Repair Of Radiation-induced DNA Damage Research Articles

Microdosimetry of proton and carbon ions.

To investigate microdosimetry properties of 160 MeV/u protons and 290 MeV/u(12)C ion beams in small volumes of diameters 10-100 nm. Energy distributions of primary particles and nuclear fragments in the beams were calculated from simulations with the general purpose code SHIELD-HIT, while energy depositions by monoenergetic ions in nanometer volumes were obtained from the event-by-event Monte Carlo track structure ion code PITS99 coupled with the electron track structure code KURBUC. The results are presented for frequencies of energy depositions in cylindrical targets of diameters 10-100 nm, dose distributions yd(y) in lineal energy y, and dose-mean lineal energies yD. For monoenergetic ions, the yD was found to increase with an increasing target size for high-linear energy transfer (LET) ions, but decrease with an increasing target size for low-LET ions. Compared to the depth dose profile of the ion beams, the maximum of the yD depth profile for the 160 MeV proton beam was located at ∼ 0.5 cm behind the Bragg peak maximum, while the yD peak of the 290 MeV/u (12)C beam coincided well with the peak of the absorbed dose profile. Differences between the yD and dose-averaged linear energy transfer (LETD) were large in the proton beam for both target volumes studied, and in the (12)C beam for the 10 nm diameter cylindrical volumes. The yD determined for 100 nm diameter cylindrical volumes in the (12)C beam was approximately equal to the LETD. The contributions from secondary particles to the yD of the beams are presented, including the contributions from secondary protons in the proton beam and from fragments with atomic number Z = 1-6 in the (12)C beam. The present investigation provides an insight into differences in energy depositions in subcellular-size volumes when irradiated by proton and carbon ion beams. The results are useful for characterizing ion beams of practical importance for biophysical modeling of radiation-induced DNA damage response and repair in the depth profiles of protons and carbon ions used in radiotherapy.

Premature aging induced by radiation exhibits pro-atherosclerotic effects mediated by epigenetic activation of CD44 expression.

Age is undoubtedly a major risk factor for heart disease. However, the reason for this is not entirely clear. In the course of our investigation into the mechanism of radiation-induced cardiovascular disease, we made several unexpected findings that inform us on this question. We observed that human coronary endothelial cells, while being able to initiate repair of radiation-induced DNA damage, often fail to complete the repair and become senescent. Such radiation-induced cellular aging occurs through a mutation-independent route. Endothelial cells that aged naturally through replication or as a result of radiation exhibited indistinguishable characteristics. The promoter regions of the CD44 gene in aging endothelial cells become demethylated, and the proteins are highly expressed on the cell surface, making the cells adhesive for monocytes. Adhesion is a cardinal feature that recruits monocytes to the endothelium, allowing them to infiltrate the vessel wall and initiate atherosclerosis. The epigenetic activation of CD44 expression is particularly significant as it causes persistent elevated CD44 protein expression, making senescent endothelial cells chronically adhesive. In addition to understanding why cardiovascular disease increases with age, these observations provide insights into the puzzling association between radiation and cardiovascular disease and highlight the need to consider premature aging as an additional risk of radiation to human health.

Inhibition of mammalian target of rapamycin by rapamycin increases the radiosensitivity of esophageal carcinoma Eca109 cells.

