Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis.

BACKGROUND: Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs. METHODS: TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using ß-aminopropionitrile (BAPN). RESULTS: LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model. CONCLUSION: LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.

Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells.

BACKGROUND: We isolated tumour endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. No report is available on biglycan expression or function in TECs. METHODS: The NEC and TEC were isolated. We investigated the biglycan expression and function in TECs. Western blotting analysis of biglycan was performed on sera from cancer patients. RESULTS: Biglycan expression levels were higher in TECs than in NECs. Biglycan knockdown inhibited cell migration and caused morphological changes in TECs. Furthermore, immunostaining revealed strong biglycan expression in vivo in human tumour vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers. CONCLUSION: These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy.

Viral-mediated stabilization of AU-rich element containing mRNA contributes to cell transformation.

E4orf6 is one of the oncogene products of adenovirus, and it also has an important role for transportation of cellular and viral messenger RNA (mRNA) during the late phase of virus infection. We previously revealed that E4orf6 controls the fate of AU-rich element (ARE) containing mRNA by perturbing the chromosome maintenance region 1-dependent export mechanism. Here, we show that E4orf6 stabilizes ARE-mRNA through the region required for its oncogenic activity and ubiquitin E3 ligase assembly. Cells that failed to stabilize ARE-mRNA after HuR knockdown were unable to produce colonies in soft agar, even when E4orf6 was expressed. Furthermore, the stabilized ARE-mRNA induced the transformation of rodent immortalized cells. These findings indicate that stabilized ARE-mRNA is necessary, if not all, for the oncogenic activity of E4orf6 and has the potential to transform cells, at least under a certain condition.

HuR keeps an angiogenic switch on by stabilising mRNA of VEGF and COX-2 in tumour endothelium.

BACKGROUND: Tumour stromal cells differ from its normal counterpart. We have shown that tumour endothelial cells (TECs) isolated from tumour tissues are also abnormal. Furthermore, we found that mRNAs of vascular endothelial growth factor-A (VEGF-A) and cyclooxygenase-2 (COX-2) were upregulated in TECs. Vascular endothelial growth factor-A and COX-2 are angiogenic factors and their mRNAs contain an AU-rich element (ARE). AU-rich element-containing mRNAs are reportedly stabilised by Hu antigen R (HuR), which is exported to the cytoplasm. METHODS: Normal endothelial cell (NEC) and two types of TECs were isolated. We evaluated the correlation of HuR and accumulation of VEGF-A and COX-2 mRNAs in TECs and effects of HuR on biological phenotypes of TECs. RESULTS: The HuR protein was accumulated in the cytoplasm of TECs, but not in NECs. Vascular endothelial growth factor-A and COX-2 mRNA levels decreased due to HuR knockdown and it was shown that these ARE-mRNA were bound to HuR in TECs. Furthermore, HuR knockdown inhibited cell survival, random motility, tube formation, and Akt phosphorylation in TECs. CONCLUSION: Hu antigen R is associated with the upregulation of VEGF-A and COX-2 mRNA in TECs, and has an important role in keeping an angiogenic switch on, through activating angiogenic phenotype in tumour endothelium.

Synovial chondromatosis of the temporomandibular joint accompanied by loose bodies in both the superior and inferior joint compartments: case report.

Synovial chondromatosis (SC) of the temporomandibular joint (TMJ) is a benign lesion characterized by the formation of metaplastic cartilaginous nodules. SC of the TMJ usually only affects the superior joint compartment of the TMJ. The authors report a rare case of SC of the TMJ affecting the inferior and superior joint compartments.

HuR is exported to the cytoplasm in oral cancer cells in a different manner from that of normal cells.

HuR, a ubiquitously expressed member of the Hu protein family that binds and stabilizes an AU-rich element (ARE)-containing mRNAs, is known to shuttle between the nucleus and the cytoplasm via several export pathways. When normal cells were treated with heat shock, HuR was exported to the cytoplasm in a chromosome maintenance region 1 (CRM1)-dependent manner. However, in this study, we demonstrate that HuR is exported to the cytoplasm in oral cancer cells even if the cells were treated with the inhibitor of the CRM1-independent export pathway. Immunohistochemical and biochemical analyses showed that HuR existed in both the cytoplasm and the nucleus in oral cancer cells, such as HSC-3 and Ca9.22, but existed entirely inside the nucleus in normal cells. AU-rich element-mRNAs were also exported to the cytoplasm and stabilised in the oral cancer cells, which were inhibited by HuR knockdown. This export of HuR was not affected by at least 7 h of treatment of leptomycin B (LMB), which is an inhibitor of the CRM1-dependent export pathway. These findings suggest that HuR is exported to the cytoplasm in oral carcinoma cells in a different manner from that of normal cells, and is likely to occur through the perturbation of a normal export pathway.

