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In this study, we explored the differences in the human adenovirus type 5 (Ad5) production efficiencies of various cell types. The rate of virus production was higher in several cell lines, such as HeLa cells, than in Saos-2 cells. The expression level of the coxsackie and adenovirus receptor (CAR) protein, an adenovirus receptor, was very similar among these cell lines. Although no significant difference in the expression of early region 1A (E1A) mRNA was detected, the amount of E1A protein in the Saos-2 cells was markedly lower than that in HeLa cells. Proteasome inhibitor treatment did not rescue the quantity of E1A in the Saos-2 cells, suggesting that their decreased E1A protein expression is not due to protein decay. To examine the different expression of E1A protein, we employed a bioinformatics approach to identify miRNA that target the 3'-untranslated region (3'-UTR) of E1A mRNA and identified miR-214 as a highly promising candidate. In Saos-2 cells, which have abundant levels of endogenous miR-214, the expression of luciferase was dramatically repressed, when the reporter gene was fused with the 3'-UTR of E1A mRNA including an miR-214 binding site. On the other hand, the activity from the same reporter was unchanged in HeLa cells, which display low-level miR-214 expression. Finally, we confirmed that the knockdown of the miR-214 upregulated the productive efficiency of the virus. These findings indicate that cellular miR-214 is capable of inhibiting adenovirus replication by regulating the translation of E1A protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in re-endothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TEC-specific marker and might be a useful therapeutic target., (© 2012 Japanese Cancer Association.)

Tumor angiogenesis is necessary for progression and metastasis of solid tumor. Tumor blood vessels are morphologically different from their normal counterparts. In this study, we isolated tumor endothelial cells (TECs) and revealed their abnormalities. We have compared the gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and found that several genes were upregulated in TECs. Expression of the chemokine receptor CXCR7 mRNA was higher in TECs than in NECs. However, information regarding the expression of CXCR7 in the tumor vessels of renal cell carcinoma is limited. CXCR7 and its ligand CXCL12 have been implicated in tumor cell survival. In this study, the expression of CXCR7 in the tumor vessels of renal cell carcinoma (RCC) was investigated. Real-time PCR revealed higher expression level of CXCR7 in cultured TECs than in cultured NECs. Furthermore, similar to mouse TECs, immunostaining revealed strong expression of CXCR7 in vivo in human tumor vessels. These findings suggest that CXCR7 is a novel TEC marker and a target for antiangiogenic therapy for RCC., (© 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

An important concept in tumor angiogenesis is that tumor endothelial cells (TECs) are genetically normal and homogeneous. However, we previously reported that TECs differ from normal ECs. Whether the characteristics of TECs derived from different tumors differ remains unknown. To elucidate this, in this study, we isolated two types of TECs from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor-derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor-derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1α, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the stemness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. These results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor endothelial cells (TECs) are therapeutic targets in anti-angiogenic therapy. Contrary to the traditional assumption, TECs can be genetically abnormal and might also acquire drug resistance. In this study, mouse TECs and normal ECs were isolated to investigate the drug resistance of TECs and the mechanism by which it is acquired. TECs were more resistant to paclitaxel with the up-regulation of multidrug resistance (MDR) 1 mRNA, which encodes the P-glycoprotein, compared with normal ECs. Normal human microvascular ECs were cultured in tumor-conditioned medium (CM) and became more resistant to paclitaxel through MDR1 mRNA up-regulation and nuclear translocation of Y-box-binding protein 1, which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in human microvascular ECs by tumor CM. We observed that tumor CM contained a significantly high level of VEGF. A VEGFR kinase inhibitor, Ki8751, and a phosphatidylinositol 3-kinase-Akt inhibitor, LY294002, blocked tumor CM-induced MDR1 up-regulation. MDR1 up-regulation, via the VEGF-VEGFR pathway in the tumor microenvironment, is one of the mechanisms of drug resistance acquired by TECs. We observed that VEGF secreted from tumors up-regulated MDR1 through the activation of VEGFR2 and Akt. This process is a novel mechanism of the acquisition of drug resistance by TECs in the tumor microenvironment., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs., (Copyright © 2011 UICC.)

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear., Methodology/principal Findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis., Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

Tumor blood vessels play an important role in tumor progression and metastasis. Thus, targeting the tumor blood vessels is an important strategy in cancer therapy. Tumor blood vessels generally arise from pre-existing vessels and have been thought to be genetically normal. However, they have been shown to differ from their normal counterparts, e.g. with regard to the morphological changes. We isolated tumor endothelial cells (TEC) from mouse tumor xenografts and showed that they were abnormal. TEC up-regulate many genes, proliferate more rapidly and migrate more than normal endothelial cells (NEC). Furthermore, the TEC in our study were cytogenetically abnormal. We concluded that TEC can acquire cytogenetic abnormalities while in the tumor microenvironment. In order to develop ideal antiangiogenic therapies, understanding the crosstalk between blood vessels and the tumor microenvironment is important. This review considers the current studies on TEC abnormalities and discusses the possible mechanism by which the tumor microenvironment produces abnormal TEC., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Approximately 85% of human malignant tumors express increased levels of telomerase. The marked association of telomerase activity with malignant tissue provides strong evidence that telomerase activity is a significant marker for the diagnosis of cancer. In this study, telomerase activity was examined in 12 benign salivary gland tumors (8 pleomorphic adenomas and 4 adenolymphomas), 24 malignant tumors (15 mucoepidermoid carcinomas, 6 adenoid cystic carcinomas and 3 acinic cell carcinomas) and 6 non-neoplastic salivary glands. The mRNA expression of the human telomerase reverse transcriptase (hTERT) and additional telomerase‑associated proteins (hTEP1, p23, Hsp90 and dyskerin) was also examined. Of the 24 malignant tumors, 15 revealed strong telomerase activity. The non-neoplastic salivary glands appeared to have a negative telomerase expression. Furthermore, telomerase activity was significantly higher in high-grade mucoepidermoid carcinomas compared to low‑grade ones (Student's t-test, p<0.05). A significant correlation was found between telomerase activity and mRNA expression of hTERT in 15 cases, including non-neoplastic salivary glands and tumors (Spearman's rank correlation test, p<0.05). Furthermore, a significant correlation was found between telomerase activity and mRNA expression of EGFR (Spearman's rank correlation test, p<0.001). The results suggest that not only hTERT, but also EGFR play a significant role in the activation of telomerase. In conclusion, the results suggest that telomerase activity and hTERT/EGFR mRNA expression are useful markers for the detection of malignant cells in salivary gland carcinomas. Moreover, our results indicated that telomerase activity determines the degree of malignancy of mucoepidermoid carcinoma.

Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor κB ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tumor metastasis to lymph nodes occurs through the lymphatic vessels located in the tumor circumference. However, few studies have focused on the phenotypes of lymphatic vessels around these tumors. We investigated the characteristics of the lymph vessels of tongue squamous cell carcinoma (SCC) and compared them to clinicopathological characteristics. A total of 43 patients diagnosed as having tongue SCC consulted Hokkaido University Hospital were examined. The lymphatic vessels were identified by antibody D2-40 and the number and diameter of tumor lymphatic vessels were measured. The proliferative activity of lymphatic endothelial cells was also examined by immunostaining using antibody MIB-1. We then measured the DNA density of lymphatic endothelial cells in normal and tumor tissues. The number of tumor lymphatic vessels significantly increased in highly metastatic cases of tongue SCC, particularly in cases with a large number of micro lymphatic vessels. A significant correlation was found between the metastatic and proliferative activity of tumor lymphatic endothelial cells. Moreover, the DNA density of tumor lymphatic endothelial cells increased compared to normal tissues. These results suggest that the phenotypes of tumor lymphatic endothelial cells are an indicator of lymph node metastasis of tongue SCC.

Patients with an oral squamous cell carcinoma (OSCC) often develop multiple malignant lesions. This report examined whether individual tumours developed in a patient show the same genetic alteration, such as p53 mutations. This case study describes three SCCs and three leukoplakias which developed simultaneously in a single 67-year-old Japanese man. A p53 mutation was detected in two of the three SCCs and one of the three leukoplakias. One SCC had a missense mutation at codon 285 (GAG>AAG, Glu>Lys) and the other a nonsense mutation at codon 336, and the leukoplakia had a missense mutation at codon 273 (CGT>CAT, Arg>His). This case showed that individual oral tumours may have different genetic changes even when they develop in a single patient. Therefore, this report provided strong evidence that in cases of multiple tumours it is necessary to design tailor-made therapies for each individual tumour rather than a single standardised therapy for all multiple tumours.

Adrenomedullin (AM) is a multifunctional 52-amino acid peptide. AM has several effects and acts as a growth factor in several types of cancer cells. Our previous study revealed that an AM antagonist (AMA) suppressed the growth of pancreatic tumors in mice, although its mechanism was not elucidated. In this study, we constructed an AMA expression vector and used it to treat renal cell carcinoma (RCC) in mice. This AMA expression vector significantly reduced tumor growth in mice. In addition, microvessel density was decreased in AMA-treated tumors. To analyze the effect of AMA on tumor angiogenesis in this model, tumor endothelial cells (TECs) were isolated from RCC xenografts. TEC proliferation was stimulated by AM and it was inhibited by AMA significantly. AM induced migration of TECs and it was also blocked by AMA. However, normal ECs (NECs) were not affected by either AM or AMA. These results demonstrate that AMA has inhibitory effects on TECs specifically, not on NEC, thereby inhibiting tumor angiogenesis. Furthermore, we showed that vascular endothelial growth factor-induced mobilization of endothelial progenitor cell (EPC) into circulation was inhibited by AMA. These results suggest that AMA can be considered a good anti-angiogenic reagent that selectively targets TECs and EPC in renal cancer.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research., (2010 Elsevier Inc. All rights reserved.)

HuR binds to AU-rich element-containing mRNA to protect them from rapid degradation. Here, we show that knockdown of HuR changes the oncogenic properties of oral cancer cells. Oral squamous cell carcinoma cell lines, HSC-3 and Ca9.22, which express HuR protein and cytoplasmic AU-rich element mRNA more abundantly than normal cells, were subjected to HuR knockdown. In the HuR-knockdown cancer cells, the cytoplasmic expression of c-fos, c-myc, and COX-2 mRNAs was inhibited compared with those in cells that had been transfected with a control small interfering RNA, and the half-lives of these mRNAs were shorter than those of their counterparts in the control cells. HuR-knockdown cells failed to make colonies in soft agar, suggesting that the cells had lost their ability for anchorage-independent cell growth. Additionally, the motile and invasive activities of the cells decreased remarkably by HuR knockdown. Furthermore, the expression of cell cycle-related proteins, such as cyclin A, cyclin B1, cyclin D1, and cyclin-dependent kinase 1, was reduced in HuR-knockdown cancer cells, and HuR bound to cdk1 mRNA to stabilize it. These findings suggest that HuR knockdown changes the features of oral cancer cells, at least in part, by affecting their cell cycle and shows potential as an effective therapeutic approach., ((c) 2010 AACR.)