The aim of the present study was to investigate whether radiation induces the mammalian target of rapamycin (Rap) (mTOR) signaling pathway in esophageal carcinoma Eca109 cells, and whether mTOR inhibition by rapamycin increases Eca109 cell radiosensitivity. Changes in the levels of mTOR signaling pathway and DNA damage-repair proteins in Eca109 cells prior to and following radiation were determined. The Eca109 cells were treated with Rap (0, 100, 200 and 400 nmol/l) in combination with radiation (0, 2, 4 and 6 Gy). The cell proliferation inhibition rate was determined by MTT assay. The optimum Rap concentration and radiation dose, which appropriately inhibited cell proliferation, were then selected for further study. An appropriate combination of Rap and radiation for the Eca109 cells was also selected and changes in the mTOR signaling pathway, apoptosis and DNA damage-repair proteins, as well as in cell clone formation, survival curves, the apoptosis rate and radiation-induced DNA damage were determined. The expression of the mTOR signaling pathway and DNA damage-repair proteins were found to increase following the irradiation of the Eca109 cells. In addition, Rap was found to inhibit the mTOR signaling pathway and the expression of the DNA damage-repair proteins. At the same radiation dose, with increasing Rap concentration, the proliferation inhibition rates of the Eca109 cells were found to improve. The clone formation and survival curves of the experimental group were less than those of the control groups. Furthermore, the cell apoptosis rate and expression of cleaved caspase-3 and bax in the experimental group were higher than those of the control groups, whereas the expression of bcl-2 was less than that of the control groups. The radiation-induced DNA damage of the experimental group was greater than that of the control group. The inhibition of mTOR by Rap was found to effectively inhibit the proliferation, survival and radiation-induced DNA damage repair of the Eca109 cells following irradiation, as well as promoting radiation-induced apoptosis, thereby increasing the radiosensitivity of the esophageal carcinoma Eca109 cells.

Highlights of This Issue

EGFR-mutations and ALK rearrangements could co-exist in non-small cell lung cancer (NSCLC). To explore the predictive biomarkers in EGFR/ALK co-altered tumors, Yang and colleagues, investigated molecular alterations of EGFR and ALK in a 977-case Chinese cohort and correlated phospho-EGFR and phospho-ALK with responses to EGFR-TKIs and crizotinib. Tumors with such co-alterations could have diverse responses to first-line EGFR TKIs, which were associated with phospho-EGFR levels. Elevated phospho-ALK levels correlated the efficacy of subsequent crizotinib treatment. This study showed the potential utility of receptors phosphorylation status to predict the treatment by EGFR-TKI or crizotinib in EGFR/ALK co-altered NSCLC.Glioblastomas are highly fatal brain tumors that are extremely radioresistant. This study identifies NVP-BEZ235 (a dual PI3K/mTOR inhibitor) as a potent inhibitor of key DNA repair enzymes, DNA-PKcs and ATM, in vivo. Treatment with this drug blocks the repair of radiation-induced DNA damage in tumors, resulting in remarkable radiosensitization and prolonging survival of brain-tumor bearing mice. Based on the results presented, blocking DNA repair with potent and bioavailable inhibitors of DNA-PKcs and ATM appears to be a feasible option for improving GBM therapy in the future.Gong and colleagues demonstrate an important activity of the natural compound, Nexrutine on inflammation-associated carcinogenesis and inhibition of fibrosis. The study elaborates on the noteworthy signaling axis among STAT3, NFκB, COX-2 and EP4. The authors show that STAT3 negatively regulates NFκB activation. Suppression of this crosstalk inhibits COX-2 transcriptional activity and subdues prostaglandins. The immediate outcome of this study can influence current modalities for pancreatic cancer treatment through the combinatorial use of Nexrutine as a dual inhibitor of NFκB and STAT3. It also has clinical implications for other inflammation and fibrosis-associated cancers.Relapse of myeloma after autologous stem cell transplantation (ASCT) is frequent. Posttransplant immunotherapy using adoptive T-cell transfers and tumor antigen vaccines may improve clinical responses if a high frequency of tumor antigen-directed immune responses can be generated. Rapoport and colleagues show that adoptive transfer of costimulated autologous T cells which are primed and boosted by a MAGE-A3 Trojan peptide vaccine combined with the TLR-3 agonist and novel vaccine adjuvant Poly-ICLC induces vaccine-directed cellular immune responses in 76% of patients after ASCT. A high frequency of vaccine-directed antibody responses also occurred but only in the presence of montanide.

