an increase of anxiety-like behaviors as measured by the open field and social interaction tests. Animals also expressed an increase in depressive-like behavior in the sucrose preference test. Notably, performance on the novel object recognition task, a memory test, improved after predatory stress. Taken as a whole, our results indicate that 15 exposures to this innovative predatory stress paradigm are sufficient to elicit robust anxiety-like behaviors with evidence of co-morbid depressive-like behavior, as well as changes in cognitive behavior in male mice.

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.

Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37 degrees Centigrade in space for 14 days before being fixed for analysis of DNA damages with the gamma-H2AX assay. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate gamma rays at 37 degrees Centigrade. Cells exposed to chronic gamma rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET (Linear Energy Transfer) protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

MicroRNA-21 (miR-21) is an oncomir overexpressed in most human tumors in that it promotes malignant growth and progression by acting on multiple targets. Here, we broaden the impact of miR-21 in cancer by showing that it regulates the formation of reactive oxygen species (ROS) that promote tumorigenesis. Key targets of miR-21 in mediating this function were SOD3 and TNFα. We found that miR-21 inhibited the metabolism of superoxide to hydrogen peroxide, produced either by endogenous basal activities or exposure to ionizing radiation (IR), by directing attenuating SOD3 or by an indirect mechanism that limited TNFa production, thereby reducing SOD2 levels. Importantly, both effects contributed to an elevation of IR-induced cell transformation. Our findings, therefore, establish that miR-21 promotes tumorigenesis to a large extent through its regulation of cellular ROS levels. Cancer Res; 72(18); 4707–13. ©2012 AACR.

Purpose S-phase cells are more resistant to low-linear energy transfer (LET) ionizing radiation (IR) than nonsynchronized and G 1 -phase cells, because both nonhomologous end-joining (NHEJ) and homologous recombination repair can repair DNA double-strand breaks (DSBs) in the S phase. Although it was reported 3 decades ago that S-phase cells did not show more resistance to high-LET IR than cells in other phases, the mechanism remains unclear. We therefore attempted to study the phenotypes and elucidate the mechanism involved. Methods and Materials Wild-type and NHEJ-deficient cell lines were synchronized using the double-thymidine approach. A clonogenic assay was used to detect the sensitivity of nonsynchronized, synchronized S-phase, and G 2 -phase cells to high- and low-LET IR. The amounts of Ku bound to DSBs in the high- and low-LET–irradiated cells were also examined. Results S-phase wild-type cells (but not NHEJ-deficient cells) were more sensitive to high-LET IR than nonsynchronized and G 2 -phase cells. In addition, S-phase wild-type cells showed less efficient Ku protein binding to DSBs than nonsynchronized and G 2 -phase cells in response to high-LET IR, although all cells at all phases showed similarly efficient levels of Ku protein binding to DSBs in response to low-LET IR. Conclusions S-phase cells are more sensitive to high-LET IR than nonsynchronized and G 2 -phase cells, because of the following mechanism: it is more difficult for Ku protein to bind to high-LET IR-induced DNA DSBs in S-phase cells than in cells at other phases, which results in less efficient NHEJ.

DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ. © 2008 The Author(s).

