Atsuhisa Kajihara

Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.

Disk Position and Temporomandibular Joint Structure Associated with Mandibular Setback in Mandibular Asymmetry Patients

OBJECTIVE To determine the changes in articular disk position and the temporomandibular joint (TMJ) structure in patients who had an asymmetric setback of the mandible performed by a bilateral sagittal split ramus osteotomy with manual positioning of the condyle. MATERIALS AND METHODS Twenty-two patients with skeletal Class III malocclusion being treated at Nara Medical University Hospital were evaluated using clinical examination and pre- and postoperative magnetic resonance images of their TMJs. RESULTS Changes in articular disk position after asymmetric setback surgery were not statistically significant, although a tendency of anterior displacement in the fossa was noted. The anterior and posterior joint spaces did not show significant changes, whereas the condyle head of the deviated or contralateral side tended to be positioned downward. CONCLUSION The asymmetric setback of the mandible with intraoperative manual positioning of the condyle does not significantly change the disk position in the fossa.

Protein microarray analysis of apoptosis-related protein expression following heat shock in human tongue squamous cell carcinomas containing different p53 phenotypes

Purpose: Hyperthermia is useful in the treatment of human head and neck cancers, because it is relatively easy to regulate temperatures when compared to tumors located in deep organs. In this study, attention was focused on p53 as a possible predictive indicator for the efficacy of hyperthermic cancer therapy. Methods: Two kinds of cell lines were used. These were derived from a human squamous cell carcinoma (SAS) and had identical genetic backgrounds except for their p53 gene status. It was previously reported that the heat sensitivity and frequency of apoptosis in wild-type p53 cells (SAS/neo) were clearly elevated when compared with mutated p53 cells (SAS/mp53). In order to study the expression of apoptosis related proteins after heat treatment, protein microarray analysis was used. Results: The expression of apoptosis inhibitory proteins such as Bcl-2, Bcl-xL, NF-κB, COX2, STAT3, IL-6, and IKKα/1 was seen to increase after heat treatment in SAS/mp53 cells, but not in SAS/neo cells. Conclusion: The result of these observations indicates that apoptosis inhibitory proteins (such as Bcl-2, Bcl-xL, IL-6, etc.) were highly induced in SAS/mp53 cells after heat treatment when compared to control SAS/neo cells.

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

Abstract Purpose: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. Materials and methods: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock. Results: Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. Conclusions: The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

The BRCA2 gene is a potential molecular target during 5-fluorouracil therapy in human oral cancer cells

5-Fluorouracil (5-FU) is widely used in clinical cancer therapy. It is commonly used either alone or in combination with other drugs and/or radiation for head and neck, and other types of cancers. 5-FU induces DNA double-strand breaks (DSBs). Inhibition of the repair of 5-FU-induced DSBs may improve the therapeutic response in many tumors to this anticancer agent. The aim of the present study was to further our understanding of the pathways which are involved in the repair of 5-FU-induced DSBs. Cell survival after drug treatment was examined with colony forming assays using Chinese hamster lung fibroblast cells or Chinese hamster ovary cell lines which are deficient in DSB repair pathways involving the homologous recombination repair-related genes BRCA2 and XRCC2, and the non-homologous end joining repair-related genes DNA-PKcs and Ku80. It was found that BRCA2 was involved in such repair, and may be effectively targeted to inhibit the repair of 5-FU-induced damage. Observations showed that knockdown of BRCA2 using small interference RNA suppression increased the sensitivity to 5-FU of human oral cancer cell lines (SAS and HSC3). These findings suggest that downregulation of BRCA2 may be useful for sensitizing tumor cells during 5-FU chemotherapy.

BRCA2 protects mammalian cells from heat shock

Abstract Purpose: Heat shock induces DNA double-strand breaks (DSBs) in mammalian cells. Mammalian cells are capable of repairing DSBs by utilising the homologous recombination (HR) pathway. Breast cancer susceptibility gene 2 (BRCA2) is known to regulate the HR pathway. Here, we investigate the role of BRCA2 in repairing DNA damage induced by heat shock. Materials and methods: Chinese hamster lung fibroblast cell lines and human tongue squamous cell carcinoma SAS cells were used. RAD51 foci formation assay was used as an HR indicator. Heat sensitivity was analysed with colony forming assays. Phosphorylated histone H2AX (γH2AX) intensity, which correlates with the number of DSBs, was analysed with flow cytometry. Results: RAD51 foci appeared with heat shock, and the number of cells with RAD51 foci was maximal at about 4 h after heat shock. Heat-induced RAD51 foci co-localised with γH2AX foci. BRCA2-deficient cells were sensitive to heat when compared to their parental wild-type cells. Heat-induced γH2AX was higher in BRCA2-deficient cells compared to parental cells. In SAS cells, cells transfected with BRCA2-siRNA were more sensitive to heat than cells transfected with negative control siRNA. Apoptotic bodies increased in number more rapidly in BRCA2-siRNA transfected cells than in cells transfected with negative control siRNA when cells were observed at 48 h after a heat treatment. In addition, cells deficient in BRCA2 were incapable of activating heat-induced G2/M arrest. Conclusion: BRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.