Modification of radiation‐induced DNA double strand break repair pathways by chemicals extracted from Podophyllum hexandrum: An in vitro study in human blood leukocytes

Radiation exposure is a serious threat to biomolecules, particularly DNA, proteins and lipids. Various exogenous substances have been reported to protect these biomolecules. In this study we explored the effect of pre-treatment with G-002M, a mixture of three active derivatives isolated from the rhizomes of Podophyllum hexandrum, on DNA damage response in irradiated human blood leukocytes. Blood was collected from healthy male volunteers, preincubated with G-002M and then irradiated with various doses of radiation. Samples were analyzed using flow cytometry to quantify DNA double strand break (DSB) biomarkers including γ-H2AX, P53BP1 and levels of ligase IV. Blood samples were irradiated in vitro and processed to determine time and dose-dependent kinetics. Semiquantitative RT-PCR was performed at various time points to measure gene expression of DNA-PKcs, Ku80, ATM, and 53BP1; each of these genes is involved in DNA repair signaling. Pre-treatment of blood with G-002M resulted in reduction of γ-H2AX and P53BP1 biomarkers levels and elevated ligase IV levels relative to non-G-002M-treated irradiated cells. These results confirm suppression in radiation-induced DNA DSBs. Samples pre-treated with G-002M and then irradiated also showed significant up-regulation of DNA-PKcs and Ku80 and downregulation of ATM and 53BP1 gene expressions, suggesting that G-002M plays a protective role against DNA damage. The protective effect of G-002M may be due to its ability to scavange radiation-induced free radicals or assist in DNA repair. Further studies are needed to decipher the role of G-002M on signaling molecules involved in radiation-induced DNA damage repair pathways.

RADIOSENSITIZATION OF CANCER STEM CELLS: TARGETING TGFβ, NOTCH OR TELOMERASE TO IMPROVE TUMOR RESPONSE O RADIOTHERAPY

Radiation resistant cancer stem cells are the main reason for treatment failure and tumor recurrence after cancer radiotherapy. Increasing biological evidences demonstrate that these cells possess the capacity to repair radiation induced DNA damage, protect themselves from radiation derived reactive oxygen species, survive and proliferate after several fractions of radiotherapy and finally, repopulate the heterogeneity of the tumor. Thus, targeting and eliminating these cells should be necessary to achieve cancer cure in radiotherapy. Three major approaches that specifically target radioresistant cancer stem cells have been recently investigated. First, inhibition of TGFβ, a major mediator of the tissue response to radiation, has been shown to induce radiosensitization of cancer stem cells by targeting the DNA damage response mechanism. Second, by preventing Notch activation during fractionated radiotherapy, cancer stem cells were depleted from their ability to repopulate the tumor after radiation. Finally, telomerase activity inhibitors have shown to specifically decrease the cancer stem cell population after radiotherapy. In the present review, we evaluate these radiosentitizing approaches and their possible effects when combined with fractionated radiotherapy as they promise to be a powerful tool in the battle against this cancer.

Sym004, a Novel Anti-EGFR Antibody Mixture, Augments Radiation Response in Human Lung and Head and Neck Cancers

Sym004 represents a novel EGF receptor (EGFR)-targeting approach comprising a mixture of two anti-EGFR antibodies directed against distinct epitopes of EGFR. In contrast with single anti-EGFR antibodies, Sym004 induces rapid and highly efficient degradation of EGFR. In the current study, we examine the capacity of Sym004 to augment radiation response in lung cancer and head and neck cancer model systems. We first examined the antiproliferative effect of Sym004 and confirmed 40% to 60% growth inhibition by Sym004. Using clonogenic survival analysis, we identified that Sym004 potently increased cell kill by up to 10-fold following radiation exposure. A significant increase of γH2AX foci resulting from DNA double-strand breaks was observed in Sym004-treated cells following exposure to radiation. Mechanistic studies further showed that Sym004 enhanced radiation response via induction of cell-cycle arrest followed by induction of apoptosis and cell death, reflecting inhibitory effects on DNA damage repair. The expression of several critical molecules involved in radiation-induced DNA damage repair was significantly inhibited by Sym004, including DNAPK, NBS1, RAD50, and BRCA1. Using single and fractionated radiation in human tumor xenograft models, we confirmed that the combination of Sym004 and radiation resulted in significant tumor regrowth delay and superior antitumor effects compared with treatment with Sym004 or radiation alone. Taken together, these data reveal the strong capacity of Sym004 to augment radiation response in lung and head and neck cancers. The unique action mechanism of Sym004 warrants further investigation as a promising EGFR targeting agent combined with radiotherapy in cancer therapy.