Purα is a multifunctional protein that plays an important roles in DNA repair and cell cycle regulation. Purα knockout mice have been successfully established but unfortunately the knockout mice die at the 4th week after birth and that impeded the functional research of Purα in nervous system. The cells originated from nervous system is difficult for the transfection with the routine approaches and the lower transfection efficiency also hindered the research of gene manipulation in the nervous system. The lentivirus provided a new thoroughfare for the research of gene function in nervous system. On the current paper we reported here that lentivirus vector with overexpression of EGFP-Purα fusion fluorescent protein and its control-lentivirus vector with EGFP have been successfully constructed. The constructs were packaged and the human glioblastoma cell, U87MG were infected with these two lentivirus and selected with puromycin to establish the stable cell lines with overexpression of EGFP-Purα fusion protein. The expression level in the established cell lines were examined with qPCR and western blotting assay, the results demonstrated that Purα can be stably expressed in the established cell lines. The further experiments were employed to detect the effects of Purα on cell growth and proliferation with cell counting assay (CCK-8), the results proved that when the cells were treated with different concentrations of HU, the rate of cell survival in Purα overexpression group is higher than that in the control group, it suggested that Purα could protect the cells from the toxicity of HU. The protective effects of Purα on DNA damage were also evaluated with examination of the changes of the amount of DNA damage associated protein, γH2AX, as well as the pulse field gel assay, all the results illustrated that when cells were treated with 2mM HU for 18 hours, the amount of γH2AX is much less in Purα overexpression cell line than that in the control group, pulse field gel assay also exhibited the less DNA damage in Purα overexpression group than that in the control groups. Above all, these results confirmed that Purα plays a protective role in the DNA damage induced by HU.

Background: One of the pathological hallmarks of Alzheimer’s disease is the presence of fibrillary amyloid-β deposits, which result from cleavage of the amyloid precursor protein. Understanding the regulatory mechanism of the amyloid precursor protein gene expression is crucial for comprehending the genesis and development of Alzheimer’s disease. The nucleic acid binding protein, Purα, is best characterized as a transcriptional factor (TF) with affinity to singlestrand G/C-rich regions. In a previous study, we demonstrated that the Purα protein can downregulate amyloid precursor protein (APP) promoter activity, but the mechanism underlying this downregulation requires further investigation. To better understand this mechanism, we analyzed the characteristic of the APP promoter and found that another transcriptional factor, namely Egr-1, can bind the APP promoter and may exert transcriptional regulatory effects on APP gene expression. Therefore, the interaction between these two transcriptional factors may explain the mechanism in regulating APP gene expression. Methodology/Principal Findings: The binding sites of Purα and Egr-1 on the APP promoter 5’-UTR were identified, and reporter plasmids in which the binding sites for Purα and Egr-1 were deleted have been constructed. A luciferase assay was performed, and the results demonstrated that both Purα and Egr-1 lost their regulatory effects when these binding sites were deleted. The luciferase results also demonstrated that Purα can suppress the effects of Egr-1 on APP promoter activities. The electrophoresis mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay results demonstrated that both Purα and Egr- 1 can competently bind to the APP promoter. The endogenous Egr-1 expression was disturbed with the HDAC inhibitor and suramin, and the Egr-1 expression level affected the APP promoter activities and APP gene expression. Purα can also suppress the endogenous expression of Egr-1. Conclusion/Significance: The mechanism through which Purα regulates AβPP gene expression may involve its interaction with Egr-1, which is a positive regulator of the APP promoter. Because both transcriptional factors possess the binding sites in the APP promoter 5’-UTR and the position of these sites are overlapped, there may exist a displacement mechanism for these two transcriptional factors. In addition, Purα also suppresses the endogenous Egr-1 expression. All of these findings explain the mechanism through which Purα regulates APP gene expression.

In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.

Caffeine inhibits ATM and ATR, two important checkpoint regulators, abolishes ionizing radiation-induced checkpoint response, and radiosensitizes cells. Radiation-induced DNA double-strand breaks (DSBs) are repaired by two major processes, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). It remains unclear which repair process, HRR or NHEJ, is affected when the checkpoint responses are abolished by caffeine. In this study we observed the effect of caffeine on gene-targeted DT40 chicken lymphoblast cells. We show that caffeine efficiently abolishes S- and G(2)-phase checkpoint responses after irradiation in all cell lines tested and greatly radiosensitizes wild-type and ATM(-/-) cells, the partially checkpoint-deficient cells. However, caffeine has a much smaller radiosensitizing effect on RAD54(-/-) cells and has no effect on RAD51-deficient cells. RAD51 and RAD54 are the important factors for HRR. Our results indicate that the checkpoint responses abolished by caffeine (S and G(2)) mainly affect HRR, which results in cell radiosensitization.

