鵜澤 一弘

Treatment protocols for malignant tumors in the oral cavity differ greatly based on the presence of cervical lymph node metastasis. We applied gene expression profiling to the pathological lymph node status and used a training-test approach to evaluate the reliability of cDNA microarray-based classifications of 15 matched resected primary oral squamous cell carcinomas (OSCCs) and corresponding normal oral tissues. The clustering of all the microarray data was separated into two groups based on metastatic node positivity and node negativity. Furthermore, a 20-gene signature was identified that differentiated the testing set (n=8) with high classification accuracy (88%). Our findings support the hypothesis that the lymph node metastasis status can be predicted using the gene expression patterns of the primary OSCC, and may be a powerful tool in identifying patients at high risk of lymph node metastasis.

Purpose: The purpose of this study was to assess the gene expression changes in oral squamous cell carcinoma (OSCC) cells after carbon ion irradiation. Methods and Materials: Three OSCC cell lines (HSC2, Ca9-22, and HSC3) were irradiated with accelerated carbon ion beams or X-rays using three different doses. The cellular sensitivities were determined by clonogenic survival assay. To identify genes the expression of which is influenced by carbon ion irradiation in a dose-dependent manner, we performed Affymetrix GeneChip analysis with HG-U133 plus 2.0 arrays containing 54,675 probe sets. The identified genes were analyzed using the Ingenuity Pathway Analysis Tool to investigate the functional network and gene ontology. Changes in mRNA expression in the genes were assessed by real-time reverse transcriptase-polymerase chain reaction. Results: We identified 98 genes with expression levels that were altered significantly at least twofold in each of the three carbon-irradiated OSCC cell lines at all dose points compared with nonirradiated control cells. Among these, SPHK1, the expression of which was significantly upregulated by carbon ion irradiation, was modulated little by X-rays. The function of SPHK1 related to cellular growth and proliferation had the highest p value (p = 9.25e-7 to 2.19e-2). Real-time reverse transcriptase-polymerase chain reaction analysis showed significantly elevated SPHK1 expression levels after carbon ion irradiation (p < 0.05), consistent with microarray data. Clonogenic survival assay indicated that carbon ion irradiation could induce cell death in Ca9-22 cells more effectively than X-rays. Conclusions: Our findings suggest that SPHK1 helps to elucidate the molecular mechanisms and processes underlying the biologic response to carbon ion beams in OSCC. (c) 2006 Elsevier Inc.

The structure and function of chromatin can be altered by modifications to histone. Histone acetylation is a reversible process governed by histone acetyltransferases and historic deacetylases (HDACs). HDAC6 is a subtype of the HDAC family that deacetylates a-tubulin and increases cell motility. We investigated the expression levels of HDAC6 mRNA and protein expression in oral squamous cell carcinoma (OSCC)-derived cell lines and human primary OSCCs to elucidate the potential involvement of HDAC6 in OSCC. Using quantitative real-time reverse transcription polymerase chain reaction and Western blots on nine OSCC-derived cell lines and normal oral keratinocytes (NOKs), HDAC6 mRNA and protein expression were commonly up-regulated in all cell lines compared with the NOKs. Immunofluorescence analysis detected HDAC6 protein in the cytoplasm of OSCC cell lines. Similar to OSCC cell lines, high frequencies of HDAC6 up-regulation were evident in both mRNA (74%) and protein (51%) levels of primary tumors. Among the clinical variables analyzed, the clinical tumor stage was found to be associated with the HDAC6 expression states. The analysis indicated a significant difference in the HDAC6 expression level between the early stage (stage I and II) and advanced-stage (stage III and IV) tumors (P=0.014). These results suggest that HDAC6 expression may be correlated with tumor aggressiveness and offer clues to the planning of new treatments.

術前にTS-1を使用した口腔扁平上皮癌患者31例を対象とし,TS-1単剤による術前補助化学療法の治療効果,副作用,ならびに臨床指標との相関について検討した.CR 6例,PR 13例で,奏効率は61.3%であった.副作用はGrade 1以上が3例,Grade 3以上は1例であった.T分類での治療効果ではT1+T2症例の奏効率75.0%,T3+T4症例の奏効率45.5%であった.複数のリンパ節転移症例(pN2症例)や,術後遠隔転移症例では奏効率が0%であった.TS-1が口腔癌に対する効果的な薬剤であることが示唆された