Requirement of STAT3 activation for maximal collagenase-1 (MMP-1) induction by epidermal growth factor and malignant characteristics in T24 bladder cancer cells.

Signal transducers and activators of transcription (STATs) are latent transcription factors that mediate cytokine- and growth factor-induced transcription. Constitutive activation of STAT3 has been shown in human cancers and transformed cell lines. We report that STAT3, but not STAT1 and STAT5, becomes phosphorylated in response to epidermal growth factor (EGF) and achieves maximal induction of collagenase-1 (MMP-1) transcription by interacting with c-JUN. Phosphorylation of STAT3 protein is biphasic: the first peak within 30 min and the second peak between 4 and 8 h. Association of STAT3 with c-JUN is detected and its constituting STAT3 is increasingly phosphorylated. The STAT and AP-1 elements are necessary for effective induction of MMP-1 promoter by EGF. Mutation of AP-1 element closely located at the STAT site abolishes the binding not only of c-JUN but also of STAT3 to MMP-1 promoter, resulting in the loss of the responsiveness to EGF. By blocking STAT3 activity with the dominant-negative form, we show the requirement of STAT3 for EGF induction of MMP-1 and MMP-10 (stromelysin-2). Furthermore, expression of the dominant-negative STAT3 is sufficient to inhibit the constitutive and EGF-inducible cell migration and invasion and the tumor formation in nude mice. These results demonstrate that STAT3 phosphorylation and its possible interaction with c-JUN are required for the strong responsiveness of MMP-1 to EGF, and STAT3 activation is crucial for exhibition of malignant characteristics in T24 bladder cancer cells.

Induction of calcification in MC3T3-E1 cells by inorganic polyphosphate.

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.

Amelanotic malignant melanomas of the oral mucosa.

Oral amelanotic melanomas are rare and the prognosis is poorer than that of pigmented melanomas because of delays in establishing the correct diagnosis and in the initiation of treatment. Amelanotic forms are also thought to be biologically more aggressive than pigmented melanomas. We have seen three cases of oral amelanotic melanomas since 1970, in two of whom the diagnosis was long delayed. Two lesions were not pigmented but one had slight pigmentation. One patient simultaneously had both an amelanotic and a pigmented melanoma in the oral cavity. Lymph node metastases and distant metastases developed in all patients, two of whom eventually died of the disease. Early diagnosis by histological examination together with immunostaining with S100 and HMB-45 are the keys to improve survival for patients with amelanotic melanoma.

Increased pre-therapeutic serum vascular endothelial growth factor in patients with early clinical relapse of osteosarcoma.

To investigate the clinical significance of circulating angiogenic factors, especially in association with early relapse of osteosarcoma, we quantified pre-therapeutic levels of vascular endothelial growth factor, basic fibroblast growth factor and placenta growth factor in the sera of 16 patients with osteosarcoma using an enzyme-linked immunosorbent assay. After a 1-year follow-up, the serum level of angiogenic factors was analysed with respect to microvessel density of the biopsy specimen and clinical disease relapse. The serum vascular endothelial growth factor levels were positively correlated with the microvessel density with statistical significance (P=0.004; Spearman rank correlation) and also significantly higher in seven patients who developed pulmonary metastasis than the remaining nine patients without detectable disease relapse (P=0.0009; The Mann-Whitney U-test). In contrast, the serum levels of basic fibroblast growth factor or placenta growth factor failed to show significant correlation with the microvessel density or relapse of the disease. Although there was no significant correlation between serum vascular endothelial growth factor levels and the tumour volume, the serum vascular endothelial growth factor levels were significantly higher in patients with a vascular endothelial growth factor-positive tumour than those with a vascular endothelial growth factor-negative tumour. These findings suggest that the pre-therapeutic serum vascular endothelial growth factor level reflects the angiogenic property of primary tumour and may have a predictive value on early disease relapse of osteosarcoma.

Expression of E1AF/PEA3, an Ets-related transcription factor in human non-small-cell lung cancers: its relevance in cell motility and invasion.

