Isao Furuta

Immunohistochemical study on overexpression of cyclooxygenase-2 in squamous cell carcinoma of the oral cavity: its importance as a prognostic predictor

Cyclooxygenase-2 (COX-2) is known as one of the critical prognostic factors in carcinomas of the various organs. However, the importance of COX-2 overexpression in oral squamous cell carcinomas has not been fully described yet. We investigated overexpression of COX-2 by immunohistochemistry in 72 surgical specimens from patients with squamous cell carcinoma of the oral cavity, and evaluated correlations between COX-2 overexpression and clinicopathologic variables. The immunoreactivity of COX-2 was cytoplasmic. COX-2 overexpression was observed in 10 (13.9%) of 72 tumours and it was well correlated with lymph node involvement at the time of surgical treatment (P=0.011) and postoperative recurrence (P=0.025), but not with the other clinicopathologic variables including age, gender, tumour stage and histological grade. In addition, COX-2 overexpression showed a close association with postoperatively disease-free survival (P=0.039) and overall survival as well (P=0.043), and multivariate analyses revealed that COX-2 overexpression was an independent predictor for disease-free survival but not for overall survival. The current study suggests that overexpression of COX-2 could impact on disease-free survival for patients with oral squamous cell carcinoma and that selective inhibition of COX-2 is a possible target for the therapeutic strategies.

Correlations of bcl-2 and p53 expression with the clinicopathological features in tongue squamous cell carcinomas

bcl-2 oncogene prolongs cell survival by inhibition of apoptosis. p53 tumor suppressor gene participates not only in cell proliferation control but also in induction of apoptosis. The expression of both bcl-2 and p53 proteins in 52 primary tongue squamous cell carcinomas (SCCs) was immunohistochemically explored in correlations with clinico-pathological features, patients prognosis and apoptosis index (AI) of this tumor type. bcl-2 and p53 expression were identified in 26/52 (50%) cases and 31/52 (60%) cases, respectively. The frequency of bcl-2 expression was associated with tumor histologic grade (P = 0.0128) and marginally with mode of tumor invasion (P = 0.0671) but not with lymph nodal involvement. The frequency of p53 expression was associated with mode of tumor invasion (P = 0.0458) and pN status (P = 0.0224) but not with tumor histologic grade. Moreover, the three combined bcl-2/p53 staining patterns of bcl-2-/p53-, bcl-2+/p53- and bcl-2-/p53+, and bcl-2+/p53+ were significantly correlated with tumor histologic grade (P = 0.0299), mode of tumor invasion (P = 0.0022) and pN status (P = 0.0024). In addition, the frequent appearance of bcl-2 protein expression was associated with a decrease in AI (P = 0.0290). Our results suggest that the combined investigation on the two biological markers may have value in assessment of tumor aggressiveness, and that the suppressing mechanism of bcl-2 oncogene in regulation of apoptosis preserves in tongue SCC.

Activation of MAP kinases, Akt and PDGF receptors in injured peripheral nerves

A number of receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol‐3‐kinase (PI3K)/Akt and mitogen‐activated protein (MAP) kinase signaling pathways have been critically involved in peripheral nerve regeneration. Here, we examined the activation of PI3K/Akt and MAP kinase pathways, and platelet‐derived growth factor receptors (PDGFRs) in the distal segments of crushed rat sciatic nerve from 3 to 28 days after injury. In Western blot analyses, the phosphorylated forms of extracellular signal‐regulated protein kinase (ERK) and c‐Jun NH2‐terminal kinases (JNKs) were highly augmented on days 3 and 7 and on days 7 and 14 after injury, respectively. Phosphorylated Akt and p38 consistently increased from 3 to 28 days after injury. Phosphorylated PDGFR‐α and ‐β were also increased from 3 to 14 days. In the immunohistological analyses, phosphorylated ERK and PDGFR‐α were co‐localized in many activated Schwann cells and regrowing axons 3 days after injury, while PDGFR‐β was localized in a few spindle‐shaped cells. The detected temporal profile of RTK signaling appears to be crucial for the regulation of Schwann cell proliferation and following redifferentiation. Furthermore, the immunohistological studies suggested a role of ERK and PDGFR‐α in axon regeneration as well.

Active Src expression is induced after rat peripheral nerve injury.

The non–receptor‐type Src tyrosine kinases are key components of intracellular signal transduction that are expressed at high levels in the nervous system. To improve understanding of the cascades of molecular events underlying peripheral nerve regeneration, we analyzed active Src expression in the crushed or cut rat sciatic nerves using a monoclonal antibody (clone 28) that recognizes the active form of Src tyrosine kinases, including c‐Src and c‐Fyn. Western blots showed that active Src expressed in the normal sciatic nerve transiently increased up to threefolds after both types of injury. Immunohistochemistry using clone 28 showed that axonal components are the primary sites of active Src expression in the normal sciatic nerve. Soon after both types of injury, active Src was abundantly expressed in Schwann cells of the segments distal to the injury site. The expression of active Src in the cells decreased with restoration of the axon‐Schwann cell relationship and eventually became depleted to very low levels after crushing, but was sustained at high levels in the cut model until the end of the experiment. Regenerated axons consistently expressed active Src throughout nerve regeneration and these eventually became the major sites of active Src expression in the crushed nerve. Among the Src tyrosine kinases, active c‐Src selectively increased after crushing according to immunoprecipitation and immunoblotting analyses. Due to its potent biological activity, the increased amounts of the active form of Src probably enhance axonal regrowth, the Schwann cell response, and axon‐Schwann cell contact for peripheral nerve regeneration. GLIA 42:184–193, 2003.