Functional proteomics is a useful method to explore changes in protein expression in human diseases, including carcinomas. To identify tumor-associated proteins as biomarkers or molecular targets of human oral squamous cell carcinomas (OSCCs), we performed two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Comparison of the protein expression profiles of OSCC cell lines and normal oral keratinocytes identified six proteins with markedly different expression levels. Of the six proteins, we found a 1D-myo-inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) protein that was down-regulated in OSCC cell lines. ITPKA phosphorylates inositol 1,4,5-trisphosphate, which regulates the calcium (Ca2+) level within the cell by releasing Ca2+ from intracellular stores, and is responsible for regulating the levels of a large number of inositol polyphosphates that are important in cellular signaling. Western blots revealed dramatically down-regulated ITPKA expression in all OSCC cell lines examined. Real-time quantitative reverse transcriptase-polymerase chain reaction showed down-regulated ITPKA mRNA expression in nine of 12 (75%) OSCC cell lines. Immunohistochemistry analysis showed that 40 of 100 OSCC clinical samples had a significant decrease in ITPKA. Poorly differentiated tumors showed significantly lower immunoreactivity of the protein compared to well- and moderately-differentiated tumors. These data suggest that ITPKA may be related to carcinogenesis by the modulation of inositol polyphosphates and Ca2+ homeostasis and that ITPKA may be a potential novel molecular target, biomarker, parameter, or all of these of cellular differentiation and of intracellular Ca2+ homeostatic characteristics in clinical medicine.

This study was designed to disclose detailed genetic mechanisms in salivary gland tumors (SGTs) for development of novel independent marker. We constructed an in-house cDNA microarray carrying 2,201 cDNA clones derived from SGT and oral squamous cell carcinoma cDNA libraries. Four cell lines that originated from the SGT-derived cell lines were analyzed using this microarray system. The genes identified by our microarray system were further analyzed at the mRNA or protein expression level in other types of human cancer cell lines and clinical samples (ten normal salivary glands [NSGs], eleven pleomorphic adenomas, ten adenoid cystic carcinomas and three adenocarcinomas). Two up-regulated genes and six down-regulated genes were identified in common when compared with the control RNA. Of the up-regulated genes, WISP-2, which plays an important role in breast carcinogenesis, was selected for further analyses. We found a higher expression of the WISP-2 gene in the SGT-derived cell lines compared with other types of human cancer cell lines. Furthermore, WISP-2 mRNA and protein expression levels in NSGs were significantly higher than those in SGTs. These results suggest that WISP-2 could be a reliable independent marker and that down-regulation or loss of the WISP-2 gene may be associated with the development of SGTs.

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 < 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. (c) 2005 Wiley-Liss, Inc.

腺様嚢胞癌(ACC)に特異的なマーカーの同定を目的にACC組織と正常唾液腺組織から抽出したタンパクを二次元ゲル電気泳動法及びマトリックス支援レーザー脱離イオン化質量分析を行い比較検討した.その結果,ACC組織より3種類の発現増強タンパク(stathmin,maspin,fibrin beta)と3種類の発現減弱タンパク(superoxide dismutase 2,aminolevulinate delta-dehydratase,proapolipoprotein)を同定した.特異的な強発現を呈した3種類のタンパクのうち抗体が入手できたstathminとmaspinについてACCの3組織型を含む5検体を対象にウエスタンブロット法を行ったところ,5検体全てにおいてstathminとmaspinの強発現が確認された.以上よりstathminとmaspinがACCのマーカーとなり得ることが示唆された

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.

We examined the therapeutic effect and adverse reaction for 31 oral squamous cell carcinoma patients who underwent neoadjuvant chemotherapy with TS-1 between April 2003 and March 2005. Among the 31 cases, 6 cases were complete response (CR), and 7 cases were partial response (PR). The response rate was 61.3% (19/3. Adverse reactions of higher than grade 1 were observed in 3 patients, and that of higher than grade 3 was observed in only one case. The response rates were: 75.0% for T1+T2 cases 45.5% for T3+T4 cases. The response rate was 0% for multiple lymph node metastasis cases (pN2 cases) and postoperative distant metastasis cases. These results suggest that TS-1 is an effective therapeutic agent for oral cancer. © 2006, Japan Society for Head and Neck Cancer. All rights reserved.

The plasma membrane Ca2+ 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.

Cisplatin (CDDP) is a widely used potent chemotherapeutic agent for many malignancies. However, the mechanism of resistance to CDDP remains unclear. To investigate the molecular mechanism, we established a CDDP-resistant cell line (H-1R) from a CDDP-sensitive cell line (H-1) which was derived from moderately differentiated squamous cell carcinoma of the lower gingiva. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay indicated that H-1R had a 10-fold greater resistance to CDDP than H-1. When we compared gene expression levels in the cell lines using an in-house cDNA microarray, which represented 2,201 genes originating from normal oral tissue, primary oral cancer, and oral cancer cell lines, 12 genes showing elevated mRNA expression in H-1R compared with H-I were identified. Among them, the upregulated expression of ATP-binding cassette transporter genes (MDR1], MRP1, and MRP2), CD55, and PGK1 and down-regulated expression of Caveolin I were further confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (PCR) or real-time PCR. Our results suggest that H-1 and H-1R cell lines could be useful for elucidating the candidate genes responsible for CDDP resistance, including the genes found in this study.