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg2+. At 0.5 mM Mg2+, where only DNA ligase IV is expected to retain activity, low levels of end joining (∼10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg2+ in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.

Replication protein-A (RPA) is a heterotrimeric single-stranded DNA-binding protein playing essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair and homologous recombination. Here, the role of RPA in the rejoining of radiation-induced DNA double-strand breaks (DSB) by non-homologous end-joining (NHEJ) was investigated.A previously described in vitro assay for DSB rejoining was employed. The assay used 'naked' genomic DNA prepared from agarose-embedded G(1)-phase A549 cells as a substrate and extracts prepared from HeLa cells as a source of enzymes. Rejoining of DSB in this assay is absolutely dependent on cell extract and proceeds, under optimal reaction conditions, to an extent similar to that observed in intact cells. For experiments, extracts were supplemented with excess purified recombinant RPA. Alternatively, RPA was removed from the extracts either by fractionation or immunodepletion.Although the rejoining of DSB in vitro was not absolutely dependent on RPA, it proceeded faster and to higher levels of completion when recombinant protein was added to the extracts. Depletion of RPA from extracts reduced the rejoining half-times and addition of purified recombinant protein restored the kinetics of DSB rejoining. Extract fractionation indicated the operation of at least two pathways in DSB rejoining, only one of which was facilitated by RPA.The results suggest that in addition to its role in homologous recombination, RPA may also have a supportive role in some forms of non-homologous end-joining.

The carcinogenic risk of high-charge and energy (HZE) particle exposure arises from its ability to both induce complex DNA damage and from its ability to evoke deleterious, non-DNA targeted effects. We investigate here whether these nontargeted effects involve dysregulation of double-strand break repair, such that a history of HZE exposure heightens the risks from future injury. We used a new human cell reporter line, in which expression of the I-SceI meganuclease stimulates both translocations on different chromosomes, and deletions on the same chromosome. Exposure to 1.0 Gy of 600 MeV/u (56)Fe ions led to a doubling in the frequency of I-SceI-mediated translocations and a smaller, but nevertheless significant, increase in the frequency of I-SceI-mediated deletions. This mutagenic repair phenotype persisted for up to two weeks and eight population doublings. The phenotype was not induced by low-linear energy transfer radiation or by a lower dose of HZE-particle radiation (0.3 Gy) indicating that the effect is radiation quality and dose dependent. The mutagenic repair phenotype was associated with the presence of micronuclei and persistent DSB repair foci, consistent with a hypothesis that genomic stress is a causative factor.

Heavy ion therapy is a promising approach for cancer treatment, particularly for locally advanced solid tumors. High linear energy transfer (LET) radiation induces greater lesions density in close proximity (clusters) compared that in low-LET radiation. Such clustered DNA damage is thought to be more difficult to repair than isolated DNA damage produced by equivalent doses of low-LET radiation and persist longer in irradiated cells. It has been known that homologous combination contributes to the repair of clustered DNA damage other than classic non-homologous end-joining (NHEJ). DNA double-strand breaks are also repaired by an alternative NHEJ pathway with PARP1, DNA ligase III and Mre11. This alternative pathway is error prone and contributes to genomic instability, such as chromosomal translocation and telomere fusion. Genetic analysis of tumors in TCGA database revealed that these genes are amplified in human tumors and their overexpression is correlated with poor overall survival of cancer patients. However, influence of alternative NHEJ pathways on the relative biological effectiveness (RBE) of heavy ions remains unclear. Here we investigated the effect of PARP-1 and DNA ligase III on RBE of heavy ion particles on cell survival and chromosomal aberration in breast cancer cells and pancreatic cancer cells. Cells were irradiated with X-ray, Carbon (290 MeV/n, 13 KeV/μM) and Iron (1 GeV/n, 150 KeV/μM). We found that overexpression of PARP-1 and DNA ligase III increased RBE of carbon and iron. PARP inhibitor reduced chromosomal aberration induced by heavy ions. This study may provide predictor of radiation responsive of tumor cells from cancer patients that would lead to patient stratification for heavy ion therapy selection Citation Format: Huichen Wang, Premkuma Saganti. Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3713.

