Effects of depletion of dihydropyrimidine dehydrogenase on focus formation and RPA phosphorylation

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase (DPYD), partially inhibits homologous recombination (HR) repair and has a radiosensitizing effect as well as enhanced sensitivity to Camptothecin (CPT). DPYD is the target protein for radiosensitization by Gimeracil. We investigated the mechanisms of sensitization of radiation and CPT by DPYD inhibition using DLD-1 cells treated with siRNA for DPYD. We investigated the focus formation of various kinds of proteins involved in HR and examined the phosphorylation of RPA by irradiation using Western blot analysis. DPYD depletion by siRNA significantly restrained the formation of radiation-induced foci of Rad51 and RPA, whereas it increased the number of foci of NBS1. The numbers of colocalization of NBS1 and RPA foci in DPYD-depleted cells after radiation were significantly smaller than in the control cells. These results suggest that DPYD depletion is attributable to decreased single-stranded DNA generated by the Mre11/Rad50/NBS1 complex-dependent resection of DNA double-strand break ends. The phosphorylation of RPA by irradiation was partially suppressed in DPYD-depleted cells, suggesting that DPYD depletion may partially inhibit DNA repair with HR by suppressing phosphorylation of RPA. DPYD depletion showed a radiosensitizing effect as well as enhanced sensitivity to CPT. The radiosensitizing effect of DPYD depletion plus CPT was the additive effect of DPYD depletion and CPT. DPYD depletion did not have a cell-killing effect, suggesting that DPYD depletion may not be so toxic. Considering these results, the combination of CPT and drugs that inhibit DPYD may prove useful for radiotherapy as a method of radiosensitization.     

Modification of radiosensitivity by the so-called tissue recovery stimulator. I. Radiosensitizing effects of solcoseryl.

The effect of solcoseryl on the growth, radiosensitization and ability of V79 cells to recover from X-ray-induced damage has been observed. Solcoseryl at 0.8 mg/ml was the optimal concentration for the stimulation of cell growth. Increased sensitivity to X-irradiation was found in the shoulder region of V79 cells treated before and after irradiation with solcoseryl (0.8 mg/ml). The Dq and extrapolation number (n) decreased. Solcoseryl treatment apparently does not reduce split dose recovery or inhibit the repair of potentially lethal damage. Flow cytofluorometry studies of the cell cycle distribution and mitotic index show that solcoseryl inhibits the expression of radiation-induced cell arrest in the G2 phase of the cell cycle. Although this action increases radiation sensitization, additional mechanisms probably exist.     

Role of DNA double-strand break repair genes in cell proliferation under low dose-rate irradiation conditions

Radiation-induced DNA double-stand breaks (DSBs) lead to numerous biological effects. To elucidate the molecular mechanisms involved in cellular responses to low dose and low dose-rate radiation, it is informative to clarify the roles of DSB repair related genes. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, it is shown that in chicken DT40 cells irradiated with gamma-rays at a low dose-rate (2.4 cGy/day), the growth delay in NHEJ-related KU70- and PRKDC (encoding DNA-PKcs)-defective cells were remarkably higher than in cells defective for the HR-related RAD51B and RAD54 genes. DNA-PKcs- defective human M059J cells also showed an obvious growth delay when compared to control M059K cells. RAD54(-/-)KU70(-/-) cells demonstrated their highest degree of growth delay after an X-irradiation with a high dose-rate of 0.9 Gy/min. However they showed a lower degree of growth delay than that seen in KU70(-/-) and PRKDC(-/-/-) cells exposed to low dose-rate irradiation. These findings indicate that cellular responses to low dose-rate radiation are remarkably different from those to high dose-rate radiation. The fact that both DT40 and mammalian NHEJ-defective cells were highly sensitive to low dose-rate radiation, provide a foundation for the concept that NHEJ-related factors may be useful as molecular markers to predict the sensitivity of humans to low dose-rate radiation.     

Dancing on damaged chromatin: functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to DNA damage

In order to preserve and protect genetic information, eukaryotic cells have developed a signaling or communications network to help the cell respond to DNA damage, and ATM and NBS1 are key players in this network. ATM is a protein kinase which is activated immediately after a DNA double strand break (DSB) is formed, and the resulting signal cascade generated in response to cellular DSBs is regulated by post-translational protein modifications such as phosphorylation and acetylation. In addition, to ensure the efficient functioning of DNA repair and cell cycle checkpoints, the highly ordered structure of eukaryotic chromatin must be appropriately altered to permit access of repair-related factors to DNA. These alterations are termed chromatin remodeling, and are executed by a specific remodeling complex in conjunction with histone modifications. Current advances in the molecular analysis of DNA damage responses have shown that the auto-phosphorylation of ATM and the interaction between ATM and NBS1 are key steps for ATM activation, and that the association of ATM and NBS1 is involved in chromatin remodeling. Identification of novel factors which function in ubiquitination (RNF8, Ubc13, Rap80, etc.) has also enabled us to understand more details of the early stages in DNA repair pathways which respond to DSBs. In this review, the focus is on the role of ATM and the RAD50/MRE11/NBS1 complex in DSB response pathways, and their role in DSB repair and in the regulation of chromatin remodeling.     