Tenascin-X (TNX), which has a molecular mass of roughly 450 kDa, is the largest member of the tenascin family. Complete deficiency of TNX in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS). TNX is expressed abundantly in a variety of tissues, especially in cardiac muscle and in perivascular stroma. Human TNX is also present in serum with an apparent molecular size of 140 kDa. In the present study, we investigated the expression levels of TNX protein in thoracic and abdominal aortic aneurysm tissues. The level of TNX was significantly increased in both aortic aneurysm tissues compared with that in adjacent normal tissues. Next, to compare TNX levels in serum from both patients with thoracic aortic aneurysm and patients with abdominal aortic aneurysm with levels in serum from healthy individuals, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using TNX-specific antibodies. Measurement of TNX serum concentrations in both aortic aneurysm patients and controls showed that the levels were almost the same. These results indicate that TNX expression is significantly elevated in both thoracic and abdominal aortic aneurysm tissues but that the increase in TNX levels in both tissues does not result in an increase in TNX serum concentration in patients with TAA or AAA.

Tumor blood vessels are thought to contain genetically normal and stable endothelial cells (ECs), unlike tumor cells, which typically display genetic instability. Yet, chromosomal aberration in human tumor-associated ECs (hTECs) in carcinoma has not yet been investigated. Here we isolated TECs from 20 human renal cell carcinomas and analyzed their cytogenetic abnormalities. The degree of aneuploidy was analyzed by fluorescence in situ hybridization using chromosome 7 and chromosome 8 DNA probes in isolated hTECs. In human renal cell carcinomas, 22-58% (median, 33%) of uncultured hTECs were aneuploid, whereas normal ECs were diploid. The mechanisms governing TEC aneuploidy were then studied using mouse TECs (mTECs) isolated from xenografts of human epithelial tumors. To investigate the contribution of progenitor cells to aneuploidy in mTECs, CD133(+) and CD133(-) mTECs were compared for aneuploidy. CD133(+) mTECs showed aneuploidy more frequently than CD133(-) mTECs. This is the first report showing cytogenetic abnormality of hTECs in carcinoma, contrary to traditional belief. Cytogenetic alterations in tumor vessels of carcinoma therefore can occur and may play a significant role in modifying tumor- stromal interactions.

Dentinogenesis imperfecta (DGI) type II is one of the most common dominantly inherited dentin defects, in which both the primary and permanent teeth are affected. Here, we report a Japanese family with autosomal-dominant DGI type II, including both molecular genetic defects and pathogenesis with histological analysis. Mutation analysis revealed a mutation (c.53T>A, p.V18D, g.1192T>A) involving the second nucleotide of the first codon within exon 3 of the dentin sialophosphoprotein (DSPP) gene. This mutation has previously been reported in a Korean family. Thus far, 24 allelic DSPP mutations have been reported, and this is the seventh mutation involving the DSPP V18 residue. Among those, only one other was shown to be caused by a de novo mutation, and that mutation also affected the V18 amino acid residue. The DSPP V18 residue is highly conserved among other mammalian species. These findings thus suggest that the V18 amino acid might be a sensitive mutational hot spot, playing a critical role in the pathogenesis of DGI.

Daxx is a regulatory protein for apoptosis signal-regulating kinase 1 (ASK1) which activates c-Jun NH2-terminal kinase (JNK) and p38 pathways in response to stressors such as tumor necrosis factor-alpha (TNFalpha). Here, we show that TNFalpha treatment induces the accumulation of Daxx protein through ASK1 activation by preventing its proteasome-dependent degradation. ASK1 directly phosphorylates Daxx at Ser(176) and Ser(184) and Daxx is required for the sustained activation of JNK. Tumorigenic mutant p53, which binds to Daxx and inhibits Daxx-dependent activation of ASK1, prevents Daxx phosphorylation and stabilization. When mutant p53 was depleted in cancer cells, Daxx was accumulated and the cell-killing effect of TNFalpha was restored. Our results indicate that Daxx not only activates ASK1 but also is a downstream target of ASK1 and that accumulated Daxx further activates ASK1. Thus, the Daxx-ASK1 positive feedback loop amplifying JNK/p38 signaling plays an important role in the cell-killing effects of stressors, such as TNFalpha. Tumorigenic mutant p53 disrupts this circuit and makes cells more tolerable to stresses, as its gain-of-function mechanism.

The polyphenol epigallocatechin-3 gallate (EGCG) in green tea suppresses tumor growth by direct action on tumor cells and by inhibition of angiogenesis, but it is not known whether it specifically inhibits tumor angiogenesis. We examined the anti-angiogenic effect of EGCG on tumor-associated endothelial cells (TEC), endothelial progenitor cells (EPC), and normal endothelial cells (NEC). EGCG suppressed the migration of TEC and EPC but not NEC. EGCG also inhibited the phosphorylation of Akt in TEC but not in NEC. Furthermore, vascular endothelial growth factor-induced mobilization of EPC into circulation was inhibited by EGCG. MMP-9 in the bone marrow plasma plays key roles in EPC mobilization into circulation. We observed that expression of MMP-9 mRNA was downregulated by EGCG in mouse bone marrow stromal cells. In an in vivo model, EGCG suppressed growth of melanoma and reduced microvessel density. Our study showed that EGCG has selective anti-angiogenic effects on TEC and EPC. It is suggested that EGCG could be a promising angiogenesis inhibitor for cancer therapy.

