Comparison of core and valence band electronic structures of bulk uracil and 5-halouracils.

Core (C, N, and O 1s regions) and valence band electronic structures of bulk uracil and 5-fluoro-, -chloro-, and -iodouracils were investigated using X-ray photoemission spectroscopy and comprehensively compared with those of 5-bromouracil measured under the same experimental conditions before. The halogenation of uracil shifted the core peaks of the 5-position carbons toward the higher binding energy side and reduced the ionization potentials depending on the type of halogen. Theoretical calculations supported these results. The alterations of electronic properties induced by the halogenation would result in the characteristic properties of 5-halouracils.

Development of a silicone-based radio-fluorogenic dosimeter using dihydrorhodamine 6G.

A silicon-based three-dimensional dosimeter can be formed in a free shape without a container and deformed because of its flexibility. Several studies have focused on enhancing its radiological characteristics and assessing its applicability as a quality assurance tool for image-guided and adaptive radiation therapy, considering motion and deformation. Here, we applied a fluorescence probe (dihydrorhodamine 6G, DHR6G) to a silicon elastomer as a new radiosensitive compound that converts nonfluorescent into fluorescent dyes using irradiation, and its fluorescence intensity increases linearly with the absorbed dose. In this study, we demonstrated a cost-effective synthesis method and optimized the composition conditions. The results showed that the DHR6G-SE prepared from 2.2 × 10-3 wt% DHR6G, 0.024 wt% pyridine, and a silicone elastomer (SE) (SILPOT TM 184, base/curing agent = 10/1) exhibited a linear increase in fluorescence with radiation exposure within a dose range of 0-8 Gy and a highly stable sensitivity for as long as 64 h. To demonstrate its container-less characteristics, the possibility of dosimetry for low-energy X-rays using DHR6G-SE was investigated.

Radiation-Induced Autophagy in Human Pancreatic Cancer Cells is Critically Dependent on G2 Checkpoint Activation: A Mechanism of Radioresistance in Pancreatic Cancer.

PURPOSE: Autophagy and cell-cycle checkpoints act in concert to confer cellular radioresistance. We investigated the functional interaction between radiation-induced autophagy and G2 checkpoint activation in highly radioresistant human pancreatic ductal adenocarcinoma (PDAC) cells. METHODS AND MATERIALS: Four human PDAC cell lines (MIA PaCa-2, KP-4, Panc-1, and SUIT-2) were analyzed. These cells were first irradiated using x-rays, and their cell cycle status, autophagy, and cell cycle checkpoint marker expression and ATP production levels were evaluated. Autophagic flux assays and siRNA knockdown were used to evaluate autophagy activity. Double thymidine block experiments were performed to synchronize the cells. Two inhibitors (MK-1775 and SCH 900776) were used to attenuate G2 checkpoint activation. Cell survival assays and animal experiments were performed to evaluate the radiosensitizing effects of the G2 checkpoint inhibitors. RESULTS: Autophagy and G2/M accumulation were synchronously induced in human PDAC cells with an activated G2 checkpoint at 12 hours after x-ray irradiation of 6 Gy. Radiation-induced autophagy produced the ATP levels required for cell survival. Double thymidine block experiments revealed that no autophagy occurred in cells that were solely in G2 phase. MK-1775 or SCH 900776 exposure attenuated not only G2 checkpoint activation but also postirradiation autophagy, indicating the dependence of radiation-induced autophagy on an activated G2 checkpoint. The inhibitors demonstrated a higher radiosensitizing effect in the PDAC cells than the autophagy inhibitor chloroquine. MK-1775 in combination with x-rays significantly suppressed the tumor growth of MIA PaCa-2 xenografts compared with other treatment groups, including radiation or drug exposure alone, to enhance the radiosensitivity of PDAC cells in vivo. CONCLUSIONS: Biological crosstalk exists between the G2 checkpoint activation and radiation-induced autophagy processes that are believed to independently contribute to the radioresistance of human PDAC cells. These findings have important implications for the development of future radiation therapy strategies for PDAC.

Field size effects on DNA damage and proliferation in normal human cell populations irradiated with X-ray microbeams.

To clarify the health risks of internal radiation exposure, it is important to investigate the radiological effects of local exposure at cell levels from radioactive materials taken up by organs. Focusing on the response of cell populations post-irradiation, X-ray microbeams are very effective at reproducing the effects of local exposure within an internal exposure in vitro. The present study aims to clarify the effects of local exposure by investigating the response of normal human cell (MRC-5) populations irradiated with X-ray microbeams of different beam sizes to DNA damage. The populations of MRC-5 were locally irradiated with X-ray microbeams of 1 Gy at 0.02-1.89 mm2 field sizes, and analyzed whether the number of 53BP1 foci as DSB (DNA double strand break) per cell changed with the field size. We found that even at the same dose, the number of DSB per cell increased depending on the X-irradiated field size on the cell population. This result indicated that DNA damage repair of X-irradiated cells might be enhanced in small size fields surrounded by non-irradiated cells. This study suggests that X-irradiated cells received some signal (a rescue signal) from surrounding non-irradiated cells may be involved in the response of cell populations post-irradiation.

