坂本 洋右

BACKGROUND: Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho family GTPase, which has been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of ECT2 in regulating oral cancer cell behavior. In this study, we investigated the involvement of ECT2 in oral squamous cell carcinoma (OSCC). METHODOLOGY/PRINCIPAL FINDINGS: We analyzed ECT2 expression in OSCC-derived cell lines and primary OSCCs compared with matched normal tissue (n = 96) by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. We then evaluated the correlation between the ECT2 expression status in primary OSCCs and the clinicopathological features. ECT2 expression was significantly up-regulated in OSCCs in vitro and in vivo (p<0.05). Among the clinical variables analyzed, higher ECT2 expression also was associated with the TNM stage grading (p<0.05). When we performed functional analyses of ECT2 in OSCC-derived cells using the shRNA system, the cellular proliferation of the ECT2 knockdown cells decreased significantly compared with the control cells (p<0.05). Cell cycle analysis by flow cytometry showed arrest of cell cycle progression at the G1 phase in the ECT2 knockdown cells. We also found up-regulation of the Cip/Kip family of the cyclin-dependent kinase inhibitors, p21(cip1) and p27(kip1), and down-regulation of cyclin D1, cyclin E, and CDK4. These data suggested that the elevated Cip/Kip family induced inhibition of the cyclin D1-CDK complex activity leading to cell cycle arrest at the G1 phase. CONCLUSIONS/SIGNIFICANCE: Our results proposed for the first time that ECT2 is an indicator of cellular proliferation in OSCCs and that ECT2 might be a potential therapeutic target for the development of new treatments for OSCCs.

The authors report a case of right mandibular metastasis from thyroid follicular carcinoma of a 71-year-old woman who suffered from painless swelling in the right retromolar region for 10 months. As pulse was detected through a mass, we suspected that the mass was hemangiosarcoma or A-V malformation. However, by an incisional biopsy, a diagnosis of a metastasis from thyroid follicular carcinoma, was suggested. Under general anesthesia, right lobe thyroid gland resection, mandible segmental resection, neck dissection, and plate reconstruction were performed. Final diagnosis of thyroid follicular carcinoma and metastasis to the right mandible was made. The postoperative period was uneventful and the patient was asymptomatic without signs of recurrence 4 years postoperatively. The present case is the uncommon case of mandibular metastasis from thyroid follicular carcinoma. © 2010 Asian Association of Oral and Maxillofacial Surgeons.

Objective: This study was performed to evaluate the usefulness of continuous-selective intra-arterial injection of antibiotics via the superior thyroid artery for osteoradionecrosis of the mandible. Patients and methods: Three osteoradionecrosis cases were treated with this innovative drug delivery system. Totals of 7200. mg, 7810. mg and 6960. mg of clindamycin (CLDM) were administered in cases 1, 2 and 3, respectively. Patients also received conventional intravenous cefozopran hydrochloride (CZOP) or ceftazidime (CAZ) or moxifloxacin (MFLX) and hyperbaric oxygen therapy (HBO). Results: Both clinical findings and laboratory data improved immediately after these treatments in these cases. Conclusion: The present study indicates that continuous-selective antibiotic injection via the superior thyroid artery might be an additional treatment option if performed in combination with conventional approaches in the refractory cases. © 2010 Asian Association of Oral and Maxillofacial Surgeons.

Serpins (serine protease inhibitors) are known as a diverse family of protease inhibitors; however, various other biological activities including tumor suppression, have been recently reported for these molecules. To clarify whether members of the serpin family are involved in OSCC (oral squamous cell carcinoma), global gene screening using microarray analysis was performed with OSCC-derived cell lines. A trend toward diminished expression was shown for some SERPIN genes located on 11q12-q13.1 and 18q21. mRNA expression of SERPIN genes at these chromosome regions was therefore analyzed using real-time quantitative RT-PCR (qRT-PCR) in 55 OSCC samples and matched normal tissue. Statistically significant decreases in expression were found for SERPINB12 (P=0.001), SERPINB13 (P=0.001), SERPINB4 (P=0.042), SERPINB3 (P<0.001), SERPINB11 (P<0.001), SERPINB7 (P=0.021) and SERPINB2 (P=0.018). All of these genes are located on 18q21, the known location of the serpin gene cluster. The results strongly suggest that this chromosome region plays a crucial role in OSCC. Some serpin members in the region might be involved in tumor suppression, or there might be unidentified tumor suppressor genes within or near the chromosome region.