Pim-1 is a serine/threonine kinase as well as a proto-oncogene that induces T-cell lymphoma. Pim-1 induces cell cycle progression in cooperation with c-Myc and acts as an inhibitor of apoptotic cell death, actions that are involved in blood cell oncogenesis. However, little is known regarding the role of Pim-1 in oral carcinogenesis. We investigated Pim-1 expression in tongue squamous cell carcinoma (SCC) and examined its clinicopathological features. Western blotting was performed in 6 oral SCC cell lines, with Pim-1 being detected in all 6 of the lines. Immunohistochemical detection of Pim-1 was carried out in 39 cases of tongue SCC and analyzed in terms of its associated clinicopathological features. Pim-1 was expressed in 17/39 cases of tongue carcinoma, and was significantly correlated with lymph node metastasis. The role of Pim-1 in cell motility was further examined in HSC3 cells using the GTP-binding assay for Rho family protein, the motility assay and siRNA treatment. Rac1 activation was observed, and cell motility was reduced when Pim-1 was knocked down by siRNA. These results indicate that Pim-1 is involved in the carcinogenesis of oral SCC and is correlated to metastasis, which is in part associated with the enhancement of cell motility.

To identify molecular signatures and establish a new diagnostic model for progressive oral squamous cell carcinoma (OSCC). Total RNAs were isolated from primary OSCCs from both node-positive and -negative patients and used in cDNA microarray analysis. To identify marker genes representing a malignant phenotype, their expression was further examined by quantitative reverse transcription-PCR (QRT-PCR) in 64 OSCC tissues. Using Fisher's linear discriminant analysis (LDA) fitted with a stepwise increment method, we created discriminatory predictor models. The stability of these models was examined using leave-one-out cross validation. Immunohistochemical analysis was performed. Among the 16,600 possible target cDNAs in the array analysis, 83 genes demonstrated significantly differential signals (>2-fold). We further identified 53 marker genes that can be implicated in the Yamamoto-Kohama's (YKs) mode of invasion for OSCCs (P < 0.06). Using LDA fitted with a stepwise increment method, we created four discriminatory predictor models based on 16- to 25-gene signatures which could best distinguish the five established grades of YKs mode of invasion. Leave-one out validation demonstrated that the stability of these models was 92-95%. For validation, we also examined an independent set of 13 primary OSCCs; the predictor models determined the invasion status from 77% to 100% (on average, 85%) fidelity with the pathological observations. TGM3 protein expression was markedly suppressed in highly invasive OSCCs. We reveal novel gene expression alterations during the progression of OSCC, and have constructed prediction models for the evaluation of the invasion status of these cancers.

A 57-year-old woman was admitted to Hokkaido University Hospital because of dysphagia. Laryngoscopy indicated hypopharyngeal tumor histologically diagnosed as squamous cell carcinoma (SCC). A combination of radiotherapy and chemotherapy was performed for 2 months, and the hypopharyngeal lesion completely regressed. After 4 months, fever, anorexia, and malaise appeared, and chest X-ray and CT indicated two large tumors in the right lung. Transbroncheal lung biopsy (TBLB) specimens were diagnosed as SCC. Laboratory data showed high levels of serum granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP). Subsequently, positron emission tomography (PET) showed multiple metastases to several organs including the liver, spine, skull, and thigh. Two months after readmission, the patient died with no success of chemotherapy. At autopsy, the lung tumor was clearly positive for both G-CSF and PTHrP on immunohistostaining. Retrospectively, examination showed that the primary pharyngeal tumor was focally positive for these two cytokines. Thus, a certain population of hypopharyngeal cancer producing G-CSF and PTHrP, spread to various organs and contributed to the rapid progression and poor prognosis. This case is presented with a discussion of several other cases in the literature.

The direct determination of lipoyllysine (LLys) in proteins was carried out by reversed-phase high-performance liquid chromatography with fluorescence (FL) detection. The proteins containing alpha-lipoic acid (LA) were first hydrolyzed with several enzymes such as pronase E and subtilisin A. The disulfide bond (-S-S-) in LLys liberated from the enzyme digestion was reduced with tris(2-carboxyethyl)phosphine to the thiol form (-SH). The reduced LLys was then labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate (SBD-F) at 50 degrees C for 1h. The resulting fluorophore, SBD-LLys, was separated by reversed-phase chromatography and fluorometrically detected at 510 nm (excitation at 380 nm). The calibration curve obtained from the peak areas versus the injection amounts of LLys showed a good linearity. The limits of detection and quantification of LLys on the chromatogram were approximately 0.13 pmol (signal-to-noise ratio (S/N)=3) and 0.44 pmol (S/N=10), respectively. A good recovery (98.9-107.1%) and precision (R.S.D.: 4.49-17.2%) of LLys were also obtained using the present procedure. The proposed method was used for the determination of LLys in spinach and animal tissues. The FL derivative was completely separated without any interference by endogenous substances in the sample and sensitively detected by the fluorimetry. The assay values of LLys per 1g wet tissues were 3.67 microg (kidney), 1.97 microg (liver), 2.09 microg (heart), 0.59 microg (brain), 0.30 microg (lung), 0.38 microg (pancreas), and 0.20 microg (spleen). The direct determination of LLys in protein using the FL labeling method is reported for the first time.

Matrix metalloproteinase (MMP) is closely involved in the degradation of extracellular matrix and confers invasive and metastatic potential to malignant tumors. MMP-2 is a type-IV collagenase secreted as a proenzyme that is activated on the surface of the tumor cell by membrane-type 1-MMP (MT1-MMP). MT1-MMP plays a critical role during tumor progression and metastasis. We investigated the expression levels of E1AF and MT1-MMP in malignant melanoma cell lines and specimens from patients in order to clarify the mechanisms responsible for the invasion and metastasis of malignant melanoma. High levels of E1AF and MT1-MMP mRNA expression were observed in melanoma cells by Northern blotting and real-time PCR. The expression level was highly correlated with an invasive potential determined by an in vitro invasion assay. The down-regulation of MT1-MMP was identified when E1AF was knocked down by RNA interference. These results suggest that E1AF plays a crucial role in the invasion and metastasis of malignant melanoma through up-regulating the MT1-MMP expression.

Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from their normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, even though these endothelial cells are structurally and functionally abnormal. To date, many anti-angiogenic drugs have been developed, but, their therapeutic efficacy is not dramatic and they have also been reported to cause toxic side effects. To develop ideal antiangiogenic therapies, understanding tumor endothelial cell abnormalities is important. We have isolated tumor endothelial cells from mouse tumor xenografts and have shown that tumor-associated endothelial cells are abnormal. Tumor-associated endothelial cells upregulate many genes, such as epidermal growth factor receptor (EGFR). Tumor-associated endothelial cells are also more sensitive to EGF. They also have relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells are cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid. They had normal centrosomes and remained cytogenetically stable in culture even up to 20 passages. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment. Questions as to whether or not tumor endothelial cells become resistant to antiangiogenic drugs are thus raised. Our preliminary data show that tumor endothelial cells are more resistant to certain chemotherapeutic drugs. Studies to evaluate the mechanism for cytogenetic abnormalities in tumor endothelial cells are underway. It is becoming quite clear that the tumor vasculature is much more complex and unpredictable than initially perceived. Here, we provide an overview of the current studies on tumor endothelial cell abnormalities.

Inorganic polyphosphate [poly(P)] is a biopolymer existing in almost all cells and tissues, although its biological functions in higher eukaryotes have not been completely elucidated. We previously demonstrated that poly(P) enhances the function of fibroblast growth factors (FGFs) by stabilizing them and strengthening the affinity between FGFs and their cell surface receptors. Since FGFs play crucial roles in bone regeneration, we further investigated the effect of poly(P) on the cell differentiation of human stem cells via FGF signaling systems. Human dental pulp cells (HDPCs) isolated from human dental pulp show the characteristics of multipotent mesenchymal stem cells (MSCs). HDPCs secreted FGFs and the proliferation of HDPCs was shown to be enhanced by treatment with poly(P). Cell surface receptor-bound FGF-2 was stably maintained for more than 40 hours in the presence of poly(P). The phosphorylation of ERK1/2 was also enhanced by poly(P). The effect of poly(P) on the osteogenic differentiation of HDPCs and human MSCs (hMSCs) were also investigated. After 5 days of treatment with poly(P), type-I collagen expression of both cell types was enhanced. The C-terminal peptide of type-I collagen was also released at higher levels in poly(P)-treated HDPCs. Microarray analysis showed that expression of matrix metalloproteinase-1 (MMP1), osteopontin (OPN), osteocalcin (OC) and osteoprotegerin was induced in both cell types by poly(P). Furthermore, induced expression of MMP1, OPN and OC genes in both cells was confirmed by real-time PCR. Calcification of both cell types was clearly observed by alizarin red staining following treatment with poly(P). The results suggest that the activation of the FGF signaling pathway by poly(P) induces both proliferation and mineralization of stem cells.

The molecular mechanisms of growth suppression by retinoic acid (RA) were examined. Our results suggest that the cytostatic effects of RA could be mediated by the activation of endogenous CBR3 gene in oral squamous cell carcinomas (OSCCs), and the expression is a potential marker for oral malignancy.

There is accumulating evidence demonstrating that HIF-1 functions as a key regulator of the adaptation responses to hypoxia in cancer tissues. To this evidence, we add that adaptation responses to glucose deprivation plus hypoxia are also necessary for the survival of tumor cells in the tumor microenvironment as cancer tissues are exposed to glucose deprivation as well as hypoxia. We found that adrenomedullin (AM), VEGF, Glut-1, Glut-3, and Hexokinase-2 among 45 hypoxia-inducible genes investigated were expressed at higher levels under glucose-deprived hypoxic conditions than under hypoxic conditions. Glucose deprivation activated the AMPK under normoxia and hypoxia. Compound C, an inhibitor of AMPK, suppressed the expressions of AM and VEGF which had already been enhanced under glucose-deprived hypoxic conditions. siRNAs for both AMPKalpha1 and AMPKalpha2 suppressed the expressions of AM and VEGF. HIF-1alpha protein level and the transcriptional activity of HIF-1 under glucose-deprived hypoxic conditions were thus found to be similar to those under hypoxic conditions. Furthermore, tumor cells in 15 out of 20 human pancreatic cancer tissue specimens were stained by anti-phospho-AMPKalpha antibody. Our results thus suggest that the enhanced expressions of those genes mediated by the activation of AMPK and HIF-1 therefore play a pivotal role in the tumor formation of pancreatic cancers.

The purpose of this study is to generate a classifier for oral squamous cell carcinoma (OSCC) and leukoplakias (LPs), and evaluate its diagnostic potential. In order to identify marker gene candidates, differential gene expression between LPs and OSCCs were examined by cDNA microarray. The expression of 118 marker gene candidates was further evaluated by quantitative reverse transcription-PCR (QRT-PCR) analyses of 27 OSCC and 19 LP tissues. We identified 12 up-regulated and 15 down-regulated marker genes in OSCCs compared to LPs. Using Fisher's linear discriminant analysis (LDA), we demonstrated that 11-gene predictors among this novel marker set could best distinguish OSCCs from LPs (>97% accuracy), whereas a further seven of these gene predictors could be utilized to distinguish higher grade (higher than moderate) from lower grade (lower than mild) dysplasias (>95% accuracy). These predictor gene sets provide multigene classifiers for the diagnosis of pre-cancerous to cancerous transition of oral malignancy.