Radiation therapy is currently the standard adjuvant approach for oral squamous cell carcinoma (OSCC) patients. Individual OSCCs display a wide range of radiosensitivity (RS). To identify genes associated with radioresistance (RR) of OSCC and establish a useful method of predicting radiotherapeutic effectiveness, we examined the gene expression patterns of OSCC cell lines that exhibited different responses to ionizing radiation (IR) by clonogenic survival assay using an in-house cDNA microarray consisting of 2,201 human genes and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). Microarray analysis showed overexpression of 7 genes in the radioresistant cell line, HSC2, and 2 genes in the radiosensitive cell line, HSC3. The changes in expression levels in 7 of 9 genes (Cytokeratin18, DTNBP1, ASNA1, Tcp20, Cyclophilin F, KIAA0218, and HBp17) were confirmed with QRT-PCR. Of these, the genetic alterations of Tcp20, whose expression was remarkably elevated in radioresistant HSC2 cells after IR, were investigated. The escalation of X-ray doses resulted in an enhanced Tcp20 expression level in HSC2 cells compared to radiosensitive HSC3 cells (P<0.05, Mann-Whitney U test). These results suggest that the identified genes, which include Tcp20, may play an important role in conferring RR to OSCC, and could also be useful in identifying cases of OSCC with more radioresistance.

Esophageal and oral carcinomas are relatively resistant to adenovirus serotype 5 (Ad5)-mediated gene transfer, primarily because expression of the cellular receptors for Ad5, the coxsackievirus and adenovirus receptor, is often dowunregulated in these types of tumor. The types of Ad in which the receptor expression is not suppressed in tumors are therefore better vectors for gene transfer into tumors. CD46, a cellular receptor for Ad subtype B2, such as Ad 11 and Ad35, is well expressed in a number of esophageal and oral tumor cells. Since the infectivity of Ad to target cells is mainly influenced by the interaction between their fibers and the cellular receptors, we examined the infectivity of chimeric Ad5, whose fiber structure was substituted with that of type 11 or 35 (Ad5/11 or Ad5/35), to 6 human oral and I I esophagus carcinoma cells. We found that the chimeric Ad, in particular Ad5/35, infected more effectively than Ad5 in all the tumors tested. However, the efficacy of Ad5/35- and Ad5/11-mediated transduction was not correlated with the expression level of CD46 or CD80/86, a cellular receptor of the Ad subtype B 1, in the target cells. These data suggest that the Ad subtype B2 are suitable vectors of gene transfer for human squamous cell carcinomas of the upper, gastrointestinal tract, and that the infectivity of the Ad subtype B2 can possibly be regulated by other receptors besides CD46.

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 38 000 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. (C) 2005 Elsevier Ltd. All rights reserved.

Chromosome 1 open reading frame 10 (C1orf10) is a recently identified gene encoding a protein with an 5100 EF-hand calcium-binding motif, and its expression is known to be down-regulated in esophageal squamous cell carcinoma. In this study, to determine whether the loss of C1orf10 gene function could contribute to the development of oral squamous cell carcinoma (OSCC), we have evaluated the expression status of this gene by reverse transcriptase-polymerise chain reaction (RT-PCR) analysis and quantitative real-time PCR analysis. A high frequency of decrease in C1 orf10 gene was detected not only in OSCC-derived cell lines but also in tumor tissues. Next, to define biological function of this gene in oral carcinogenesis, we transfected C1orf10 with an Ecdysone-inducible system in OSCC cell lines and analyzed the effects of its overexpression. Induction of C1orf10 expression resulted in a significant decline in the rate of cell proliferation, and in an arrest in the G, phase of the cell cycle, with a down-regulation of Cyclin D1 expression. However, we could not detect significant difference in the percentage of apoptotic cells. Thus, our results suggest that the down-regulation of C1 orf10 gene plays a role in oral carcinogenesis, and that its expression may negatively regulate OSCC cell proliferation by arresting the cell cycle. (C) 2005 Elsevier Ltd. All rights reserved.