High charge and energy (HZE) particles are a component of galactic cosmic rays. They cause complex damage to DNA and other cellular components, leading to both direct and indirect biological effects. Here, we investigate the hypothesis that one of these indirect effects is to compromise the accuracy of the DNA repair machinery, reducing the ability to cope with subsequent genotoxic insults. To this end, we used a new human reporter cell line with single-copy integrated fluorescent reporter cassettes that allow measurement of the frequency of mutagenic repair. Introduction of the rare cutting I-SceI nuclease stimulates both translocations (joining of two I-SceI sites on different chromosomes), and deletions (joining of two I-SceI sites after deletion of an intervening fragment) [ 1]. These can be measured simultaneously in the same cell population using different color reporter genes. To test the effect of HZE exposure on the frequency of translocations and deletions in this assay, cells were exposed to 600 MeV/u 56Fe ions or 1000 MeV/u 48Ti ions at doses of 0.3 or 1.0 Gy. They were allowed to recover and challenged with I-SceI at 1, 7, 14, 21 and 28 days post-irradiation. Results showed that HZE particle irradiation significantly increased the frequency of I-SceI translocations and deletions above baseline levels. There was an increase in translocations by up to threefold, seen in both 56Fe- and 48Ti-treated populations. There was also a more modest, but significant increase in I-SceI-mediated deletions seen in a population that received the higher dose (1.0 Gy) of 56Fe particles. The increased frequency of I-SceI-induced translocations and deletions persisted for 2–3 weeks with 56Fe (but not with 48Ti). The increased frequency of translocations and deletions was not observed in populations treated with low-LET radiation at doses up 3 Gy. Thus, the phenomenon, which we term the ‘mutagenic repair phenotype’ depends on both dose and radiation quality. The mutagenic repair phenotype was associated with an elevated frequency of micronuclei and excess DNA repair foci, suggesting that persistent genomic stress might be one causative factor [ 2]. To further explore the mechanism underlying the mutagenic DNA repair phenotype, we performed genome-wide expression profiling on cells that were harvested 7 days post-56Fe ion exposure. Results showed significant alterations in 234 genes. Many of the most highly induced genes encode secreted proteins that have previously been implicated in cellular senescence or pro-inflammatory processes. These findings suggest that a paracrine mechanism (induction of a set of senescence or inflammation-related proteins) may also contribute to the mutagenic repair phenotype. Altered regulation of cellular double-strand break repair, with an increased reliance on an error-prone pathway, is a novel mechanism whereby an indirect effect of HZE exposure may amplify cancer risk.

Improving frequency interference is the primary purpose of network planning and optimization in TD_SCDMA. In this paper, a new algorithm to improve frequency interference, named Ant Colony Algorithm (ACA), was put forward to; in addition existent methods were enriched. We described ACA principle, and then a new specific scenario was investigated and introduced into the frequency planning. Afterwards, by applying the algorithm into the mainstream planning software APOX® we provided some comparative actual projects of the two algorithms: ACA and graph coloring. In APOX®, frequency pollution was defined as the number of prime frequency with frequency compared to RSCP in the scope of 3db.So, after comparative analysis of the actual results of TD-SCDMA network planning projects, it comes to the conclusion that frequency interference is improved to some extent, at the same time frequency reuse distance is expanded by applying the new algorithm.