E1AF was first identified as a transcription factor that binds to enhancer motifs of the adenovirus E1A gene and is thought to be a human homologue of mouse PEA3, one of the ets oncoprotein families. Here we show the effect of E1A on the gene expression and function of E1AF. E1A repressed the activity of E1AF promoter, and the N-terminal region of E1A, which is involved in the oncogenic activity of E1A, was essential for this repression. The ability as a transcription factor of E1AF, as well as those of the other PEA3 subfamily members ER81 and ERM, was also repressed by E1A via the same oncogenic domain. Furthermore, E1AF repressed the transformation activity of E1A cooperating with E1B, whereas the other ets family Ets-1 enhanced this activity. These results suggest that E1AF is one of the targets of E1A.

A 14-year-old Japanese girl with a progressing combined immunodeficiency had developed non-Hodgkin's diffuse large B cell lymphoma. Her molecular analysis showed a compound heterozygote of novel mutations in the LIG4 gene, M249V substitution and a five nucleotides deletion from nucleotide position 1,270-1,274. She had also a set of characteristic clinical features of LIG4 syndrome. Mutations in the LIG4 gene, which plays a critical role in the repair of DNA double-strand breaks, imply a correlation with malignancies and several cases with leukemia or lymphoma have already been reported. We report here on a case of LIG4 syndrome complicated with distinct EBV-associated B-cell lymphoma.

Although hypovasculature is an outstanding characteristic of pancreatic cancers, the tumor cells survive and proliferate under severe hypoxic, glucose-deprived conditions caused by low blood supply. It is well known that the hypoxia-inducible factor-1 pathway is essential for the survival of pancreatic cancer cells under hypoxic conditions. To discover how pancreatic cancer cells adapt to glucose deprivation as well as hypoxia, we sought glucose deprivation-inducible genes by means of a DNA microarray system. We identified 63 genes whose expression was enhanced under glucose-deprived conditions at >2-fold higher levels than under normal glucose conditions. Among these genes, asparagine synthetase (ASNS) was studied in detail. Although it is known to be associated with drug resistance in leukemia and oncogenesis triggered by mutated p53, its function is yet to be determined. In this study, we found that glucose deprivation induced the overexpression of ASNS through an AMP-activated protein kinase-independent and activating transcription factor-4-dependent manner and that ASNS protects pancreatic cancer cells from apoptosis induced by glucose deprivation itself. ASNS overexpression also induced resistance to apoptosis triggered by cisplatin [cis-diammine-dichloroplatinum (CDDP)] and carboplatin, but not by 5-fluorouracil, paclitaxel, etoposide, or gemcitabine. We show that glucose deprivation induces the activation of c-jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase (SAPK) in a mock transfectant but not in an ASNS transfectant. Consequently, an inhibitor of JNK/SAPK decreased the sensitivity of pancreatic cancer cells to apoptosis by glucose deprivation and CDDP. These results strongly suggest that ASNS is induced by glucose deprivation and may play a pivotal role in the survival of pancreatic cancer cells under glucose-deprived conditions.

E1AF is a member of the ETS oncogene family and is thought to be a human homologue of mouse PEA3. We have isolated a genomic clone of E1AF and analyzed the promoter activity of its 5'-flanking region. We identified a variation in exon 1, which depends on the cell type. There was no typical TATA box in the 5'-flanking region, but putative binding sites of a number of transcription factors including PEA3 as well as CAAT boxes were seen. A luciferase reporter assay indicated that the 5'-flanking region possesses promoter activity. Northern blot studies demonstrated significant expression of the E1AF gene in restricted tissues such as the pituitary gland, placenta, and fetal kidney. Moreover, the E1AF promoter was activated by E1AF itself and estrogen receptor. These findings suggest that E1AF is a housekeeping gene, whose expression is controlled in specific tissues.

We describe the psychophysical features of vocal amusia in a professional tango singer caused by an infarction mainly involving the superior temporal cortex of the right hemisphere. The lesion also extended to the supramarginal gyrus, the posterior aspect of the postcentral gyrus and the posterior insula. She presented with impairment of musical perception that was especially pronounced in discriminating timbre and loudness but also in discriminating pitch, and a severely impaired ability to reproduce the pitch just presented. In contrast, language and motor disturbances were almost entirely absent. By comparing her pre- and post-stroke singing, we were able to show that her singing after the stroke lacked the fine control of the subtle stress and pitch changes that characterized her pre-stroke singing. Such impairment could not be explained by the impairment of pitch perception. The findings suggest that damage to the right temporoparietal cortex is enough to produce both perceptive and expressive deficits in music.

E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

E1AF is an ets-oncogene family transcription factor that has been shown to up-regulate multiple MMPs whereas MMP-2, a potent extracellular matrix degrading enzyme, is not up-regulated. We investigated the activation mechanism of MMP-2 in oral squamous cell carcinoma. Chloramphenicol acetyltransferase (CAT) assay was utilized to investigate whether E1AF is able to up-regulate membrane type-1 matrix metalloproteinase (MT1-MMP), which is known to activate MMP-2. Expression of the CAT reporter gene under the control of the MT1-MMP promoter was increased approximately 40-fold by co-transfection with the E1AF expression vector. Real-time RT-PCR study was carried out in 25 patients with tongue squamous cell carcinoma, and the mRNA expression level of E1AF and MT1-MMP was synergistically increased. These results indicate that E1AF positively regulates transcription from MT1-MMP genes, which plays an important role in invasion and metastasis of squamous cell carcinoma of the tongue by converting pro-MMP-2 into active-MMP-2.

E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function.