Oxidative stress results in damage to cellular structures and has been linked to numerous diseases, including cancer. Extracellular superoxide dismutase (EC-SOD) is a principal enzymatic antioxidant in extracellular space. The purpose of this study was to determine whether the expression of EC-SOD protein is altered in the carcinogenetic process of oral squamous-cell carcinoma (OSCC). Immunohistochemical analysis was carried out in matched normal and tumour specimens collected from 58 OSCCs and 20 oral premalignant lesions (OPLs). Correlations between the EC-SOD expression levels and clinicopathological features of OSCC patients were evaluated by Fisher's exact test. Although EC-SOD protein was consistently expressed on the plasma membrane of cells in normal tissues, plasma membranous EC-SOD expression was lost in almost all the OSCC specimens examined (98%). Instead, positive EC-SOD expression was detected in the cytoplasmic compartments of cancerous cells in both OPLs (65%) and OSCCs (52%), together with a high incidence of lymph node metastasis (p=0.0397). These results suggest that the dysregulation of EC-SOD protein expression is a frequently occuring and early event in oral carcinogenesis, and that cytoplasmic EC-SOD may contribute to the increased aggressiveness of OSCC.

1980年〜2009年迄の30年間に受診した顎顔面骨折1247例中、頬骨骨折109例(8.7%)を対象に、Knight&North分類(KN分類)に従い、骨折型を検索し、併発骨折の有無や治療法を含め臨床的に検討した。性別は男性76名(69.7%)、女性33名(30.3%)で圧倒的に男性が多く男女比は2.3:1であった。年齢別の頻度は20歳代が41例(37.6%)と最も多く、次いで30歳代17例で、両者で全体の53.2%を占め、60歳代・70歳代を除き、各年齢層で男性の占める割合が多かった。受傷原因は交通事故が55例(50.5%)と最も多く、転倒18例、殴打・スポーツ各14例であった。年代別にみると、1980年代に比べ、2000年代では交通事故で受傷した症例が半減していた。受診までの期間は2日後が17例と最も多く、1週間までに66例(60.6%)が受傷した。頬骨単独骨折はKN分類I型とII型に属し、頬骨の偏位の程度が大きくなるIII型以降は併発骨折も増加し、観血的整復固定術を施行することが多かった。

PURPOSE: The purpose of this study was to characterize changes in the expression of copper-zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD (Mn-SOD) in oral squamous-cell carcinoma (OSCC). METHODS: Real-time quantitative reverse transcriptase-polymerase chain reaction analysis of Cu/Zn-SOD and Mn-SOD mRNA expression was carried out in 50 pairs of OSCC tissue specimens and corresponding normal tissues. Mn-SOD protein expression was evaluated further in 65 OSCC tissue samples and 33 oral premalignant lesions (OPLs) using immunohistochemistry. RESULTS: Significant (P < 0.001) upregulation of Mn-SOD mRNA expression was observed in OSCC tissues compared with the normal tissue counterparts, whereas no significant difference was detected in Cu/Zn-SOD expression. Significant increases in Mn-SOD protein expression were seen in both OPLs (P < 0.001) and OSCC tissue (P < 0.001) together with a high incidence of lymph node metastasis (P = 0.04). CONCLUSIONS: Our findings suggested that Mn-SOD overexpression is a frequent and early event during oral carcinogenesis and could contribute to aggressive OSCC.

1998年1月〜2008年6月に上顎骨骨折あるいは頬骨骨折と診断された21例(男19名、女2名、年齢15〜67歳)を対象に検討を行った。受傷原因は交通事故8例38.1%、転倒6例28.6%が多く、骨折部位は上顎骨単独、頬骨単独および上顎骨と下顎骨の合併が各5例、23.8%、上顎骨と頬骨の合併4例19.0%、上顎骨と頬骨および鼻骨の合併が2例9.5%であった。治療法は観血的整復固定術14例66.7%、非観血的整復固定術6例28.6%であった。上顎骨骨折あるいは頬骨骨折に随伴した合併症状は咬合異常13例61.9%、顔貌変形11例52.4%、眼窩下神経障害11例52.4%および複視2例9.5%であった。咬合異常、顔貌変形、複視は手術により早期に治癒したが、眼窩下神経障害の回復は29日〜5年以上を要し、平均で278.5日であった。中顔面骨骨折において眼窩下神経障害は重要な合併症であり、対応が治療上の鍵となる事が示唆された。