EGFR-Inhibition Enhances Apoptosis in Irradiated Human Head and Neck Xenograft Tumors Independent of Effects on DNA Repair

Epidermal growth factor receptor (EGFR) inhibition using cetuximab improves the efficacy of radiotherapy in only a subgroup of head and neck squamous cell carcinoma (HNSCC) patients. Therefore, to improve patient selection a better understanding of tumor characteristics that affect treatment is necessary. Here, we investigated the effect of cetuximab on repair of radiation-induced DNA damage in a HNSCC xenograft model, which shows a synergistic effect to cetuximab and radiotherapy (SCCNij185) and a HNSCC model, which shows no additive effect of cetuximab to radiotherapy (SCCNij153). In both tumor models, clear increases were seen in the number of 53BP1 and Rad51 foci after irradiation. 53BP1 foci were present at comparable levels in hypoxic and normoxic tumor areas of the tumor xenografts, while the number of Rad51 foci was significantly higher in normoxic areas compared to hypoxic areas (P < 0.05). In both SCCNij185 and SCCNij153 xenografts an increased number of 53BP1 foci was observed in tumors treated with cetuximab and radiotherapy compared to radiotherapy alone. In SCCNij185 this increase was statistically significant in normoxic tumor areas (P = 0.04) and in SCCNij153 in both hypoxic and normoxic areas (P = 0.007 and P = 0.02, respectively). The number of Rad51 foci was not significantly different when cetuximab was added to radiotherapy compared to radiotherapy alone. Levels of pEGFR and pERK1/2 were decreased when cetuximab was added to radiotherapy in SCCNij185, but not in SCCNij153. Apoptosis was also only increased in SCCNij185 tumors at 4 days after cetuximab and radiotherapy treatment (P < 0.01). In conclusion, cetuximab inhibited DNA repair in both HNSCC models, but this effect was not predictive for the radiosensitizing effect of cetuximab in vivo. This lack of correlation may be related to differential effects of cetuximab and radiotherapy on ERK1/2 signaling and a decreased induction of apoptosis by cetuximab and radiotherapy in the resistant model.

X-Ray Induced DNA Damage and Repair in Germ Cells of PARP1−/− Male Mice

Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1−/− and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1−/− and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1−/− cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1−/− mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.

Nimotuzumab Enhances the Radiosensitivity of Cancer Cells In Vitro by Inhibiting Radiation-Induced DNA Damage Repair

BackgroundNimotuzumab is a humanized IgG1 monoclonal antibody specifically targeting EGFR. In this study, we aimed to investigate the molecular mechanisms of nimotuzumab in its effects of enhancing cancer cell radiosensitivity.Principal FindingLung cancer A549 cells and breast cancer MCF-7 cells were pretreated with or without nimotuzumab for 24 h before radiation to perform the clonogenic survival assay and to analyze the cell apoptosis by flow ctyometry. γ-H2AX foci were detected by confocal microscopy to assess the effect of nimotuzumab on radiation induced DNA repair. EGFR activation was examined and the levels of DNA damage repair related proteins in A549 cells at different time point and at varying doses exposure after nimotuzumab and radiation treatment were examined by Western blot. Pretreatment with nimotuzumab reduced clonogenic survival after radiation, inhibited radiation-induced EGFR activation and increased the radiation-induced apoptosis in both A549 cells and MCF-7 cells. The foci of γ-H2AX 24 h after radiation significantly increased in nimotuzumab pretreated cells with different doses. The phosphorylation of AKT and DNA-PKcs were remarkably inhibited in the combination group at each dose point as well as time point.ConclusionsOur results revealed that the possible mechanism of nimotuzumab enhancing the cancer radiosensitivity is that nimotuzumab inhibited the radiation-induced activation of DNA-PKcs through blocking the PI3K/AKT pathway, which ultimately affected the DNA DSBs repair.