The nucleic acid-binding protein Puralpha is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Puralpha interacts with HIV-1 Tat, which regulates homologous recombination-directed DNA repair (HRR).We investigated Rad51 and HRR regulation in mouse embryo fibroblasts (MEFs) from PURA -/- knockout mice that lack Puralpha.Rad51 was induced in PURA -/- MEFs but was repressed when Puralpha was ectopically expressed in these cells. Similarly Rad51 inversely correlated with the level of Puralpha in normal postnatal mouse brain. HIV-1 Tat stimulated HRR DNA repair of I-SceI induced DNA DSBs and the nuclear appearance of Rad51 foci. In contrast, Puralpha suppressed HRR DNA repair, Rad51 expression, and Rad51 foci formation.Tat stimulates the Rad51 promoter involving both Puralpha-dependent and Puralpha-independent mechanisms. Interaction between Puralpha and Tat may have opposing effects on Rad51 expression. The effects on HRR may contribute to HIV-1 associated pathogenesis.

Puralpha is a sequence-specific nucleic acid binding protein that is involved in multiple cellular functions including regulation of transcription, initiation of DNA replication, cell cycle progression, and neuronal cell differentiation. Its potential role in DNA repair has not been explored. We have now analyzed the role of Puralpha in the cellular response to replication-associated DNA repair of double-strand breaks (DSBs) using Puralpha knockout mouse embryo fibroblasts (MEFs). We found that Puralpha negative cells are hypersensitive to the DNA replication inhibitor, hydroxyurea (HU), and that HU induces excessive DSBs, which delayed the resumption of cell cycle progression after HU treatment. Reintroduction of Pura into Pura null cells reduced the accumulation of DSBs and enhanced DNA end joining. These results suggest a role for Puralpha as a caretaker protein that is involved in the repair of DSBs induced by stalled replication forks.

Inflammatory cytokines have been implicated in the regulation of radiation-induced genomic instability in the hematopoietic system and have also been shown to induce chronic DNA damage responses in radiation-induced senescence. We have previously shown that human bronchial epithelial cells (HBEC3-KT) have increased genomic instability and IL-8 production persisting at day 7 after exposure to high-LET (600 MeV/nucleon (56)Fe ions) compared to low-LET (320 keV X rays) radiation. Thus, we investigated whether IL-8 induction is part of a broader pro-inflammatory response produced by the epithelial cells in response to damage, which influences genomic instability measured by increased micronuclei and DNA repair foci frequencies. We found that exposure to radiation induced the release of multiple inflammatory cytokines into the media, including GM-CSF, GROα, IL-1α, IL-8 and the inflammation modulator, IL-1 receptor antagonist (IL-1RA). Our results suggest that this is an IL-1α-driven response, because an identical signature was induced by the addition of recombinant IL-1α to nonirradiated cells and functional interference with recombinant IL-1RA (Anakinra) or anti-IL-1α function-blocking antibody, decreased IL-8 production induced by radiation exposure. However, genomic instability was not influenced by this pathway as addition of recombinant IL-1α to naive or irradiated cells or the presence of IL-1 RA under the same conditions as those that interfered with the function of IL-8, did not affect micronuclei or DNA repair foci frequencies measured at day 7 after exposure. While dose-response studies revealed that genomic instability and IL-8 production are the consequences of targeted effects, experiments employing a co-culture transwell system revealed the propagation of pro-inflammatory responses but not genomic instability from irradiated to nonirradiated cells. Collectively, these results point to a cell-autonomous mechanism sustaining radiation-induced genomic instability in this model system and suggest that while molecules associated with these mechanisms could be markers for persisting damage, they reflect two different outcomes.

Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characterization of NHEJ has significantly benefited from in vitro plasmid end-joining assays that can complement and extend information obtained from genetic studies. There is evidence that several factors involved in DNA-PK-dependent NHEJ remain to be identified. In addition, under certain circumstances, cells utilize backup pathways of NHEJ that depend on unknown factors to remove DSBs. Characterization of these putative factors will benefit from plasmid-based assays of DNA end-joining. Here, we describe a protocol for in vitro end-joining using plasmid DNA as substrate. The required procedures include: (1) preparation of HeLa-cell nuclear extract; (2) preparation of plasmid substrate DNA; (3) assembly of in vitro DNA repair reactions; and (4) product analysis by gel electrophoresis. The assay is powerful and easy to perform, but one should be aware that it represents an oversimplification, as it does not consider the in vivo organization of DNA into chromatin.

Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characterization of NHEJ has significantly benefited from in vitro plasmid end-joining assays that can complement and extend information obtained from genetic studies. There is evidence that several factors involved in DNA-PK-dependent NHEJ remain to be identified. In addition, under certain circumstances, cells utilize backup pathways of NHEJ that depend on unknown factors to remove DSBs. Characterization of these putative factors will benefit from plasmid-based assays of DNA end-joining. Here, we describe a protocol for in vitro end-joining using plasmid DNA as substrate. The required procedures include: (1) preparation of HeLa-cell nuclear extract; (2) preparation of plasmid substrate DNA; (3) assembly of in vitro DNA repair reactions; and (4) product analysis by gel electrophoresis. The assay is powerful and easy to perform, but one should be aware that it represents an oversimplification, as it does not consider the in vivo organization of DNA into chromatin.

Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV–deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.

ATR is one of the most important checkpoint proteins in mammalian cells responding to DNA damage. Cells defective in normal ATR activity are sensitive to ionizing radiation (IR). The mechanism by which ATR protects the cells from IR-induced killing remains unclear. DNA double-strand breaks (DSBs) induced by IR are critical lesions for cell survival. Two major DNA DSB repair pathways exist in mammalian cells: homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). We show that the doxycycline (dox)-induced ATR kinase dead (ATRkd) cells have the similar inductions and rejoining rates of DNA DSBs compared with cells without dox induction, although the dox-induced ATRkd cells are more sensitive to IR and have the deficient S and G2 checkpoints. We also show that the dox-induced ATRkd cells have a lower HRR efficiency compared with the cells without dox induction. These results indicate that the effects of ATR on cell radiosensitivity are independent of NHEJ but are linked to HRR that may be affected by the deficient S and G2 checkpoints.

In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.

In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.

We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause-effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.

Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.

After exposure to ionizing radiation, proliferating cells actively slow down progression through the cell cycle through the activation of checkpoints to provide time for repair. Two major complementary DNA double-strand break (DSB) repair pathways exist in mammalian cells, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). The relationship between checkpoint activation and these two types of DNA DSB repair pathways is not clear. Caffeine, as a nonspecific inhibitor of ATM and ATR, abolishes multi-checkpoint responses and sensitizes cells to radiation-induced killing. However, it remains unknown which DNA repair process, NHEJ or HRR, or both, is affected by caffeine-abolished checkpoint responses. We report here that caffeine abolishes the radiation-induced G(2)-phase checkpoint and efficiently sensitizes both NHEJ-proficient and NHEJ-deficient mammalian cells to radiation-induced killing without affecting NHEJ. Our results indicate that caffeine-induced radiosensitization occurs by affecting an NHEJ-independent process, possibly HRR.

Reactive oxygen species (ROS) are involved in amplifying the radiation-induced damage to macromolecules as well as in downstream early responses leading to DNA repair or cell death and in delayed responses leading to tissue damage and/or tumorigenesis. We found in immortalized normal human bronchial epithelial cells (HBEC-3KT) following exposure to 1 Gy low (X-rays) or high (Fe ions) LET radiation that ROS levels persist elevated for up to eight population doublings (2 weeks) in the progeny of cells that repaired the radiation-induced damage and survived. To evaluate the function of persistently elevated ROS levels, we interrogated cells over the same period for genomic instability, proliferation and senescence biomarkers and evaluated whether ROS are functionally related to any of these phenotypes. We found that increased ROS production overlapped temporally with the persistence of reporters for genomic instability, increased micronucleus frequency and the presence of γH2AX-53BP1 positive ionizing radiation-induced foci. Although low LET radiation at high doses and high LET radiation induced a senescence-like phenotype dependent on ATM and p38 MAPK activity, this pathway was not a causative factor in ROS generation and did not affect cell proliferation rates at day 7 post-irradiation. On the contrary, when this pathway was inhibited, ROS levels increased further and micronucleus formation frequency was reduced. Because these results suggest a positive effect for increased ROS levels in the resolution of persistent micronucleus formation, their role was further confirmed by exposure to exogenous hydrogen peroxide, which resulted in a reduced micronucleus frequency. These results implicate ROS as an effector in the resolution of genomic instability and suggest that the senescence phenotype induced by high LET radiation or high low LET radiation dose promotes genomic instability by interfering with the cellular mechanism regulating ROS levels or the DNA repair machinery.