In this study, we performed two- dimensional electrophoresis ( 2- DE) and matrix- assisted laser desorption/ ionisation time of fly mass spectrometry to identify the protein( s) associated with the development of oral squamous cell carcinomas ( OSCCs) by comparing patterns of OSCC- derived cell lines with normal oral keratinocytes ( NOKs), and found that downregulation of ubiquitous mitochondrial creatine kinase ( CKMT1) could be a good candidate. Decreased levels of CKMT1 mRNA and protein were detected in all OSCC- derived cell lines examined ( n = 9) when compared to those in primary normal oral keratinocytes. Although no sequence variation in the coding region of the CKMT1 gene with the exception of a nonsense mutation in exon 8 was identified in these cell lines, we found a frequent hypermethylation in the CpG island region. CKMT1 expression was restored by experimental demethylation. In addition, when we transfected CKMT1 into the cell lines, they showed an apoptotic phenotype but no invasiveness. In clinical samples, high frequencies of CKMT1 downregulation were detected by immunohistochemistry ( 19 of 52 ( 37%)) and quantitative real- time RT - PCR ( 21 of 50 ( 42%)). Furthermore, the CKMT1 expression status was significantly correlated with tumour differentiation ( P &lt; 0.0001). These results suggest that the CKMT1 gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression, which may lead to block apoptosis.

Adenoid cystic carcinoma (ACC) is one of the most common malignant tumors of the salivary glands. It tends to grow slowly but is associated with a poor prognosis compared to other malignant salivary gland tumors. To identify specific markers of ACC, we examined protein expression profiling in ACC xenograft and normal salivary glands (NSG) using fluorescent 2-dimensional differential in-gel electrophoresis (2-D-DIGE), an emerging technique for comparative proteomics, that improves the reproducibility and reliability of differential protein expression analysis between the samples. To identify the proteins, matrix-assisted laser desorption/ionization time-of-flight peptide mass fingerprinting was carried out. Using these strategies, we detected 4 upregulated proteins and 5 downregulated proteins in ACC xenograft. Maspin and stathmin were selected for further analyses. Western blotting and immunohistochemical staining showed a higher expression of these proteins in ACC xenograft and clinical ACC tissue compared to NSG. Furthermore, Expression of these proteins was correlated with the histologic grading of ACC (n = 10). Therefore, our data indicate that maspin and stathmin may be not only useful biomarkers of ACC but also markers of biologic behavior in this tumor.

The human salivary glands have a variety of histologic features such as intercalated duct cells, myoepithelial cells and acinar cells. A neoplastic human salivary intercalated duct cell line (HSG) and its derivatives, HSG with a myoepithelial cell phenotype (HSG-AZA1) and HSG with an acinar cell phenotype (HSG-AZA3) induced by 5-aza-2'-dC treatment of HSG cells, have been reported. To identify characterization of intercalated duct cells, myoepithelial cells and acinar cells in the salivary gland, we selected HSG, HSG-AZA1 and HSG-AZA3 cell lines to perform two-dimensional electrophoresis analysis. We used a fluorescent two-dimensional differential in-gel electrophoresis (2-D-DIGE) for comparative proteomics, which improved the reproducibility and reliability of differential protein expression analysis between the samples. Furthermore, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting (PMF) was used to identify the proteins. These methods were combined to approach the protein profiles associated with characterization between HSG, HSG-AZA1 and HSG-AZA3 cells. Using these strategies, we identified seven HSG associated proteins, such as actin-beta, hydrocephalus inducing protein, L-plastin, KIAA0657 protein, septin 6 isoform A, lamin A/C isoform 2 and superoxide dismutase 2, three HSG-AZA1 associated proteins such as ubiquitin carboxyl-terminal esterase L1, myosin light chain 2 and muscle creatine kinase, and two HSG-AZA3 associated proteins, microtubule-associated protein 6 and Annexin A3. These results suggest that the proteins are associated with characterization of the salivary gland.

The plasma membrane Ca(2+) ATPase (PMCA) is an essential regulator of free intracellular calcium. Recent studies have reported aberrant expression of the PMCA1 gene, a member of the PMCA family, in several cancer cell types. To elucidate the contribution of PMCA1 to oral carcinogenesis, we analyzed genetic and epigenetic changes and mRNA and protein expression in primary oral squamous cell carcinomas (OSCCs), oral premalignant lesions (OPLs), and OSCC-derived cell lines. The PMCA1 gene was epigenetically inactivated, but not mutated in the eight OSCC-derived cell lines tested. In clinical samples, frequent down-regulation of PMCA1 protein expression was found not only in primary OSCCs (43%), but also in OPLs (40%). Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. These results suggest that inactivation of the PMCA1 gene is a frequent and early event during oral carcinogenesis, and gene expression may be regulated by an epigenetic mechanism.