STAT3 as a target for sensitizing prostate cancer cells to irradiation

Radioresistance of prostate cancer (PCa) is a major factor leading to local failure of radiotherapy. STAT3 is an oncogenic protein that was recently found to be activated in PCa tumors. This study aimed to investigate the radiosensitization effect of targeting STAT3 in PCa tumors. Here, the radiosensitization effect of STAT3 blockade was investigated by clonogenic assay, flow cytometry and western blot analysis in human PCa cells in vitro and in vivo. We demonstrated that STAT3 blockade with a STAT3 inhibitor or siRNA increased the radiosensitivity of PCa cells and that radiation together with STAT3 blockade induced more apoptosis and double-strand breaks (DSBs) than radiation alone in LNCaP cells. In addition, radiation induced STAT3 activation and survivin expression in PCa cells, which was inhibited by STAT3 blockade. Transfection with survivin cDNA attenuated the radiosensitization effect of STAT3 blockade. These effects were further confirmed by in vivo studies, which showed that the STAT3 inhibitor enhanced the treatment efficacy of radiation on LNCaP xenografts with decreased STAT3 activation and survivin expression.These findings suggest that STAT3 blockade radiosensitizes PCa cells through regulation of survivin. Thus, our study has revealed STAT3 as a potential sensitizer for irradiation in PCa cells. Its clinical application as an adjuvant in radiotherapy of PCa should be explored in the future.     

DNA damage recognition proteins localize along heavy ion induced tracks in the cell nucleus

To identify the repair dynamics involved in high linear energy transfer (LET) radiation-induced DNA damage, phospho-H2AX (gammaH2AX) foci formation was analyzed after cellular exposure to iron ions (Fe-ions, 500 MeV u(-1), 200 KeV microm(-1)). The foci located at DNA damage sites were visualized using immunocytochemical methods. Since H2AX is phosphorylated at sites of radiation-induced double strand breaks (DSB), gammaH2AX foci were used to detect or illuminate tracks formed by DSB after exposure to various doses of ionizing radiation. Additional DSB-recognition proteins such as ATM phospho-serine 1981, DNA-PKcs phospho-threonine 2609, NBS1 phospho-serine 343 and CHK2 phospho-threonine 68 all co-localized with gammaH2AX at high LET radiation induced DSB. In addition, Fe-ion induced foci remained for longer times than X-radiation induced foci. These findings suggest that Fe-ion induced damage is repaired more slowly than X-radiation induced damage, possibly because Fe-ion induced damage or lesions are more complex or extensive. Antibodies for all these phosphorylated DNA DSB recognition proteins appear to be very effective for the detection and localization of DSB.     

Positional cloning and functional analysis of the gene responsible for Nijmegen breakage syndrome, NBS1

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, combined immunodeficiency, and a high incidence of lymphoid tumor. Cells from NBS patients show chromosomal instability, hypersensitivity to ionizing radiation and abnormal p53-mediated cell cycle regulation. We cloned the underlying gene for NBS, designated NBS1, by complementation-assisted positional cloning from the candidate region 8q21. Large genomic sequencing, as well as a search using computer programs, provides a powerful approach for identifying the underlying gene for a disease. The NBS1 gene encodes a protein of 754 amino acids that has FHA and BRCT domains which often are conserved in cell-cycle checkpoint proteins. The gene has weak homology to the yeast (Saccharomyces cerevisiae) Xrs2 protein in the N-terminus region. Like yeast Xrs2, the NBS1 protein forms a complex with hRAD50/hMRE11, and the complex is condensed as foci in the nucleus after irradiation, indicative that this triple-complex is a crucial factor in DNA repair. Functional analysis of the NBS1 protein is in progress and it should provide further clues to understanding the repair mechanism of radiation-induced DNA double-strand breaks.     

Protective effect of triphlorethol-A from Ecklonia cava against ionizing radiation in vitro

We studied the cytoprotective effect of triphlorethol-A against gamma-ray radiation-induced oxidative stress. In this study, hydrogen peroxide, which is a reactive oxygen species (ROS), was detected using 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) assay. Triphlorethol-A reduced intracellular hydrogen peroxide generated by gamma-ray radiation. This compound provided protection against radiation-induced membrane lipid peroxidation and cellular DNA damage which are the main targets of radiation-induced damage. Triphlorethol-A protected the cell viability damaged by the radiation through inhibition of apoptosis. Triphlorethol-A reduced the expression of bax and activated caspase 3 induced by radiation, but recovered the expression of bcl-2 decreased by radiation. Taken together, the results suggest that triphlorethol-A protects cells against oxidative damage induced by radiation through reducing ROS.     