Microbeam irradiation of C. elegans nematode in microfluidic channels

To perform high-throughput studies on the biological effects of ionizing radiation in vivo, we have implemented a microfluidic tool for microbeam irradiation of Caenorhabditis elegans. The device allows the immobilization of worms with minimal stress for a rapid and controlled microbeam irradiation of multiple samples in parallel. Adapted from an established design, our microfluidic clamp consists of 16 tapered channels with 10-μm-thin bottoms to ensure charged particle traversal. Worms are introduced into the microfluidic device through liquid flow between an inlet and an outlet, and the size of each microchannel guarantees that young adult worms are immobilized within minutes without the use of anesthesia. After site-specific irradiation with the microbeam, the worms can be released by reversing the flow direction in the clamp and collected for analysis of biological endpoints such as repair of radiation-induced DNA damage. For such studies, minimal sample manipulation and reduced use of drugs such as anesthetics that might interfere with normal physiological processes are preferable. By using our microfluidic device that allows simultaneous immobilization and imaging for irradiation of several whole living samples on a single clamp, here we show that 4.5-MeV proton microbeam irradiation induced DNA damage in wild-type C. elegans, as assessed by the formation of Rad51 foci that are essential for homologous repair of radiation-induced DNA damage.

Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin

Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

Effects of Low-Dose Ionizing Radiation and Menadione, an Inducer of Oxidative Stress, Alone and in Combination in a Vertebrate Embryo Model

Prior work has established the zebrafish embryo as an in vivo model for studying the biological effects of exposure to low doses of ionizing radiation. One of the known effects of radiation is to elevate the levels of reactive oxygen species (ROS) in tissue. However, ROS are also produced as by-products of normal metabolism and, regardless of origin, ROS produce similar chemical damage to DNA. Here we use the zebrafish embryo model to investigate whether the effects of low-dose (0-1.5 Gy) radiation and endogenous ROS are mechanistically distinct. We increased levels of endogenous ROS by exposure to low concentrations of the quinone drug, menadione. Imaging studies in live embryos showed that exposure to 3 μM or higher concentrations of menadione dramatically increased ROS levels. This treatment was associated with a growth delay and morphologic abnormalities, which were partially or fully reversible. By contrast, exposure to low doses of ionizing radiation had no discernable effects on overall growth or morphology, although, there was an increase in TUNEL-positive apoptotic cells, consistent with the results of prior studies. Further studies showed that the combined effect of radiation and menadione exposure are greater than with either agent alone, and that attenuation of the expression of Ku80, a gene important for repair of radiation-induced DNA damage, had only a slight effect on menadione sensitivity. Together, results suggest that ionizing radiation and menadione affect the embryo by distinct mechanisms.