The tissue response to hat-stacked carbon nanofibers (H-CNFs) was evaluated. H-CNFs were implanted in the subcutaneous tissue of rats. Histological and ultrastructural investigations were carried out by transmission electron microscopy. Although many macrophages and foreign body giant cells were seen around H-CNFs, no severe inflammatory response such as necrosis was observed. Some H-CNFs were observed in lysosomal vacuoles of phagocytes. These results showed that H-CNFs were not strong prophlogistic substances and were englobed in vivo.

Objective: N-Acetylglucosaminyltransferase V (GnT-V) is a key enzyme in the formation of branching asparagine-linked oligosaccharides and is linked to tumor invasion and metastasis in colon and breast cancers. In normal esophageal epithelium, beta1,6-branched asparagine-linked oligosaccharides synthesized by GnT-V are seen in the basal cell layers but not in the superficial cell layers, and its presence has been shown in invasive esophageal cancers. However, neither GnT-V expression nor its clinical significance has been previously examined in human normal, premalignant and malignant esophageal tissues., Methods: GnT-V expression was studied by immunohistochemistry using a specific monoclonal antibody in 121 surgically resected specimens of esophageal squamous cell carcinomas (SCCs) and adjacent tissues, and was analyzed statistically in relation to various characteristics., Results: GnT-V expression was observed in none (0%) of the 19 normal epithelial tissues, 1 (2%) of the 43 hyperplastic tissues, 30 (54%) of the 56 mildly dysplastic tissues, 27 (63%) of the 43 moderately dysplastic tissues, 21 (44%) of the 48 in situ SCCs and 29 (26%) of the 110 invasive SCCs (p<0.005). GnT-V expression was observed significantly more frequently in mildly and moderately dysplastic tissues when compared with normal epithelial and hyperplastic tissues (p<0.005), and its frequency was decreased in in situ and invasive SCCs (p<0.005). GnT-V expression was frequently observed in SCCs of small size and without distant metastasis or lymph node metastasis., Conclusions: Increased expression of GnT-V is associated with the early event of esophageal tumorigenesis., (Copyright (c) 2005 S. Karger AG, Basel.)

Multiwalled carbon nanotubes (MWCNTs) were used as the active elements for the first time for affinity-based elimination of ionic dyes. MWCNTs were encapsulated in cross-linked alginate (ALG) microvesicles using Ba2+ as the bridging ion. The Ba2+-alginate matrix constitutes a cage which holds the physically trapped MWCNTs. The cage carries negative charges on its surface. The cage restricts the access of anions of large molecular weight, such as humic acids, because of electrostatic repulsion. The cage also restricts the access of colloids of large size, because of size exclusion. Ionic dyes partition into the cage and then are captured by MWCNTs probably on the basis of van der Waals interactions occurring between the hexagonally arrayed carbon atoms in the graphite sheet of MWCNTs and the aromatic backbones of the dyes. As a result of these interactions the target species, namely, the ionic dyes, are eliminated efficiently by the MWCNTs of Ba2+-ALG/MWCNT composite adsorbents. The adsorptive capacities for elimination of acridine orange, ethidium bromide, eosin bluish, and orange G (the model species used for this study) were found as high as 0.44, 0.43, 0.33, and 0.31 micromol, respectively, for 1.0 mg of the caged MWCNTs. Adsorptive experiments with carbon nanofibers and activated carbons as the adsorbents were also performed. The MWCNT-based adsorbents provided the best capability for the affinity-based elimination of these targeted species. Biocompatibility experiments performed in vitro and in vivo provided promising results, suggesting potential applications of the caged MWCNTs in in situ environmental remediation.

E1AF is an ets-oncogene family transcription factor. E1AF was shown to upregulate multiple matrix metalloproteinase (MMP) genes and contribute to the malignant phenotype of cancer cells by inducing invasive and metastatic activities. E1AF is upregulated by hepatocyte growth factor (HGF) stimulation, which indicates that E1AF would participate in cell motility by HGF/scatter factor. On the other hand, E1AF upregulates p21waf1/cip1 to induce cell cycle arrest when cells are exposed to stress. EWS/ETS fusions are frequently observed in Ewing's sarcoma, and we have revealed that EWS/ETS chimeric protein activates telomerase activity by upregulating hTERT. However, substitution ets binding site (EBS) mutants did not affect the responsiveness to EWS/E1AF. DNA-IP assay showed that the complexes contained EWS/E1AF bound to the hTERT promoter, which suggested that EWS/ETS functions as a co-activator for TERT transcription. Our findings that EWS/ETS acts as a transcriptional co-factor may imply that the transcription pathway is regulated by the interaction of transcription factors.

Purpose: To evaluate the safety and efficacy of a modified version of cyclophosphamide, doxorubicin, vincristine, prednisone (pirarubicin, cyclophosphamide, vincristine, and prednisone [THP-COP]) plus radiotherapy for early-stage aggressive non-Hodgkin's lymphoma of the head and neck., Methods and Materials: Between December 1993 and December 1999, 41 patients with early-stage non-Hodgkin's lymphoma with intermediate-grade histologic features were enrolled in our study. The mean patient age was 51 years. Of the 41 patients, 27 had Stage I and 14 Stage II disease. The primary site was Waldeyer's ring, a neck node, or an extranodal site in 14, 11, and 16 patients, respectively. The immunophenotype was B cell in 29 and T cell in 12 patients. All patients were in the low-risk category according to the International Prognostic Index. Chemotherapy consisted of 40 mg/m(2) i.v. pirarubicin (THP-Adriamycin), 750 mg/m(2) i.v. cyclophosphamide, and 1.0 mg/m(2) i.v. vincristine, on Day 1 and 40 mg/m(2) p.o. prednisone on Days 1-5. The combination chemotherapy was given twice at a 14-day interval. Radiotherapy was given to involved areas at a fraction size of 2.0-2.5 Gy up to a total of 40 Gy within 4-5 weeks. The mean follow-up period was 63 months., Results: The 5-year overall survival rate was 89%. The 5-year cause-specific survival and progression-free survival rate was 90% and 81%, respectively. The 5-year progression-free survival rate for patients with Waldeyer's ring primaries was 93%. Patients with tumor <5 cm in size had greater 5-year progression-free survival than those with tumor >5 cm in size (85% vs. 33%, p <0.05, log-rank test). Grade 4 neutropenia was seen in 12% of patients; however, 93% of patients (38 of 41) received chemotherapy as scheduled with the support of granulocyte colony-stimulating factor., Conclusion: Biweekly THP-COP plus radiotherapy is feasible and effective for Stage I-II low-risk non-Hodgkin's lymphoma.