Effects of expression level of DNA repair-related genes involved in the NHEJ pathway on radiation-induced cognitive impairment

Cranial radiation therapy can induce cognitive decline. Impairments of hippocampal neurogenesis are thought to be a paramountly important mechanism underlying radiation-induced cognitive dysfunction. In the mature nervous system, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) pathways. It has been demonstrated that NHEJ deficiencies are associated with impaired neurogenesis. In our study, rats were randomly divided into five groups to be irradiated by single doses of 0 (control), 0 (anesthesia control), 2, 10, and 20 Gy, respectively. The cognitive function of the irradiated rats was measured by open field, Morris water maze and passive avoidance tests. Real-time PCR was also used to detect the expression level of DNA DSB repair-related genes involved in the NHEJ pathway, such as XRCC4, XRCC5and XRCC6, in the hippocampus. The influence of different radiation doses on cognitive function in rats was investigated. From the results of the behavior tests, we found that rats receiving 20 Gy irradiation revealed poorer learning and memory, while no significant loss of learning and memory existed in rats receiving irradiation from 0–10 Gy. The real-time PCR and Western blot results showed no significant difference in the expression level of DNA repair-related genes between the 10 and 20 Gy groups, which may help to explain the behavioral results, i.e. DNA damage caused by 0–10 Gy exposure was appropriately repaired, however, damage induced by 20 Gy exceeded the body''s maximum DSB repair ability. Ionizing radiation-induced cognitive impairments depend on the radiation dose, and more directly on the body''s own ability to repair DNA DSBs via the NHEJ pathway.     

Mitotic cells can repair DNA double-strand breaks via a homology-directed pathway

The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.     

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.     

Comparison of in vivo translocation frequencies with in vitro G2 radiosensitivity in radiation workers occupationally exposed to external radiation

A group of retired workers from the British Nuclear Fuels plc facility at Sellafield who had been studied for in vivo translocation frequencies in blood lymphocytes were resampled and analysed for in vitro chromosomal radiosensitivity. Significant variation in response to a dose of 0.5 Gy given at the G(2) stage of the cell cycle was observed between individuals (P < 0.001). In a regression analysis that included age, cumulative occupational radiation dose and in vitro G(2) radiation-induced aberration frequencies as independent variables, only cumulative occupational radiation dose had a significant influence on chromosomal translocation frequency (P = 0.0036). G(2) in vitro radiosensitivity is assumed to be a marker for genetic polymorphic variation in DNA damage recognition and repair genes. Therefore, since in vivo translocation frequencies can be considered a surrogate for cancer risk, this lack of association with G(2) in vitro radiosensitivity suggests that such genetic variation has no impact on the response to low dose chronic exposure.     

Positive and negative regulation of radiation-induced apoptosis by protein kinase C

Indicators such as clonogenic survival, transformation, and chromosomal aberrations are used to evaluate the effects of radiation on cells. Apoptosis, another such indicator, is a mode of cell death, and radiation-induced apoptosis contributes to eliminating damaged cells and preventing malformation and carcinogenesis. Understanding radiation-induced apoptosis will assist in radiotherapy for cancer and treatment of patients in accidental radiation exposure. Protein kinase C (PKC) is a serine/threonine kinase that is related to cell proliferation, differentiation, metabolism, and apoptosis, and has many roles in the radiation-induced cellular responses involving apoptosis. This review describes the functions of PKC, including its relationship with other signaling networks and oxidative stress in the regulation of radiation-induced apoptosis. Such information might provide clues for evaluating the effects of radiation and for identifying clinical applications.     

ERp29 is a radiation-responsive gene in IEC-6 cell

ERp29 is a resident protein of the endoplasmic reticulum (ER) lumen, which is thought to be involved in the folding of secretory proteins. In our previous work, it was found that, when treated with ionizing radiation (IR), the ERp29 expression was increased in mouse intestinal epithelia and cultured IEC-6 cells, which suggested that ERp29 might be a radiation-induced gene. The current work is to confirm the induction of ERp29 by IR and to analyze its role in irradiated IEC-6 cells. Our results showed that ERp29 expression was elevated by IR in IEC-6 cells at mRNA and protein levels in a time-dependent manner. IEC-6 cells with different exogenous ERp29 expression were obtained by transfection with sense and antisense expression vectors of ERp29 coding region. As ERp29 expression was inhibited, these cells exhibited more serious radiation injury and more sensitivity to IR-induced apoptosis. To further elucidate the induction of ERp29, we analyzed the XBP1 expression after IR. Results showed that the spliced form of XBP1 mRNA rapidly reached a peak at 3 hours after irradiation, which indicated that UPR sensor was involved in radiation and might be a reason to induce ERp29 expression. Our results demonstrate that ERp29 is a radiation associated protein and plays an important role in protecting cells from IR.     

Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on yeast Saccharomyces cerevisiae

Sanazole/DNA repair/Hypoxic radiosensitization/DNA polymerases/Saccharomyces cerevisiae Yeast Saccharomyces cerevisiae can exist in two physiological states, namely anaerobic and aerobic. They differ in their response to gamma- radiation and radiomodification. We report hereon our results concerning radiosensitization by Sanazole (AK-2123), a well-known hypoxic radio sensitizer, whose mechanism of action has been studied extensively. The results have revealed that Sanazole (1 mM) when present during irradiation could specifically sensitize wild-type anaerobic yeast cells with a DMF of 2.4. In a radiation-sensitive mutant which lacks a DNA repair pathway specific for the recovery from gamma-radiation induced DNA damage, the extent of sensitization was considerably lower and the DMF was only 1.3. Studies on the liquid holding recovery of cells of both wild- type and rad52 yeast cells exposed to radiation in presence of Sanazole revealed that sensitization by Sanazole is due to a preferential increase in the DNA damage, and not by impairing DNA repair. This system thus holds promise for screening potential hypoxic chemical radiosensitizers.     

Modulation of ganglioside expression in human melanoma cell lines; increased resistance to chemo- and radiation treatment.

Cell surface gangliosides in human melanoma cell lines were modulated by pretreatment and adaptation to 6-thioguanine and 5-bromo-deoxyuridine. Chemo- and radiation sensitivities were compared in original cell lines and modulated cells by the human tumor colony-forming assay. Modulated cells showed decreased expression of cell surface GM2 and GD2 gangliosides. This reduction was correlated with increased resistance to bleomycin, vincristine, cisplatin and radiation treatment. These results suggest that cell surface GM2 and GD2 ganglioside expression in human melanoma cells is intimately associated with several cellular biological properties, such as drug or radiation sensitivity and cellular differentiation.     

Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB induction and conversion induced by gamma rays and helium ions

Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to ⁶⁰Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1–4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.     

Evaluation of chemical phototoxicity,focusing on phosphorylated histone H2AX

Histone H2AX is a minor component of nuclear histone H2A. The phosphorylation of histone H2AX at Ser 139, termed γ-H2AX, was originally identified as an early event after the direct formation of DNA double-strand breaks (DSBs) by ionizing radiation. Now, the generation of γ-H2AX is also considered to occur in association with secondarily formed DSBs by cellular processing such as DNA replication and repair at the site of the initial damage, including DNA adducts, crosslinks, and UV-induced photolesions. Therefore, γ-H2AX is currently attracting attention as a new biomarker for detecting various genotoxic insults. We have determined the toxic impact of various environmental stresses such as chemicals, light and/or their coexposure using γ-H2AX, and found that the γ-H2AX assay exhibited high sensitivity and a low false-positive rate as a detection system of genotoxic potential. In this review, we introduced our recent findings concerning the evaluation of chemical phototoxicity, focusing on γ-H2AX.     

Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.     

Molecular mechanism of the recruitment of NBS1/hMRE11/hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain

DNA double-strand breaks represent the most potentially serious damage to a genome, and hence, many repair proteins are recruited to DNA damage sites by as yet poorly characterized sensor mechanisms. We clarified that NBS1 physically interacts with gamma-H2AX to form nuclear foci at DNA damage sites. The fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT) of NBS1 are essential for this physical interaction and focus formation of NBS1 in response to DNA damage. The inhibition of this interaction by introduction of anti-gamma-H2AX antibody into cells abolishes NBS1 foci formation in response to DNA damage. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for re-localization of the NBS1/hMRE11/hRAD50 complex to the vicinity of DNA damage. Moreover, the foci formation of DNA repair-related proteins containing BRCT domain, such as BRCA1, requires the interaction with gamma-H2AX in response to DNA damage. These findings indicate that the physical interaction between gamma-H2AX and DNA repair-related proteins is indispensable for the recruitment of these proteins. Further, it was recently reported that the NBS1/hMRE11/hRAD50 complex has a crucial role for both the recruitment of ATM to DNA damage sites and the subsequent activation of ATM. Therefore, both gamma-H2AX and the NBS1/hMRE11/hRAD50 complex might function for the initial recognition of DNA damage.