Oral squamous cell carcinomas (OSCCs), a major public health problem worldwide, are the most common neoplasms of the head and neck. The most important prognostic indicator for patients with OSCC is metastasis to cervical lymph nodes or distant organs. Galectin-9 is correlated with cellular adhesion and aggregation in melanoma cells. To investigate expression levels of galectin-9 mRNA and protein, we performed qRT-PCR and Western blot analyses on OSCC cell lines (Ca9-22, HSC-2, and HSC-3) and normal oral keratinocytes (NOKs). Galectin-9 mRNA and protein were commonly down-regulated in OSCC cell lines compared with NOKs. We further analyzed Ca9-22, which had the lowest expression of galectin-9. We then transfected the galectin-9 cDNA into Ca9-22 cells to examine whether overexpression of galectin-9 increases cellular adhesion in vitro. An adhesion assay using a fibronectin and collagen I coating plate revealed an increased cellular adherence ratio in overexpressed galectin-9 cells compared with nontransfected cells (p < 0.05). The data suggest that galectin-9 is correlated with oral cancer cell-matrix interactions and may therefore play an important role in the metastasis of OSCCs.

Purpose: Cisplatin (CDDP) is widely used for chemotherapy of oral squamous cell carcinoma (OSCC). However, the mechanism of resistance to CDDP is unclear. Recently, caveolin-1 was identified as being associated with both metastasis and multidrug resistance. In the present study, we showed that caveolin-1 expression is significantly related to chemosensitivity in OSCC. Methods: We established a CDDP-resistant cell line, H-1R, from the parental OSCC cell line, H-1. Caveolin-1 expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in both cell lines. We analyzed expression of caveolin-1 in 30 OSCC biopsy specimens and investigated the relationship between expression of caveolin-1 and patients' clinicopathological parameters and chemotherapeutic responses. Results: The 3-(3,4-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that H-1R has a ten-times greater resistance to CDDP than H-1 has. The level of caveolin-1 expression in H-1R was significantly decreased in comparison with that in H-1 by real-time RT-PCR analysis. Positive caveolin-1 immunostaining correlated positively with a complete response (16/20, 80.0%). However, negative immunostaining was found in 6/7 (85.7%) cases with no response. Positive immunohistochemical staining of caveolin-1 correlated positively with chemosensitivity to CDDP-based combination chemotherapy (P=0.02). Conclusions: These results suggest that overexpression of the caveolin-1 gene may provide novel diagnostic markers associated with CDDP sensitivity in OSCC.

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 >= 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 fingerprinting. 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 downregulated 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.

This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT-PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis.

下顎歯肉扁平上皮癌50例について腫瘍原発巣と所属リンパ節転移巣および正常歯肉組織のKAI1遺伝子発現を検索し,発現減弱の頻度を調べた.正常歯肉組織にはKAI1遺伝子の強発現が認められた.原発巣は39例(78%)で発現が減弱していた.リンパ節転移をきたした20例の転移巣は全例で発現が減弱していた.リンパ節転移を伴う原発巣も全て発現が減弱していた.下顎歯肉部白板症20例の前癌病変組織について同様の検索を行ったところ,KAI1遺伝子の発現は9例(45%)で減弱していた

下顎歯肉扁平上皮癌40例と白板症20例の組織およびこれらの近接部正常歯肉60サンプルの組織についてSarcoendoplasmic reticulum Ca2+ ATPase 2タンパク(SERCA2タンパク)の発現を検索した.組織別にみた発現頻度は扁平上皮癌群55%,白板症群75%,正常歯肉群100%で,有意差が認められた.扁平上皮癌例のみを対象に腫瘍の大きさとSERCA2発現との関係を検討すると,2cm以下の5例ではすべて発現を認めたのに対し,2cmを超える症例の発現率は50%程度にすぎなかった.病期分類別ではステージが進むほど発現率は低下し,腫瘍の分化度別では悪性度が比較的低い高分化型の発現頻度が60%,悪性度の高い低分化型が0%であった.所属リンパ節への転移とSERCA2発現との間には明らかな関連は認められなかった

Deleted in malignant brain tumors 1 (DMBT1) gene was recently isolated on chromosome 10q25.3-26.1 and has been proposed as a putative candidate tumor suppressor for brain, esophageal, gastric, colorectal, and lung cancer. However, little is known about the association of DMBT1 with oral squamous cell carcinoma (OSCC). To study the role of DMBT1 gene in OSCC oncogenesis, we examined 9 OSCC derived cell lines and 45 primary OSCC tissue specimens with respective normal tissues. Semi-quantitative reverse transcriptase chain reaction (RT-PCR) analysis revealed down-regulation or deletion of DMBT1 expression in all of the 9 cell lines and in IS (40%) of 45 primary OSCC tissues. Additionally, 57 OSCC tissue specimens were examined by immunohistochemical staining of protein showing down-regulation of DMBT1 protein in 31 (56.1%) of the 57 primary OSCC tissue specimens. To assess restoration of DMBT1 expression by demethylation of promoter region, the 9 cell lines were treated with 5-aza-2-deoxycytidine (5-Aza-C), one of the DNA demethylating 7 agents. Six (66.7%) of 9 cell lines demonstrated restoration of DMBT1 expression after 5-Aza-C treatment. These results suggest that DMBT1 gene is involved in OSCC oncogenesis and/or progression and that methylation of promoter region is one of the important mechanisms suppressing the DMBT1 gene expression.