Radiation Dose-Rate: Engelward and Yanch Respond

Melzer raises many interesting points regarding our study of low dose-rate radiation (Olipitz et al. 2012). Responding to his letter gives us the opportunity to clarify the rationale behind some of our approaches and interpretations. Melzer points out that sample sizes in our study varied from 6 to 60. This is absolutely true because it was necessary to adjust sample sizes according to the end point being analyzed. Larger cohorts are required under conditions where there is higher variance, which is the case for the FYDR (fluorescent yellow direct repeat) mice. Smaller cohorts are sufficient when the variance is lower, such as for micronuclei. Melzer notes that we used transgenic mice for one end point and normal mice for others. In our study, all of the animals were isogenic (C57Bl6), with the only difference being the insertion of the reporter transgene into the FYDR mice. We have not observed any biological impact of this insertion, and the insertion was made in only one of the two copies of chromosome 1, making it even less likely to affect the biology of the animal. Even if there were an impact, this would not compromise the approach because each end point of the study is appropriately internally controlled. Because each end point was evaluated relative to an isogenic control cohort, the approach did not weaken the ability to detect effects but actually strengthened the method. In his letter, Melzer correctly points out that data of Tanaka et al. (2009) show a statistically significant increase in chromosome aberrations in cells from mice exposed to 1 mGy/day up to a total of 1,000 mGy. However, after exposure to that same dose-rate for a longer period (up to 8,000 mGy), there was no statistically significant change in the number of chromosome aberrations. Furthermore, Tanaka et al. (2009) stated that Regression coefficients (b4 and b3) in the equations for Dic by FISH at low dose rates of 20 mGy/day and 1 mGy/day at doses less than 8,000 mGy were not statistically significant. Tanaka et al. also stated that It remains to be clarified whether the dose–response relationship for Dic+Rc, UA or Dic by FISH was significantly different for dose-rates of 1 mGy/day and 20 mGy/day or whether the linear dose–response relationship at 1 mGy/day was significantly different from the spontaneous level. Melzer is correct that we cannot rule out the possibility that genetic changes might have been observed if the exposures had been carried out for a longer period. However, because cells have the capacity to repair radiation-induced DNA damage, it is possible that DNA damage would not accumulate with time. Melzer is correct that a recent report suggested that exposure to radiation from CT (computed tomography) scans affects the risk of cancer in exposed children. An important difference is that CT scans are an acute exposure at a high dose-rate, which is very different from the low dose-rate conditions in our study. Nevertheless, we agree that it is very important to consider the fact that children have increased sensitivity to radiation damage. One of Melzer’s final points is that inflammation might affect radiation sensitivity. Although we did not test the impact of inflammation in our study, it is important to note that inflammation is a highly genotoxic process itself, leading to levels of DNA damage orders of magnitude higher than levels we calculated in response to the low dose-rate we used. Finally, Melzer raises the issue of internal exposure by ingestion versus external sources. The body handles ingested radionuclides according to the chemical behavior of the element. Although we have examined the effect of radiation dose as delivered by internal or external photon- or beta-emitters, we did not consider the internal pattern of radionuclide uptake in our study.

Expression and localization of Artemis serine 516 phosphorylation in human scalp skin

Artemis phosphorylation at serine 516 (Ser516) has important regulatory functions in the repair of radiation-induced DNA damage, V(D)J recombination, p53-dependent apoptosis and cell cycle control. Accordingly, Artemis mutations can lead to Omenn syndrome, which is associated with human radiosensitive severe combined immunodeficiency syndrome and alopecia. In this study, we investigated the expression of Ser516 phosphorylation of Artemis in the epidermis and epidermal appendages in normal human scalp skin. Immunofluorescence analysis revealed Ser516 phosphorylation of Artemis in the upper and middle portion of anagen hair follicle [including outer root sheath (ORS), inner root sheath but not stratum basale], hair matrix, sebaceous glands (secretory and ductal portions), eccrine sweat glands (secretory and ductal portions) and epidermis (stratum basale and stratum granulosum), respectively. Artemis phosphorylation at Ser516 was most prominent in ORS keratinocytes. Therefore, we suggest that phosphorylation of Artemis at Ser516 could be involved in regulation of human epidermal appendages.

Dasatinib, a multi-kinase inhibitor increased radiation sensitivity by interfering with nuclear localization of epidermal growth factor receptor and by blocking DNA repair pathways

Background and purposeAlthough inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect. Materials and methodsSix HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells. ResultsAll six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity. ConclusionsOur data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.

