Masato Hareyama

Preventing oxidative stress: a new role for XBP1

Antioxidant molecules reduce oxidative stress and protect cells from reactive oxygen species (ROS)-mediated cellular damage and probably the development of cancer. We have investigated the contribution of X-box-binding protein (XBP1), a major endoplasmic reticulum stress-linked transcriptional factor, to cellular resistance to oxidative stress. After exposure to hydrogen peroxide (H2O2) or a strong ROS inducer parthenolide, loss of mitochondrial membrane potential (MMP) and subsequent cell death occurred more extensively in XBP1-deficient cells than wild-type mouse embryonic fibroblast cells, whereas two other anticancer agents induced death similarly in both cells. In XBP1-deficient cells, H2O2 exposure induced more extensive ROS generation and prolonged p38 phosphorylation, and expression of several antioxidant molecules including catalase was lower. Knockdown of XBP1 decreased catalase expression, enhanced ROS generation and MMP loss after H2O2 exposure, but extrinsic catalase supply rescued them. Overexpression of XBP1 recovered catalase expression in XBP1-deficient cells and diminished ROS generation after H2O2 exposure. Mutation analysis of the catalase promoter region suggests a pivotal role of CCAAT boxes, NF-Y-binding sites, for the XBP1-mediated enhancing effect. Taken together, these results indicate a protective role of XBP1 against oxidative stress, and its positive regulation of catalase expression may at least in part account for this function.

Ability to repair DNA double-strand breaks related to cancer susceptibility and radiosensitivity

Traditional radiobiology has aimed at elucidating the mechanism of radiosensitivity of cancer cells and normal cells. Because the mechanism of DNA double-strand break (DSB) repair, which is inherently important to radiosensitivity, was unknown, it has been difficult to obtain results applicable to clinical radiotherapy from traditional radiobiology research. Today, however, the molecular mechanism of DNA DSB repair has been elucidated because of the rapid advances in molecular biology. In DNA DSB repair, at least two major repair mechanisms, homologous recombination and nonhomologous end joining (NHEJ) have been reported. In the NHEJ pathway, DSBs are directly, or after processing of the DNA ends, rejoined at an appropriate chromosomal end. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA DSB repair by NHEJ. We have investigated how the ability of repair of DNA DSB influences cancer susceptibility and the radiosensitivity of tumors and normal tissues by focusing on the activity of DNA-PK. In the near future, research on DNA DSB repair mechanism will be able to be applied to research on carcinogenesis, prediction of radiosensitivity of tumors and normal cells, and sensitization of tumor cells.

IL12RB2 and ABCA1 Genes Are Associated with Susceptibility to Radiation Dermatitis

Purpose: Severe acute radiation dermatitis is observed in approximately 5% to 10% of patients who receive whole-breast radiotherapy. Several factors, including treatment-related and patient-oriented factors, are involved in susceptibility to severe dermatitis. Genetic factors are also thought to be related to a patients susceptibility to severe dermatitis. To elucidate genetic polymorphisms associated with a susceptibility to radiation-induced dermatitis, a large-scale single-nucleotide polymorphism (SNP) analysis using DNA samples from 156 patients with breast cancer was conducted. Experimental Design: Patients were selected from more than 3,000 female patients with early breast cancer who received radiotherapy after undergoing breast-conserving surgery. The dermatitis group was defined as patients who developed dermatitis at a National Cancer Institute Common Toxicity Criteria grade of ≥2. For the SNP analysis, DNA samples from each patient were subjected to the genotyping of 3,144 SNPs covering 494 genes. Results: SNPs that mapped to two genes, ABCA1 and IL12RB2, were associated with radiation-induced dermatitis. In the ABCA1 gene, one of these SNPs was a nonsynonymous coding SNP causing R219K (P = 0.0065). As for the IL12RB2 gene, the strongest association was observed at SNP-K (rs3790568; P = 0.0013). Using polymorphisms of both genes, the probability of severe dermatitis was estimated for each combination of genotypes. These analyses showed that individuals carrying a combination of genotypes accounting for 14.7% of the Japanese population have the highest probability of developing radiation-induced dermatitis. Conclusion: Our results shed light on the mechanisms responsible for radiation-induced dermatitis. These results may also contribute to the individualization of radiotherapy.