The adenomatous polyposis coli gene (APC gene) originally was identified as a tumor suppressor gene in colon cancer. We reported previously that APC is mutated and/or deleted in primary oral squamous cell carcinoma (OSCC) tissues and suggested that loss of APC function contributes to carcinogenesis in the oral region. In this study, we examined 50 OSCC tissue samples, which had been fixed in 10% buffered formaldehyde solution and embedded in paraffin, and eight cell lines, which were derived from OSCC, to analyze the expression level of the APC gene. Significant downregulation of APC was detected by immunohistochemistry in 15 (30.0%) of 50 tissue samples and by the reverse transcriptase-polymerase chain reaction in five (62.5%) of eight cell lines. We then investigated the status of APC gene promoter methylation and restoration of the APC gene mRNA. Hypermethylation of the APC promoter CpG island was detected in two of eight (25%) OSCC-derived cell lines, and APC gene mRNA was restored in all OSCC-derived cell lines showing down-regulation of gene expression (n=5) after treatment with 5-aza-2'-deoxycytidine, a DNA demethylating agent. Thus, the contribution of down-regulated APC expression to the development of human OSCC was about 30%, and hypermethylation of the gene promoter CpG island was confirmed to be a significant mechanism of inactivation of the APC gene in oral carcinogenesis.

Cisplatin (CDDP) is widely used for chemotherapy of many malignancies, especially of oral squamous cell carcinoma (SCC). However, because the mechanism of resistance to CDDP is unclear, we established a CDDP-resistant cell line, Sa-3R, from a CDDP-sensitive cell line, Sa-3, which was derived from moderately differentiated SCC of the lower gingiva. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazoliurn bromide assay indicated that Sa-3R has 7.5-fold greater resistance to CDDP than Sa-3. Comparing gene expression levels in the cell lines using an in-house cDNA microarray, which represented 2,201 oral disease origin genes, many differentially expressed genes were identified. The ATP-binding cassette transporter genes (MDR-1, MRP-1, and MRP-2), and FANCONI, GRP.58, FLJ12089, and SPINT-2 were up-regulated, whereas FOSL1, MRPS27, and PGK-1 were down-regulated. These results were confirmed by semi-quantitative reverse transcriptase-polymerase chain reaction. The Sa-3/Sa-3R cell lines could be useful to identify the candidates responsible for the mechanism of CDDP-resistance and the up- or down-regulated genes identified by the gene expression profiles in the Sa-3R cell line may be, in part, associated with the mechanism.

The objective of our study was to assess the possibility of prognostication and monitoring of oral squamous cell carcinoma by microsatellite blood assay. DNA from normal and tumorous tissues and serum DNA obtained at three time points (preoperatively, postoperatively, and 4 weeks postoperatively) from 64 patients with oral squamous cell carcinoma was examined at nine microsatellite loci. Thirty-eight (59%) DNA samples from tumorous tissues and 52% from serum showed allelic imbalances in at least one locus. Patterns of allelic imbalances in serum DNA were matched to those detected in tumor DNA. Of them, allelic imbalances were frequently detected preoperatively (44%, 28/64), and postoperatively (20%, 13/64). Moreover, among 12 cases with allelic imbalances during the postoperative period, six had no evidence of an allelic imbalance 4 weeks postoperatively, and they had no recurrence and were disease free. In contrast, six patients with allelic imbalance-positive DNA 4 weeks postoperatively have died with distant metastasis within 44 weeks. Thus, our results suggest that the assessment of microsatellite status in serum DNA could be a useful predictive tool to monitor disease prognosis. © 2005, THE SOCIETY FOR HARD TISSUE REGENERATIVE BIOLOGY. All rights reserved.

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.