Mechanism of enhancement of radiation-induced cytotoxicity by sorafenib in colorectal cancer

Sorafenib, an orally available multikinase inhibitor, combined with radiation has shown potential as an anticancer treatment in an in vitro and in vivo colon cancer model. In this study, we investigated the mechanism of enhancement of radiation-induced cytotoxicity by sorafenib in colorectal cancer. The effects of sorafenib on radiation-induced cytotoxicity of DLD-1 and HT-29 were evaluated via clonogenic assay. The impact of sorafenib on radiation-induced cell cycle kinetics and on apoptosis was analyzed using flow cytometry. Cyclin B1 was examined by western blot. As a measure of DNA damage after treatment, γ-H2AX foci and nuclear fragmentation were determined as a function of time after irradiation plus sorafenib combination. Tumor growth delay was used to evaluate the effects of sorafenib on in vivo radiation-induced cytotoxicity. Exposure of each cell line to sorafenib combined with irradiation resulted in an increased radiation-induced cytotoxicity with dose enhancement factors at a surviving fraction of 0.37 ranging from 1.13 to 1.76. Sorafenib strengthened radiation-induced accumulation of tumor cells in the G2-M phase with attenuated expression of cyclin B1, but had no effect on radiation-induced apoptosis. Exposure to sorafenib and radiation resulted in a greater number of remaining γ-H2AX foci and fragmented nuclei than radiation alone. In vivo tumor xenograft study confirmed that administration of sorafenib results in significant tumor growth inhibition when combined with radiation. These results indicate that sorafenib enhances radiation-induced cytotoxicity in colorectal cancer and suggest that the mechanism is associated with delaying repair of radiation-induced DNA damage and down-regulation of cyclin B1.

189 Establishing a Link Between Genomic Patterns of Radiation-Induced DNA Damage Repair in Human Astrocytes and Somatic Mutations in Gliomas

189 Establishing a Link Between Genomic Patterns of Radiation-Induced DNA Damage Repair in Human Astrocytes and Somatic Mutations in Gliomas

Activation of the AKT/cyclin D1/Cdk4 survival signaling pathway in radioresistant cancer stem cells

Radioresistance, which is a major cause of failure of radiotherapy (RT), is proposed as one of the intrinsic characteristics of cancer stem cells (CSCs) whose unique DNA damage response (DDR), efficient DNA repair and resistance to apoptosis are thought to confer the phenotype. We have isolated surviving CSCs by exposure to long-term fractionated radiation for 82 days from HepG2 and A172 cells (82FR-31NR cells). 82FR-31NR cells exhibited CSC properties, such as high expression of CSC marker CD133 and the ABC transporters (MDR1 and BCRP1), and high tumorigenic potential after transplantation into nude mice. The advantage of our isolated CSCs is that they can proliferate in as the same growth medium as that of parental cells without loss of CSC properties. Therefore, we can analyze DDR of non-stem cells and CSCs without any influences caused by different culture conditions. 82FR-31NR cells showed efficient DNA repair of radiation-induced DNA damage and radioresistance with activation of the AKT/cyclin D1 survival signaling pathway. In contrast, DNA damage persisted for a long time after irradiation in parental cells compared with isolated CSCs. Persisted DNA damage induced apoptosis in parental cells without activation of the AKT/cyclin D1 pathway. Therefore, inhibition of the AKT/cyclin D1 pathway by an AKT inhibitor, API-2, or cyclin D1 siRNA resulted in a loss of efficient DNA repair and radiosensitization of 82FR-31NR cells. Furthermore, knockdown of Cdk4 by its siRNA or a Cdk4 inhibitor was sufficient to suppress radioresistance of CSCs. In this study, we present a newly discovered DDR regarding the AKT/cyclin D1/Cdk4 pathway in response to radiation in CSCs. Combination of fractionated RT and reagents targeting the AKT/cyclin D1/Cdk4 pathway to eradicate CSCs would be effective therapeutic modality.

Radiation-induced alterations in histone modification patterns and their potential impact on short-term radiation effects

Detection and repair of radiation-induced DNA damage occur in the context of chromatin. An intricate network of mechanisms defines chromatin structure, including DNA methylation, incorporation of histone variants, histone modifications, and chromatin remodeling. In the last years it became clear that the cellular response to radiation-induced DNA damage involves all of these mechanisms. Here we focus on the current knowledge on radiation-induced alterations in post-translational histone modification patterns and their effect on the chromatin accessibility, transcriptional regulation and chromosomal stability.