A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis

The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 µM (37°C), heat shock (44°C per 20 min) alone or a combination of the two, then incubated at 37°C for 6 h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCδ were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCδ from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria–caspase dependent pathway and the translocation of PKCδ to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.

Gene expression in enhanced apoptosis of human lymphoma U937 cells treated with the combination of different free radical generators and hyperthermia.

The effects of various free radicals derived from 6-formylpterin (6-FP), α-phenyl-tert-butyl nitrone (PBN) and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) combined with hyperthermia, on gene expression in similarly enhanced apoptosis of human lymphoma U937 cells were investigated using cDNA microarrays containing approximately 16,600 genes and computational gene expression analysis tools. When the cells were treated for 10 min at 44°C (15% apoptosis level), 39 up-regulated and 3 down-regulated genes were identified. In the up-regulated genes, apoptosis- and unfolded protein response-associated genes were contained. The combined treatment with heat and either chemical enhanced apoptosis level (approximately 30%) and showed a chemical-specific gene expression pattern. Furthermore, the expression levels of selected genes were confirmed by a real-time quantitative PCR. The present results will provide a basis for further understanding the molecular mechanisms in enhancement of heat-induced apoptosis by different intracellular oxidative stress.

High JC virus load in tongue carcinomas may be a risk factor for tongue tumorigenesis

AbstractThe John Cunningham virus (JCV) asymptomatically infects a large proportion (~90%) of the population worldwide but may be activated in immunodeficient patients, resulting in progressive multifocal leukoencephalopathy. Recent reports demonstrated its oncogenic role in malignancies. In this paper, the presence of JCV-targeting T antigen was investigated in tongue carcinoma (TC, n = 39), dysplastic tongue epithelium (DTE, n = 15) and glossitis (n = 15) using real-time polymerase chain reaction (PCR) and in situ PCR and immunohistochemistry, and JCV copies were analyzed with the clinicopathological parameters of TCs. The results demonstrated that glossitis and DTEs had significantly lower copies of JCV (410.5 ± 44.3 and 658.3 ± 53.3 copies/μg DNA respectively) than TCs (981.5 ± 14.0, p  < 0.05). When they were divided into three groups with 0–200 copies/μg DNA (low), 201–1,000 (moderate) and more than 1001 (high), TCs showed 3 (7.6%) in the low group, 21 (53.8%) in the moderate group and 15 (38.4%) in the high group and glossitis showed 11 (73.3%) in the low group, 0 (0%) in the moderate group and 4 (26.6%) in the high group. The DTEs occupied an intermediate position between them (p < 0.001). In situ PCR demonstrated that the nuclei of TC and DTE cells are sporadically T-antigen positive but not in nasal turbinate epithelial cells. Immunohistochemistry for T-antigen protein revealed four positive cases only in TCs. The existence of JCV T-antigen DNA was not associated with the clinicopathological variables of TCs. In conclusion, the presence of JCV may be a risk factor of tongue carcinogenesis.

Thymidine phosphorylase expression in oral squamous cell carcinoma

Thymidine phosphorylase (TP), as an enzyme involved in DNA synthesis, catalyzes the reversible conversion of thymidine to thymine. It is also identical to the angiogenic factor, platelet-derived endothelial cell growth factor. We examined TP expression using immunohistochemistry in 66 archival samples obtained from the patients with primary oral squamous cell carcinoma (SCC) and investigated its relation to tumor vascularity, cell proliferation, apoptosis, clinicopathological features and survival. TP expression was identified in cytonucleus and/or cytoplasm in carcinomas, but was not identified in histologically normal epithelia distant to tumor in most cases. No significant difference of microvessel density (MVD) was found between the carcinomas with high TP expression (H-TP) and low TP expression (L-TP). The percentages of proliferative cells marked by Ki-67 staining in H-TP carcinomas was significantly higher than that in L-TP carcinomas (P=0.0222). The apoptotic indice (AI) in H-TP carcinomas tended to be lower than that in L-TP carcinomas (P=0.0723). Moreover, the level of TP expression was significantly correlated the pattern of tumor invasion (P=0.0146) and marginally correlated with lymph nodal metastasis (P=0.0804). Our results suggested that TP enzyme may play a role in promotion of tumor growth in oral SCC, and that its expression can be indicative of tumor aggressiveness in this tumor type.

Prognostic significance of p34cdc2 expression in tongue squamous cell carcinoma

We evaluated the prognostic significance of p34cdc2 expression in 50 tongue squamous cell carcinomas (SCC) using immunohistochemical methods. The p34cdc2 protein was expressed in 33 cases of 50 tumor tissues (66.0%), compared with 15 cases of 42 controlled epithelia (35.7%). The expression of p34cdc2 was significantly correlated with the histological grade of tongue carcinoma (P<0.01). In addition, on evaluation of prognosis of tumor, the p34cdc2 protein was overexpressed in recurrent tumors or in those with lymph node metastasis. Statistics showed a significant reduction in the 5-year accumulative survival rate of p34cdc2 positive cases compared with p34cdc2 negative cases (P<0.01). Namely, the p34cdc2 positive patients had worse prognosis. The results suggested that the expression of p34cdc2 suited to the histological grade might reflect the malignant degree of tongue carcinoma biologically. Therefore, the evaluation of p34cdc2 expression was of benefit in elucidating the nature of tumor malignancy and the prognostic prediction of tongue SCC.

Mucosa-associated Lymphoid Tissue Lymphoma of the Palate

Abstract This report is of a 60-year-old woman with mucosa-associated lymphoid tissue lymphoma of the palate. A moderate dose of radiotherapy and anti-CD20 monoclonal antibody chemotherapy were administered after tumour resection. There was no sign of recurrence 14 months after treatment.