To analyze gene expression in oral cancer, we produced a specialized in-house cDNA microarray. The cDNA library was constructed from surgical specimens of oral squamous cell carcinoma (SCC) using an oligo-capping method. cDNA clones (n=4,800) were randomly selected and their 5'-end nucleotide sequences were determined. Overlapping clones were excluded, and 1,423 independent clones were selected and used for microarray production. Compared to the public nucleotide sequence database, 61% of our cDNA clones were full-length. By correlating expression patterns across SCC cell lines, we identified 53 genes (7 up-regulated and 46 down-regulated) that are differentially expressed in SCC cell lines compared to normal mucosa. Semi-quantitative RT-PCR analysis confirmed these findings and validity of our cDNA microarray. Using specimens from SCC patients, we investigated the expression status of the IL-1ra gene, which showed the down-regulated gene by microarray analysis. Gene expression clearly fell in the SCC specimens relative to their references, which indicated that our in-house cDNA microarray system rapidly identified and characterized candidate biomarkers for clinical use.

A novel inhibitor of apoptosis survivin plays a,, role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis we, investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines In immunohistochemistry, 58% of tumors and 37% of premalignant lesions examined were positive for survivin while no immunoreaction was observed, in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore of the 9 normal oral tissues with no, survivin gene expression 4 showed methylation of, the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.

症例1:59歳男,症例2:64歳男で,それぞれ左下側切歯唇側肉腫脹,右下第二大臼歯遠心歯肉部腫脹を主訴とした.症例1は右上肺野腫瘤を,症例2は左肺野の異常陰影を指摘されており,転移が疑われた.いずれの症例も摂食障害,会話障害等が出現したため外部照射および口腔腫瘍摘出術を行った.その結果,摂食・会話障害等は改善したが,その後呼吸不全にて死亡した.なお,死亡迄の間,口腔内腫瘍の再発はみられなかった.病理組織診断は,症例1は肺腺癌の下顎骨転移,症例2は肺大細胞癌の下顎歯肉転移であった

Allelic deletions on the short arm of chromosome 8 (8p) are frequent events in several human malignancies, including oral cancer. We have examined and found two common regions of deletion on 8p (8p12, 8p22) in oral squamous cell carcinomas (SCC)s. The possible involvement of FEZ1/LZT1 (FEZ1) gene, a candidate tumor suppressor gene, mapped at 8p22, was also evaluated. Here we analyzed whether FEZ1 alterations play a role in the development and progression of oral SCCs. In the present study, we examined FEZ1 expression in 31 primary oral SCCs and 8 SCC-derived cell lines by reverse transcription-PCR (RT-PCR). Thirty-five percent of tumors (11 of 31) and 100% of cell lines (8 of 8) showed absent or reduced mRNA gene expression. To investigate the mechanism for silencing, cells were cultured with 5-aza-2'-deoxycytidine and all the cell lines showed restoration by the demethylating agent. These findings suggest that inactivation of the FEZ1 gene may contribute to the development of oral SCCs.

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both a chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues. (C) 2003 Elsevier Science (USA). All rights reserved.

The present study was designed to investigate the potential relationship between CDKN2A (p16) gene hypermethylation, which has reported to be frequently observed in oral squamous cell carcinomas (OSCCs), and expression of human DNA methyltransferases (DNMTs: DNMT1, DNMT3A and DNMT3B). Twenty-five pairs of primary OSCCs and matched normal oral mucosa tissues were examined. The p16 gene was hypermethylated (48%) in the tumors showing significant down-regulation of both mRNA and protein expressions. A demethylation assay on 8 OSCC-derived cell lines was also performed by means of treatment with the demethylating agent, 5-aza-2'-deoxycytidine. Four of 5 cell lines showing down-regulation of the p16 gene, revealed re-activation of gene expression after the treatment. In contrast, frequent over-expression of DNMT mRNA expression, also found in the expression of the proteins, was detected: DNMT1 at 72% and DNMT3A at 56%, and DNMT3B at 64%, respectively. However, we could not identify any statistical significance between p16-hypermethylation status in individual tumors and the expression of any of the three DNMTs. These data suggest that hypermethylation of the p16 gene and up-regulation of DNMTs are involved in oral carcinogenesis, but they may be through different mechanisms.

白板症17例における染色体微少変異をpolymerase chain reaction-loss of heterozygosity assay(PCR-LOH assay)により検索した.口腔扁平上皮癌組織において高頻度に欠失が集積されていることが既に報告されている9ヶ所のmicrosatellite領域を調べた.白板症の17例中11例(64.7%)において,1つ以上のmicrosatellite領域にLOHを認めた.LOHが同定された領域とその頻度は,D9S104(染色体9p21)で35.3%(6/17症例),IFNA(染色体9p22)で29.4%(5/17症例),D11S1356(染色体11q23)で11.8%(2/17症例),D13S273(染色体13q14.3-21.3)で11.8%(2/17症例)であった

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both a chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues. (C) 2003 Elsevier Science (USA). All rights reserved.