Mutations in the FHA-domain of ectopically expressed NBS1 lead to radiosensitization and to no increase in somatic mutation rates via a partial suppression of homologous recombination.

Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy.

Benzyl isothiocyanate sensitizes human pancreatic cancer cells to radiation by inducing apoptosis.

Isothiocyanates are a class of naturally occurring chemopreventive agents known to suppress proliferation of cancer cells in culture. The present study was undertaken in order to examine the effects of benzyl isothiocyanate (BITC), one of the common dietary isothiocyanates, on the radiosensitivity of human pancreatic cancer cells and to gain insights into the underlying molecular mechanism of BITC-induced radiosensitization. Two human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2, were treated with BITC and irradiated with X-rays. Radiation sensitivity, apoptosis, and protein levels were determined by a clonogenic assay, fluorescence microscopic analysis with DAPI staining and Western blotting, respectively. MIAPaCa-2 cells were relatively more sensitive to BITC treatment compared with PANC-1 cells. Radiosensitization was observed in both PANC-1 and MIAPaCa-2 cells incubated with BITC at 5 to 10 µM and 2.5 to 5 µM for 24 h, respectively. The combination treatments with BITC and X-rays also revealed an increased percentage of apoptotic cells. In addition, treatment with BITC and X-rays resulted in a decrease in the protein levels of the X-linked inhibitor of apoptosis (XIAP), inhibitor of apoptosis (IAP) family protein, and in a marked increase in the apoptosis protease activating factor-1 (Apaf-1), essential for activation of caspase-9 in stress-induced apoptosis. BITC may be a useful radiosensitizer for radiotherapy of pancreatic cancers.

Scriptaid, a novel histone deacetylase inhibitor, enhances the response of human tumor cells to radiation.

A group of histone deacetylase (HDAC) inhibitors has been shown to suppress the growth of a variety of human tumor lines in vitro and in vivo and they are among the most promising candidates for anti-cancer therapeutic agents. We investigated the ability of scriptaid, a novel HDAC inhibitor and trichostatin A (TSA) to enhance cell killing by radiation in radioresistant SQ-20B cells derived from human head and neck squamous carcinoma. SQ-20B cells were treated with scriptaid or TSA in combination with radiation. Cell survival was determined by a colony formation assay and protein levels were examined by Western blotting. DNA double strand breaks were measured by a gamma-H2AX focus assay. Radiosensitization was observed for SQ-20B cells incubated with scriptaid at 5 microM or TSA at 0.1 microM for 24 h. Radiosensitization by scriptaid was accompanied by a prolonged retention of gamma-H2AX foci, suggesting that the enhancement of radiation cell killing by scriptaid involved inhibition of DNA double strand break repair. In addition, treatment with scriptaid suppressed expression of Ku80, but not Ku70. Scriptaid may be a useful radiosensitizer in the treatment of radioresistant human carcinomas.

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.

Ki-67 labeling index as a marker of malignancy in ocular surface neoplasms.

PURPOSE: To evaluate the relationships among histopathological type, clinical malignant grade, and Ki-67 labeling index (LI) in sebaceous gland carcinoma (SGC), conjunctival squamous cell carcinoma (SCC), and conjunctival intraepithelial neoplasia (CIN), with pterygium and normal conjunctiva as controls. METHODS: This retrospective study was conducted at the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. We used tissue specimens obtained from 20 patients (four SGC, four SCC, four CIN, four pterygium, and four normal conjunctiva). Ki-67 immunohistochemical analysis was performed in all 20 cases. RESULTS: The Ki-67 labeling index (LI) was 46.1 +/- 3.0% (average +/- SD) in SGC, 28.4 +/- 4.5% in SCC, 20.0 +/- 7.2% in CIN, 9.0 +/- 2.2% in pterygium, and 6.8 +/- 2.3% in normal conjunctiva. Ki-67 LI was significantly (Mann-Whitney U test, P < 0.05) higher in SGC than in SCC, and higher, but not significantly, in SCC than in CIN. Ki-67 LI was significantly (P < 0.05) higher in SCC and CIN than in pterygium. CONCLUSIONS: These results suggest that Ki-67 LI may be a sensitive marker for ocular malignant tumor grading.