Human cell transformation is a key step for oncogenic development, which involves multiple pathways; however, the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC), we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung, breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and, OCT4, in turn, stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover, we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a, promotes p63 but suppresses p53 expression, which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and, most likely, also to the iPSC process.

Rejoining of ionizing radiation (IR) induced DNA DSBs usually follows biphasic kinetics with a fast (t(50): 5-30 min) component attributed to DNA-PK-dependent non-homologous endjoining (NHEJ) and a slow (t(50): 1-20 h), as of yet uncharacterized, component. To examine whether homologous recombination (HR) contributes to DNA DSB rejoining, a systematic genetic study was undertaken using the hyper-recombinogenic DT40 chicken cell line and a series of mutants defective in HR. We show that DT40 cells rejoin IR-induced DNA DSBs with half times of 13 min and 4.5 h and contributions by the fast (78%) and the slow (22%) components similar to those of other vertebrate cells with 1000-fold lower levels of HR. We also show that deletion of RAD51B, RAD52 and RAD54 leaves unchanged the rejoining half times and the contribution of the slow component, as does also a conditional knock out mutant of RAD51. A significant reduction (to 37%) in the contribution of the fast component is observed in Ku70(-/-) DT40 cells, but the slow component, operating with a half time of 18.4 h, is still able to rejoin the majority (63%) of DSBs. A double mutant Ku70(-/-)/RAD54(-/-) shows similar half times to Ku70(-/-) cells. Thus, variations in HR by several orders of magnitude leave unchanged the kinetics of rejoining of DNA DSBs, and fail to modify the contribution of the slow component in a way compatible with a dependence on HR. We propose that, in contrast to yeast, cells of vertebrates are 'hard-wired' in the utilization of NHEJ as the main pathway for rejoining of IR-induced DNA DSBs and speculate that the contribution of homologous recombination repair (HRR) is at a stage after the initial rejoining.

To investigate the role of DNA-dependent protein kinase (DNA-PK) in the rejoining of ionizing radiation-induced DNA double-strand breaks (dsb).This study employed previously described in vitro assays that utilize nuclei or 'naked' DNA prepared from agarose-embedded cells as a substrate and S-HeLa cell extracts as a source of enzymes. Rejoining of dsb in these assays is absolutely dependent on cell extract and it proceeds, under optimal reaction conditions, to an extent similar to that observed in intact cells. Results were confirmed in a plasmid-based assay for in vitro rejoining of dsb.It is shown that concentrations of wortmannin completely inhibiting DNA-PK activity profoundly affect the rejoining of dsb in vivo, but have no effect on dsb rejoining in vitro. Furthermore, fractionation of cell extracts using ammonium sulphate precipitation, generates protein fractions that are able to support dsb rejoining, despite the fact that they do not contain detectable amounts of either DNA-PKcs or Ku80. Efficient rejoining of dsb in vitro is also observed with extracts of MO59J cells that lack DNA-PK activity. Finally, rejoining of dsb remains unaffected by wortmannin in a plasmid-based assay, and is also detectable with extracts of MO59J cells.These findings are in contrast with genetic studies demonstrating a requirement for DNA-PK activity for efficient rejoining of dsb in vivo. The difference between in vitro and in vivo results may not be attributed to chromatin structure since wortmannin was without an effect when using nuclei as a substrate. It is speculated that the differences between in vivo and in vitro results can be explained either by assuming the operation of multiple pathways in dsb rejoining, some of which do not require DNA-PK, or by postulating a purely regulatory/damage-sensing role for DNA-PK in intact cells but no direct involvement in dsb rejoining.