頭頸部腫瘍術後疼痛に対するPCAポンプを用いた塩酸モルヒネの静脈内持続注入による鎮静法の有効性および副作用について検討した.検討により,(1)先取り鎮痛効果が期待できる,(2)患者自身による追加投与が可能で,疼痛出現から鎮痛剤使用迄のタイムラグをなくすことができる,(3)低用量であるため,呼吸抑制等の深い鎮静がない,(4)除痛のための疼痛や不快感を伴わない,(5)疼痛に対する個人差が大きく投与量の初期設定が患者にとっての至適濃度でなかった場合があった,(6)術後腸管蠕動抑制が出現することが多く,早期に抑制を改善する必要がある,との結果が得られた.以上より,PCAポンプを用いた塩酸モルヒネの静脈内持続注入法は頭頸部腫瘍術後疼痛に効果的で安全な優れた鎮痛法であると考えられた

OBJECTIVE: Differential diagnosis of cementifying fibroma, ossifying fibroma and fibrous dysplasia by histological evaluation is often difficult. The aim of this study was to examine the immunoreactivities for keratan sulfate (KS) and chondroitin-4-sulfate (C4S) glycosaminoglycans of the histological samples obtained from mandibles of patients with these diseases. MATERIALS AND METHODS: The samples were collected from three patients with cementifying fibroma, two with ossifying fibroma and three with fibrous dysplasia and were subjected to immunohistochemical analyses. RESULTS: The results demonstrated that a significant immunoreactivity for KS was found in lacunae housing cells in the cementum-particles of cementifying fibromas, while both ossifying fibromas and fibrous dysplasias showed no significant immunoreactivity for KS. For C4S, while the former showed little immunoreactivity, the latter two cases exhibited intensive immunostaining in the pre- and poorly mineralized matrices. CONCLUSIONS: These results suggest that cementifying fibromas could be distinguished from these fibro-osseous tumors by using immunohistochemical analysis for KS and C4S.

To evaluate the role of chromosome 2 deletions in human oral squamous cell carcinoma (SCC) progression and to define the precise location of putative tumor suppressor genes, we examined 40 primary tumors and seven lymph node metastatic tumors from 40 patients with oral SCC by the polymerase chain reaction (PCR)-loss of heterozygosity (LOH) assay, using 10 different polymorphic loci on the long arm of chromosome 2. LOH was observed in 67.5% of the patients at one or more loci on the chromosome 2q. Two commonly deleted regions with high frequency of LOH, D2S1327 region at 2q32-35 (31.6%) and D2S206 region at 2q36 (36.7%), were identified by the deletion mapping of chromosome 2q, suggesting the presence of putative tumor suppressor genes associated with oral SCC. Examination of seven metastatic tumors also revealed four commonly deleted regions, D2S436, D2S1327, D2S155, and D2S164. Of these four regions D2S1327 region has no significant increase in the frequency of LOH between in primary tumors and in metastatic tumors. However, at other three regions the frequencies were much increased in metastatic tumors, comparing the results in primary tumors. Especially, very high frequencies of LOH in metastatic tumors were detected at two regions on 2q35, 100.0% at D2S155 and 57.1% at D2S164, suggesting the significant relationship between lymph node metastasis and LOH at these two regions. Our results indicate that LOH on chromosome 2q is a common event in oncogenesis and/or progression of oral SCC, and also suggest that the LOH at 2q35 play a significant role in the lymph node metastasis. (C) 2002 Elsevier Science Ltd. All rights reserved.

To evaluate the role of chromosome 2 deletions in human oral squamous cell carcinoma (SCC) progression and to define the precise location of putative tumor suppressor genes, we examined 40 primary tumors and seven lymph node metastatic tumors from 40 patients with oral SCC by the polymerase chain reaction (PCR)-loss of heterozygosity (LOH) assay, using 10 different polymorphic loci on the long arm of chromosome 2. LOH was observed in 67.5% of the patients at one or more loci on the chromosome 2q. Two commonly deleted regions with high frequency of LOH, D2S1327 region at 2q32-35 (31.6%) and D2S206 region at 2q36 (36.7%), were identified by the deletion mapping of chromosome 2q, suggesting the presence of putative tumor suppressor genes associated with oral SCC. Examination of seven metastatic tumors also revealed four commonly deleted regions, D2S436, D2S1327, D2S155, and D2S164. Of these four regions D2S1327 region has no significant increase in the frequency of LOH between in primary tumors and in metastatic tumors. However, at other three regions the frequencies were much increased in metastatic tumors, comparing the results in primary tumors. Especially, very high frequencies of LOH in metastatic tumors were detected at two regions on 2q35, 100.0% at D2S155 and 57.1% at D2S164, suggesting the significant relationship between lymph node metastasis and LOH at these two regions. Our results indicate that LOH on chromosome 2q is a common event in oncogenesis and/or progression of oral SCC, and also suggest that the LOH at 2q35 play a significant role in the lymph node metastasis. © 2002 Elsevier Science Ltd. All Rights reserved.