Gimeracil sensitizes cells to radiation via inhibition of homologous recombination

BACKGROUND AND PURPOSEn5-Chloro-2,4-dihydroxypyridine (Gimeracil) is a component of an oral fluoropyrimidine derivative S-1. Gimeracil is originally added to S-1 to yield prolonged 5-FU concentrations in tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We found that Gimeracil by itself had the radiosensitizing effect.nnnMETHODS AND MATERIALSnWe used various cell lines deficient in non-homologous end-joining (NHEJ) or homologous recombination (HR) as well as DLD-1 and HeLa in clonogenic assay. gamma-H2AX focus formation and SCneo assay was performed to examine the effects of Gimeracil on DNA double strand break (DSB) repair mechanisms.nnnRESULTSnResults of gamma-H2AX focus assay indicated that Gimeracil inhibited DNA DSB repair. It did not sensitize cells deficient in HR but sensitized those deficient in NHEJ. In SCneo assay, Gimeracil reduced the frequency of neo-positive clones. Additionally, it sensitized the cells in S-phase more than in G0/G1.nnnCONCLUSIONSnGimeracil inhibits HR. Because HR plays key roles in the repair of DSBH caused by radiotherapy, Gimeracil may enhance the efficacy of radiotherapy through the suppression of HR-mediated DNA repair pathways.

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination

Gimeracil (5‐chloro‐2, 4‐dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5‐fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S‐1 to yield prolonged 5‐fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation‐induced focus formation of various kinds of proteins involved in HR was carried out. siRNA for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil‐treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD‐depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR‐mediated DNA repair. (Cancer Sci 2011; 102: 1712–1716)

The association of DNA-dependent protein kinase activity of peripheral blood lymphocytes with prognosis of cancer

Background:Repair of various types of DNA damages is critical for genomic stability. DNA-dependent protein kinase (DNA-PK) has an important role in DNA double-strand break repair. We examined whether there may be a correlation between DNA-PK activity in peripheral blood lymphocytes (PBLs) and survival percentages in various cancer patients. We also investigated the changes of DNA-PK activity in PBLs after radiotherapy.Methods:A total of 167 of untreated cancer patients participated in this study. Peripheral blood was collected, separated, and centrifuged. DNA-PK activity was measured by DNA-pull-down assay. Chromosomal aberrations were examined by cytogenetic methods.Results:DNA-PK activity of PBLs in advanced cancer patients was significantly lower than that in early stage. The patients with lower DNA-PK activity in PBLs tended to have the lower disease-specific survivals and distant metastasis-free survivals than those with higher DNA-PK activity in advanced stages. There was also a tendency of inverse correlation between DNA-PK activity and excess fragments. The DNA-PK activity of PBLs in most patients decreased in response to radiation as the equivalent whole-body dose increased.Conclusion:Cancer patients in advanced stage, with lower DNA-PK activity of PBLs might have higher distant metastasis and exhibit poorer prognosis. Therefore, DNA-PK activity in PBLs could be used as a marker to predict the chromosomal instability and poorer prognosis.

Brachytherapy for Oral Tongue Cancer: An Analysis of Treatment Results with Various Biological Markers

OBJECTIVEnLow-dose-rate (LDR) brachytherapy is an effective treatment for tongue cancer. However, little is known about the biological mechanism underlying this therapy, characterized by delivery of continuous exposures of LDR irradiation. It is reported that lower microvessel density (MVD), lower Ki-67 index or higher expression of endogenous hypoxic markers such as carbonic CA IX and Glut-1 are related to the poor control of tumors treated with external irradiation. To elucidate the biological characteristics of LDR brachytherapy, we analyzed our results in cases of tongue cancer treated with LDR brachytherapy by using immunohistochemical stainings with antibodies against Ki-67 and MVD, Glut-1 and CA IX.nnnMETHODSnThe prognostic value of Ki-67 index, MVD and the expression of CA IX and Glut-1 was assessed in 68 tongue cancers treated with LDR brachytherapy. The specimens were taken from tongue cancers before radiation therapy and immunohistochemical staining was performed.nnnRESULTSnThe local recurrence-free survival rates were significantly different between T1+T2 and T3 (P = 0.00067), but not between low and high Ki-67 indexes (P = 0.54), between low and high MVD (P = 0.071), low and high CA IX indexes (P = 0.062) or low and high Glut-1 indexes (P = 0.107). T stage, the size of the tumor was the only significant factor for local control in multivariate analyses (P = 0.0377).nnnCONCLUSIONnLDR could overcome the radioresistence of non-cycling and hypoxic cells; however, we cannot draw firm conclusions due to the limited number of patients.