Bisphenol A (BPA) is one of the endocrine-disrupting chemicals (EDCs) that possess estrogen-like biologic activity. Many dental materials have been reported to release BPA. However, there are few reports available on the release of BPA from dental polycarbonates. The purpose of this study was to investigate the release of BPA from dental polycarbonate crowns and to evaluate the estrogenic activity of BPA. Polycarbonate crowns were immersed in five solvents (water, ethanol, n-hepthane, acetic acid, and acetonitrile) at 37 or 65 degrees C for 24 h. The elution from the material was analyzed by high-performance liquid-chromatography (HPLC) and mass-spectrometry (MS) analysis. BPA release was detected corresponding to the degradation of dental polycarbonates under the some storage conditions (ethanol, acetic acid, and acetonitrile). A previous report proved that estrogen increased human telomerase catalytic subunit (hTERT) mRNA, whereas the effect of EDCs on the hTERT promoter has never been reported. The estrogenic activity of BPA was analyzed by luciferase assay with the use of the hTERT promoter. This assay revealed that BPA was a positive regulator of hTERT transcription. In addition, quantitative real-time PCR analysis showed that BPA increased the expression level of hTERT mRNA in MCF7 cells. Herein, it is demonstrated that hTERT is a new target of BPA.

Production of nitric oxide (NO) in the hypothalamic paraventricular nucleus (PVN) was examined by microdialysis in rats subjected to immobilization (IMO) stress. A dialysis probe was implanted in the posterior magnocellular subdivision of the PVN and nitrite (NO(2)(-)), an oxidized product of NO, was measured continuously. NO(2)(-) concentration in dialysate was enhanced to 156% after 30 min of IMO compared with the NO(2)(-) level before IMO. Intraperitoneal administration of N(G)-monomethyl-l-arginine (10 mg/kg), a NO synthase inhibitor, before IMO completely inhibited the increase of NO production that IMO was to induce. Depletion of catecholamines innervating the PVN by an injection of 6-hydroxydopamine into the lateral ventricle before the microdialysis had no suppressive effect on the increase of NO production by IMO. In contrast, NO(2)(-) levels in the PVN were lowered by continuous perfusion of the solution containing the ionotropic glutamate receptor antagonists 2-amino-5-phosphonovaleric acid (500 microm) and 6-cyano-7-nitroquinoxaline-2, 3 dione (50 microm) through the dialysis probe, and the IMO-induced increase of NO production was attenuated by the treatment. These results suggest that catecholaminergic drive to the hypothalamus is not necessary for the IMO-induced increase of NO production and that ionotropic glutamate receptors play a role in the basal and IMO-induced NO production.

Relatively large amounts of inorganic polyphosphate [poly(P)] (400 microM) have been found in normal osteoblasts. The effect of poly(P) with an average chain length of 65 phosphate residues on cell calcification was therefore investigated with the use of MC3T3-E1 cells. Expression of both osteopontin and osteocalcin was induced by poly(P) (0.1 approximately 1 mM), and cells treated with poly(P) were strongly stained by alizarin red. In addition, the level of alkaline phosphatase activity induced in poly(P)-treated cells was two-fold higher than that in either orthophosphate-treated or control cells but not higher than that in cells treated with beta-glycerophosphate and ascorbic acid. In contrast, however, polyphosphatase activities were activated by poly(P) treatment to levels up to six-fold greater than that in controls. MC3T3-E1 cells may utilize poly(P) as a phosphate source for calcification rather than phosphate sources that are mainly produced by ALPase. Poly(P)-dependent induction of polyphosphatase activities may therefore promote calcification in MC3T3-E1 cells.

EWS/ETS is a chimeric protein identified in most Ewing's sarcomas. Although EWS/ETS has been shown to activate transcription as a transcription factor, the detailed targets of EWS/ETS in transformed cells have not been clarified. Herein, we demonstrate that telomerase is a new target of EWS/ETS fusions. Both telomerase activity and the expression level of telomerase reverse transcriptase (TERT) mRNA were up-regulated in NIH3T3 cells transformed by EWS/E1AF and EWS/FLI1 as well as in two Ewing's sarcoma cell lines. Luciferase assay using the TERT promoter revealed that EWS/E1AF and EWS/FLI1 function as positive regulators of TERT transcription in an ETS binding site-independent manner. EWS/ETS appeared to be included in the initiation complex of TERT transcription and to cooperate with CREB-binding protein (CBP)/p300. When EWS/FLI1 was knocked down in Ewing's sarcomas cells by RNA interference, the expression level of TERT mRNA and the telomerase activity were significantly decreased. These findings indicate that EWS/ETS fusion proteins activate human telomerase activity in Ewing's tumors through up-regulation of TERT gene expression, probably as a transcriptional coactivator.

The adenovirus E4orf6 is a viral oncoprotein known to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventually leading to the inhibition of apoptotic activity.