Adenoid cystic carcinoma (ACC) of the salivary gland often has a variable clinical course with a poor prognosis. To investigate DNA copy number aberrations associated with ACCs, we compared comparative genome hybridization data from ACCs (n = 6) with other types of salivary gland tumors such as adenocarcinomas (n = 3) and pleomorphic adenomas (n = 6). While 15 gain loci (1q32, 6p25, 6q21-q24, 7q11.2, 7q31, 10q11.2, 11p12-q12, 12q13, 12q14, 13q24, 16p13.3-13.2, 18p11.3, 18q23, 19q13.4, and Xq28) were detected, no DNA loss locus was evident. To examine the expression status of genes on the ACC-associated loci, transcriptional measurements of approximately 38000 human genes then were monitored using Affymetrix U133 Plus 2.0 GeneChips. A total of 4431 genes were found differentially expressed by at least two-fold between ACCs and normal salivary glands. Of them, 3162 genes were up-regulated and 1269 genes were down-regulated in ACCs. After obtaining locus information about the RNA transcripts from the Affymetrix database, we found 262 ACC-associated genes with increased expression on ACC-associated loci. To investigate functional network and gene ontology, the 262 genes were analyzed using Ingenuity Pathway Analysis Tool. The function with the highest P value was a cancer-related function (P = 2.52e-4 to 4.71e-2). In addition, we identified pituitary tumor-transforming gene 1 and transformation related protein 63 genes that were up-regulated by increasing DNA copy number and modulated expression of oncogenes. These results suggested that the combination of copy number and gene expression profiling provides an improved strategy for gene identification in salivary gland ACCs.

A number of protein markers for oral cancer are still not applicable in large populations. Proteomic technologies provide excellent tools for rapid screening of a large number of potential biomarkers in malignant cells. To gain insight into the molecular mechanisms of carcinogenesis and to identify potential biomarkers for oral squamous cell carcinomas (OSCCs), we performed proteomic profiling between human normal oral keratinocytes (HNOKs) and OSCC-derived cell lines (HSC-2 and HSC-3) using fluorescent two-dimensional difference in-gel electrophoresis. Proteins with a > or =2-fold change in expression were considered significant. The spots of interest were digested and identified by matrix-assisted laser desorption/ionization time-of-flight peptide mass finger-printing. Twenty-two proteins were identified as differentially expressed between the HNOKs and OSCC-derived cell lines. Of these, 9 spots were up-regulated and 13 were down-regulated in OSCC-derived cell lines compared to the HNOKs. These spots included the cancer-related proteins; annexin A1, heat shock protein 27, lamin A/C, interleukin 1 receptor antagonist, serine proteinase inhibitor clade B5, stathmin 1, and superoxide dismutase 2. Our results are a first step toward identifying a protein profile of HNOKs and OSCC-derived cell lines. The identified proteins in this experiment may be used in future studies of carcinogenesis or as diagnostic markers and therapeutic targets for OSCC.

Cementum is a specialized mineralized tissue covering root surface of the tooth. Although the tissue's composition resembles bone, there are distinct structural and functional differences between the two mineralized tissues. In this study, the genes that are differentially expressed in putative cementoblasts (human cementum-derived cells [HCDCs]) compared with preosteoblastic cells (human bone marrow stromal cells [BMSCs]) were screened by two independent microarray systems, and some of the selected genes were further analyzed by quantitative real-time RT-PCR. The gene encoding glucose transporter 1 [GLUT1], which showed the greatest difference between the two groups by the latter analysis, was subjected to further analyses. High levels of the GLUT1 protein in HCDCs, but not in BMSCs, were detected by Western blotting and immunocytochemistry. Furthermore, intense immunoreactivities for GLUT1 were observed in cementoblasts and cementocytes but not in osteoblasts or osteocytes in human periodontal tissues. These results indicate that GLUT1 may play a role in cementogenesis and could serve as a biomarker to differentiate between cells of cementoblastic and osteoblastic lineage.

Mice with a heterozygous deletion of the Atp2a2 gene (Atp2a2(+/-)) encoding SERCA2 spontaneously develop SCCs of the skin and upper digestive tract, including the oral cavity. To elucidate the contribution of ATP2A2 to human oral carcinogenesis, we analyzed genetic and epigenetic changes as well as mRNA and protein expression in primary OSCCs and OPLs. With the exception of one OSCC-derived cell line showing a 12 bp deletion of ATP2A2, we found no mutations in the coding sequence of the gene in primary OSCCs (n = 52), OPLs (n = 32) and cell lines (n = 8). In immunohistochemistry, however, high frequencies of ATP2A2 downregulation were evident not only in primary OSCCs (42%, 42/100) but also in OPLs (31%, 10/32). Real-time quantitative RT-PCR data were consistent with the protein expression status. Aberrant DNA methylation within ATP2A2 also was detected in 9 of 30 ATP2A2-downregulated OSCCs. Moreover, restoration or elevated expression of the ATP2A2 protein was induced in most of the cell lines showing ATP2A2 methylation after treatment with 5-aza-2'-dC, a DNA demethylating agent. These results suggest that inactivation of the ATP2A2 gene is a frequent and early event during oral carcinogenesis and that loss of expression may be regulated partly by an epigenetic mechanism.