Nobuhiro Yamakawa

Evidence for the Involvement of Double-Strand Breaks in Heat-Induced Cell Killing

To identify critical events associated with heat-induced cell killing, we examined foci formation of γH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of γH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5°C to 45.5°C, we observed a linear increase with time in the number of γH2AX foci. An inflection point at 42.5°C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of γH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of γH2AX foci formation. We also found that thermotolerance was due to a depression in the number of γH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.

High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation.

Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation‐induced apoptosis. Human gingival cancer cells (Ca9‐22 cells) containing a mutated p53 (mp53) gene were irradiated with X‐rays, C‐ion (13–100 KeV/µm), or Fe‐ion beams (200 KeV/µm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase‐3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET‐dependent manner. Both RBE curves reached a peak at 100 KeV/µm, and then decreased at values over 100 KeV/µm. When cells were irradiated with high LET radiation, Caspase‐3 was cleaved and activated, leading to poly (ADP‐ribose) polymerase (PARP) cleavage. In addition, Caspase‐9 inhibitor suppressed Caspase‐3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase‐8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase‐3 through Caspase‐9, even in the presence of mp53. (Cancer Sci 2008; 99: 1455–1460)

Development of thermotolerance requires interaction between polymerase-β and heat shock proteins

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat‐induced DNA double‐strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat‐labile repair proteins such as DNA polymerase‐β (Polβ). Here the authors show that thermotolerance was partially suppressed in Polβ−/– mouse embryonic fibroblasts (MEF) when compared to the wild‐type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat‐induced γH2AX was suppressed in the thermotolerant cells. These results suggest that Polβ at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat‐induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat‐labile proteins besides Polβ. The present novel findings provide strong evidence that Polβ functions as a critical element involved in thermotolerance and exerts an important role in heat‐induced DSB. (Cancer Sci 2008; 99: 973–978)

Preoperative concurrent chemoradiotherapy for stages II–IV oral squamous cell carcinoma: a retrospective analysis and the future possibility of this treatment strategy

This study evaluated survival in 154 patients with stages II-IV oral squamous cell carcinoma (OSCC) treated with preoperative concurrent chemoradiotherapy and assessed the future use of this treatment strategy. 14 patients exhibited advanced stage II, 73 exhibited stage III and 67 exhibited stage IV. All patients received 40Gy irradiation and concurrent cisplatin-based chemotherapy in two courses. Radical surgery was undertaken after 2-6 weeks. The clinical tumour response, histopathologic regression grade, residual tumour grade (RGrade) in the primary tumour and the level of residual pN+ were associated with prognosis. 90% of patients with complete response and 73% of patients with good partial response in the primary tumour were RGrade 0 (no residual tumour cells) or RGrade 1 (viable tumour cells remained within central superficial portion). In patients with complete response in the neck, residual pN+ was only seen in levels IB (8%) and IIA (8%); the higher the level of residual pN+, the lower the survival rate (p<0.0001). This treatment strategy was excellent for stages II-IV OSCC. It may be possible to perform minimally invasive surgery in which the extent of resection in primary tumour and neck is reduced in patients who achieve good response following preoperative chemoradiotherapy.

Mandibular reconstruction with vascularised fibular osteocutaneous flaps using prefabricated stereolithographic mandibular model

modifications to avoid transverse scars. The resulting method is very simple because it requires no manipulation of flaps. Tight stitches are essential for prevention of scar widening, however, if that should occur then later correction is facilitated by the straight line of closure. Further follow-up is necessary, however the initial scar contracture is minimal in comparison with other conventional methods after more than one year follow-up.

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.

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.

Prognostic and staging implications of mandibular canal invasion in lower gingival squamous cell carcinoma

A multi‐institutional study was undertaken to determine whether mandibular canal (MC) invasion and mandibular medullary bone invasion are independent factors in lower gingival squamous cell carcinoma (SCC). A total of 345 patients with lower gingival SCC were retrospectively reviewed. Mandibular bone invasion was categorized into three types; no bone invasion; invasion through cortical bone (medullary); and MC invasion. The overall survival rate and factors affecting local, regional, and distant failures were assessed by Cox proportional hazards regression analysis and Kaplan–Meier estimates. Bone invasion was present in 201 (58%) patients, of whom 107 (31%) had medullary invasion and 94 (27%) had MC invasion. Using the International Union Against Cancer (UICC) staging system and American Joint Committee on Cancer (AJCC) system, 171 (50%) patients were classified as T4a. When the bone invasion criteria were excluded from the UICC/AJCC system definition, 152 T4a tumors were downstaged and reclassified to T1 in 12 (3%), to T2 in 98 (28%), and to T3 in 42 (12%). In Cox multivariate analysis, MC invasion was an independent predictor of overall survival but medullary bone invasion was not. Medullary bone invasion was an independent variable for distant control. The current T staging system has restricted prognostic utility. The authors recommend a modified T staging system, whereby tumors with MC invasion instead of medullary bone invasion are classified as T4a, and tumors are first classified as T1 to T3 based on size and then upstaged by one T classification in the presence of medullary invasion.

Alveolar Osteonecrosis of the Mandible after Varicella Zoster Infection of the Trigeminal Nerve

Abstract A 76-year-old man presenting with necrosis of a segment of mandibular alveolar bone and spontaneous exfoliation of the corresponding teeth 1 month after acute varicella zoster infection of the mandibular branch of the trigeminal nerve is reported. Local surgical therapy was successful. Awareness of this potential complication of varicella zoster of the trigeminal nerve is highlighted.

Significant prognostic factors affecting treatment outcomes of salivary gland carcinoma: a multicenter retrospective analysis

The purpose of this study was to investigate the prognostic factor in salivary gland carcinoma patients. Clinical and pathological data of 211 consecutive patients who treated with curative intent were analyzed. The overall survival (OS) rate, local control rate, and distant metastasis rate were calculated. To examine a prognostic factor in salivary gland carcinoma patients, a multivariate analysis was performed. The 5-year-OS rate was 84.0%, and 10-year was 69.2%. The 5-year-local control rate was 84.6%, and 10-year was 70.1%. The 5-year-distant metastasis rate was 16.9%, and 10-year was 21.1%. In a multivariate analysis, the OS rate was affected by pN(+), high-grade malignancy, and primary tumor size. The local control was affected by the primary tumor size, high-grade malignancy, and the status of the surgical margin. The primary tumor size and pN(+) were associated with the distant metastasis. The results of this study suggested that pN(+), malignancy grade, primary tumor size, and the margin status might affect the prognosis of salivary gland carcinoma patients. Postoperative radiotherapy and adjuvant chemotherapy were suggested the possibility of contribution to the good prognosis of salivary gland carcinoma patients.