The breast and ovarian cancer susceptibility gene BRCA1, is a nuclear phosphoprotein which functions as a tumor suppressor in human breast cancer cells. BRCA1 protein contains an amino-terminal zinc finger motif and a carboxy-terminal acidic region. Recently, the carboxy-terminal region of BRCA1 and the amino-terminal region of BRCA2 proteins were shown to function as transactivation domains when fused to GAL4 DNA binding domain. We have recently isolated and characterized two new naturally occurring variants of BRCA1 (BRCA1a/p110 and BRCA1b/p100) which are phosphoproteins containing phosphotyrosine that associate with E2F transcriptional factors, cyclins and cyclin dependent kinases indicating a role for BRCA1 proteins in cell-cycle regulation. Here we show for the first time that the amino-terminal region of BRCA1a (BNT) but not BRCA1b can also function as a transcriptional activator when fused to GAL4 DNA binding domain. Thus, BRCA1/1a proteins contain two autonomous transcriptional activation domains, one at the amino-terminal region (BNT) and the other at the carboxy-terminal region (BCT). BRCA1b retains only the BCT domain since it has lost part of the potential BNT domain as a result of alternative splicing. Our results also suggest the presence of an inhibitory domain at the carboxy terminal region of BRCA1 and BRCA1a proteins (BID). Thus, BRCA1b protein may function as a dominant negative variant that could regulate the transcriptional activity of BRCA1/BRCA1a proteins and hence may serve as a marker for identifying individuals with greater potential for developing breast cancer. It may be possible that loss of transcriptional activation or protein-protein interactions in patients with mutations in the amino terminal zinc finger domain could deprive the cell of an important mechanism for regulating cell proliferation leading to the development of breast cancer.

The tumor suppressor gene BRCA1, is a nuclear phosphoprotein which associates with RNA polymerase II holoenzyme. CBP is a component of the holoenzyme. Previously, we have characterized two new BRCA1 splice variants BRCA1a/p110 and BRCA1b/p100. In the present study, the carboxy-terminal domain of transcription factor CBP interacts both in vivo and in vitro with full length BRCA1a and BRCA1b proteins as demonstrated by mammalian two- hybrid assays, co-immunoprecipitation/western blot studies, GST binding assays and histone acetyl transferase (HAT) assays of BRCA1 immunoprecipitates from human breast cancer cells. Our results suggest that one of the mechanisms by which BRCA1 proteins function is through recruitment of CBP associated HAT/FAT (transcription factor acetyl-transferase) activity for acetylation of either themselves or general transcription factors or both to specific promoters resulting in transcriptional activation.

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.

Ionizing radiation induces DNA damage and oxidative stress, leading cell death during cancer therapy or genomic instability and carcinogenesis amentoflavone is a purified component extracted from medicine plants that are used to treat various diseases. Here we investigated the role of amentoflavone in radiation response in hippocampal neurons (HT22) and glioblastoma cells (U87 MG) following exposure to high linear transfer (LET) radiation and low LET radiation We found that Amentoflavone protects hippocampal neurons from radiation and enhances radiosensitivity of glioblastoma cells.Treatment of amentoflavone increased survival fractions in hippocampal neuronal cells following exposure to X-Ray radiation and 56Fe particles, though amentoflavone enhanced radiosensitivity of glioblastoma cells. Amentoflavone attenuated reactive oxygen species (ROS) and regulated DNA damage repair in irradiated hippocampal neurons through superoxide dismutase 1 (SOD1) and Foxkhead box O3 a (FOXO3a) pathways. Pretreament of amentoflavone also increased the survival rate of mice from whole body gamma irradiation. This suggests that amentoflavone may have a potential function on cancer chemo- radiotherapy and radiation protection from cancer risks. The mechanism needs to be further studied. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2678. doi:10.1158/1538-7445.AM2011-2678