24歳男.舌下部の無痛性腫脹を主訴とした.口腔内所見では口底正中部に正常粘膜で被覆された境界明瞭な半球状,弾性軟の腫瘤を認めた.MRI所見では脂肪防御造影T1強調像で口底前方に信号増強効果を示した.以上より舌下腺腫瘍を疑い腫瘤摘出術を行った.病理組織学的に腫瘍は著明な毛細血管の増生と炎症細胞浸潤を伴う肉芽組織で,間質に紡錘形細胞の不規則な錯綜像がみられたが,異型な核分裂像は認めず,結節性筋膜炎と診断された.術後3年経過した現在,再発はみられず,経過良好である

通院加療中の関節リウマチ患者117人(男17,女100,2〜79歳)における顎関節障害を調査した.117人中50人(42.7%)に顎関節症状を認めた.顎関節症状は,雑音が多く,次いで疼痛,開口障害であった.開口障害が重度なほど赤沈値が高値を示した.罹病期間が長いほど下顎頭は扁平化し,破壊されている傾向がみられた.下顎頭の骨破壊の程度と関節痛の程度には,有意差はなかったが,骨破壊が進行しているほど赤沈値が高値であった.経口ステロイド剤の累積使用量が多いほど下顎頭の形態は扁平になる傾向を示した.咬合状態に著明な影響を受けた患者は認められなかった

Recently a tumor suppressor gene, a deleted in malignant brain tumor gene (DMBT1), was detected on chromosome 10. In some types of tumors, the frequent deletion of DMBT1 locus have been reported as well as loss of heterozygosity (LOH) on chromosome 10. However, little is known relating to human oral squamous cell carcinoma (OSCC). To study the genetic aberrations on chromosome 10 in OSCC, we performed polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 16 loci, containing 2 DMBT1 loci. We examined 38 oral primary squamous cell carcinoma (SCC) tissues and corresponding normal tissues. Microsatellite instability (MI) was detected at least on 1 of the 16 loci in 15 (39.5%) of 38 cases, and loss of heterozygosity (LOH) at least I of the 16 loci was also observed in 28 (73.7%) of 38 cases. LOH was accumulated at D10S202 (34.6%) and D10S217 (28.6%), suggesting the presence of two putative tumor suppressor genes associated with OSCC. The 2 DMBT1 loci, D10S209 and D10S587, had comparatively high frequent LOH (20.0 and 22.7%, respectively), maybe indicating the important role of DMBT1 in OSCC. No significant correlation between histological differentiation and LOH was found. These results suggest that genetic aberrations on chromosome 10 play important roles in the oncogenesis of OSCC.

Purpose: KAII was originally identified in prostate cancer as a metastasis suppressor gene. Recent studies have shown a frequent down-regulation of KAII expression in many tumor types, whereas mutation or hypermethylation of the gene is infrequent. The aim of the present study was to examine whether loss of KAII expression that might be caused by genetic or epigenetic alterations could contribute to oral carcinogenesis. Experimental Design: We analyzed mutational and methylation status of the KAII gene and both the mRNA and protein level in a series of oral tumors [28 precancerous lesions, 101 primary oral squamous cell carcinomas (OSCCs), and 30 metastatic OSCCs] and OSCC-derived cell lines. We also examined p53 protein expression, which has been reported to be a candidate activator for the KAII gene. Results: With the exception of three microsatellite instabilities in the KAII gene, we found no mutations in the coding sequence of the KAII gene, no loss of heterozygosity, and no hypermethylation of the KAII promoter region in all samples investigated. By immunohistochemistry, however, high frequencies of KAII down-regulation were evident not only in the metastatic OSCCs [29 of 30 (97%)] but also in the primary OSCCs [83 of 101 (82%)] and in the precancerous lesions [13 of 28 (46%)]. There was a significant relationship between down-regulation of KAII protein expression and primary tumors associated with lymph node metastases; (P = 0.0115), whereas there was no statistical correlation between p53 status and KAII expression. Taken together, reverse transcription- PCR data were consistent with the protein expression status in 16 patients from whom mRNA was available. Conclusions: Our data suggest that whereas loss of KAII protein expression is associated with primary tumors with lymph node metastases, the down-regulation of KAII is an early event in the progression of human oral cancer. The down-regulation of KAII is not associated with either mutation, allelic loss, methylation of the promoter, or p53 regulation.