The combination of hyperthermia or chemotherapy with gimeracil for effective radiosensitization

Purpose5-chloro-2,4-dihydroxypyridine (gimeracil) is a component of the oral fluoropyrimidine derivative S-1. Gimeracil was originally added to S-1 to yield prolonged 5-fluorouracil (5-FU) concentrations in serum and tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We previously demonstrated that gimeracil enhances the efficacy of radiotherapy through the suppression of homologous recombination (HR) in DNA double strand repair. The goal of this paper was to examine the effects of gimeracil on the sensitivity of anticancer drugs and hyperthermia in order to obtain effective radiosensitization.Materials and methodsVarious cell lines, including DLD 1 (human colon carcinoma cells) and cells deficient in HR or nonhomologous end-joining (NHEJ), were used in clonogenic assays. The survival of these cells after various treatments (e.g., drug treatment, heat treatment, and radiation) was determined based on their colony-forming ability.ResultsGimeracil enhanced cell-killing effects of camptothecin (CPT), 5-FU, and hydroxyurea. Gimeracil sensitized effects of CPT or 5-FU to cells deficient in HR or NHEJ to a similar extent as in other cells (DLD1 and a parent cell), indicating that its sensitizing mechanisms may be different from inhibition of HR or NHEJ. Combination of gimeracil and CPT or 5-FU sensitized radiation more effectively than each modality alone. Gimeracil also enhanced heat sensitivity at 42°C or more. The degree of heat sensitization with gimeracil increased as the temperature increased, and the combination of gimeracil and heat-sensitized radiation was more effective than each modality alone.ConclusionGimeracil enhanced sensitivity of CPT, 5-FU, and hyperthermia. Combination of these modalities sensitized radiation more efficiently than each modality alone.ZusammenfassungZiel5-Chlor-2,4-Dihydroxypyridin (Gimeracil) ist eine Komponente des oralen Fluoropyrimidin-Derivatsxa0S-1. Gimeracil wird ursprünglich zu S-1 hinzugefügt, um die 5-FU-Konzentrationen in Blutserum und Tumorgewebe länger aufrechtzuerhalten. Dies beruht auf einer Hemmung der Dihydropyrimidin-Dehydrogenase, die 5-FU abbaut. In früheren Untersuchungen konnten wir zeigen, dass Gimeracil die Wirksamkeit der Strahlentherapie durch Unterdrückung der homologen Rekombination (HR) bei der Reparatur von DNA-Doppelstrangbrüchen verbessert. Im vorliegenden Beitrag haben wir die Wirkung von Gimeracil auf die Empfindlichkeit gegenüber Chemotherapeutika und Hyperthermie untersucht, um eine effektive Strahlensensibilisierung zu erzielen.Material und MethodikWir benutzten verschiedene Zelllinien einschließlich DLD-1 (humane Kolonkarzinomzellen) und Zellen mit HR- bzw. einer NHEJ (nichthomologen Endverknüpfung)-Defizienz in klonogenen Tests. Das Überleben dieser Zellen wurde nach verschiedenen Behandlungen wie medikamentöser Behandlung, Wärmebehandlung und Bestrahlung anhand der Koloniebildungsfähigkeit bestimmt.ErgebnisseGimeracil verstärkte die zelltötende Wirkung von Camptothecin (CPT), 5-Fluorouracil (5-FU) und Hydroxyurea. In HR- bzw. NHEJ-defizienten Zellen verstärkte Gimeracil die zelltötende Wirkung von CPT und 5-FU im gleichen Maß wie in anderen Zellen (DLD-1 und Elternzellen). Dies deutet darauf hin, dass hier andere sensibilisierende Mechanismen als bei der Hemmung von HR oder NHEJ zum Tragen kommen. Der kombinierte Einsatz von Gimeracil und CPT bzw. 5-FU bei der Strahlentherapie führte zu einer wirksameren Strahlensensibilisierung als jede Behandlungsmodalität für sich allein. Gimeracil erhöhte auch die Wärmeempfindlichkeit bei Temperaturen ab 42°C. Der Grad der Wärmeempfindlichkeit unter Gimeracil stieg mit zunehmenden Temperaturen. Die Kombination von Gimeracil mit Wärme über 42°C führte zu einer stärkeren Strahlensensibilisierung als jede einzelne Behandlungsmodalität für sich allein.SchlussfolgerungGimeracil erhöhte die Empfindlichkeit für CPT, 5-FU und Hyperthermie. Eine Kombination dieser Modalitäten sensibilisiert für Strahlung effizienter als jede Modalität allein.