Cell invasion and metastasis characterize the malignant potential of non-small-cell lung cancers (NSCLCs). We have previously reported that E1AF, a member of the Ets-related transcription factor family, confers invasive phenotype on breast cancer and oral squamous-cell carcinoma cell lines. In our study, we analyzed the E1AF expression in cell lines and resected tumors of NSCLCs by Northern blot and in situ hybridization analyses and found that 15 of 17 cell lines and 12 of 19 tumors expressed E1AF mRNA while normal lung tissue and concomitant normal cells within tumors did not. To examine the biologic importance of E1AF in NSCLCs, we introduced the E1AF gene into VMRC-LCD and NCI-H226, NSCLC cell lines lacking E1AF expression, and examined cell motility and invasion activities. E1AF-transfected VMRC-LCD cells showed increased cell motility that was 2-fold that of parental and vector-transfected control cells (p < 0.01), and both cell motility and invasion were increased 1.6-fold in NCI-H226 (p < 0.01). Furthermore, hepatocyte growth factor (HGF), which is one of the most effective cell-scattering factors, stimulated the motile and invasive activities in E1AF-transfected VMRC-LCD and NCI-H226 cells but not in their parental or vector-transfected control cells. Ets-1 mRNA expression was found in E1AF-transfected VMRC-LCD cells but not in parental or vector-transfected cells. HGF further induced expression of the Ets-1 and urokinase-type plasminogen activator (uPA) genes specifically in E1AF-transfected cells. These findings suggest that E1AF plays a substantial role in the cell motility and invasion of NSCLCs.

nm23-H1 suppresses invasion of oral squamous cell carcinoma-derived cell lines without modifying matrix metalloproteinase-2 and matrix metalloproteinase-9 expression.

nm23-H1 is a candidate gene for the suppression of cancer metastasis. Several studies on human breast, hepatocellular, gastric, ovarian, and colon carcinomas and melanomas have shown that reduced nm23-H1 expression was closely related to metastatic progression with poor prognosis. However, the biochemical mechanism by which nm23-H1 suppresses the metastasis has yet to be elucidated. In this study, we analyzed the correlation between nm23 expression, cell motility, and the invasive abilities of six different oral squamous cell carcinoma cell lines (HSC2, HSC3, HSC4, KB, OSC19, and OSC20). Reduced mRNA/protein expression of the nm23-H1 was observed in three cell lines (HSC2, HSC3, and HSC4). These cell lines exhibited increased cell motility and an invasive character on organotypic raft culture. On the other hand, the cell lines (KB, OSC19, and OSC20) that showed a higher expression of nm23-H1 exhibited a threefold to fivefold reduced motility and also reflected fewer invasions compared to the former three cell lines. Because the HSC3 cells demonstrated the lowest nm23-H1 expression with the highest cell motility and invasive character, we established nm23-H1-transfected HSC3 cell lines to investigate whether exogenous nm23-H1 protein could inhibit cell migration and invasive activity. These transfectants showed a significant reduction in cell motility with exogenous nm23-H1 in a dose-dependent manner, and exhibited a noninvasive character. An immunofluorescence study demonstrated a distinct stress-fiber distribution at peripheral region of these transfectants. However, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 expression was observed between mock transfectant and nm23-H1-transfected cells. These findings suggest that nm23-H1 inhibits the invasive activity of oral squamous cell carcinoma by suppression of cell motility without altering the MMP-2 and MMP-9 status.

Development of squamous cell carcinoma from pre-existent oral leukoplakia: with respect to treatment modality.

The present study was undertaken in order to determine whether surgical treatment of oral leukoplakia reduces the risk of the subsequent development of carcinoma. This study included 142 patients with oral leukoplakia who received or did not receive surgical treatment. All subjects were followed-up for more than 6 months with a mean follow-up period of 4 years. Malignant transformation rate was lower among patients who received surgical excision (1/75) than among those who did not receive surgical treatments (4/51). However, the malignant transformation rates were high in patients who received cryosurgery (3/12) or cryosurgery plus surgical excision (1/4). There was no obvious relation between the grade of epithelial dysplasia and the rate of malignant transformation. Our results suggest that surgical excision of oral leukoplakia may reduce the risk of the subsequent development of carcinoma.

[Evaluation of efficiency of ACD-CPR and STD-CPR; a multi-institutional study].

We compared the efficacy of ACD-CPR and STD-CPR based on 64 multi-institutional reports. No significant differences were observed in the rate of restoration of spontaneous circulation (ROSC) and in cardiopulmonary parameters during CPR using the two methods. There were 5 cases in which cardiopulmonary parameters improved after switching from STD-CPR to ACD-CPR and, eventually, in two of them spontaneous circulation was restored. In the ROSC cases of both groups, ETCO2 and values of SpO2, PaO2, and systolic BP at 30 minutes were higher than those of non-ROSC cases. ETCO2 never exceeded 20 mmHg in the non-ROSC cases, but it was higher in the ROSC cases. ACD-CPR is a good choice when trained persons are present or when extra hands are available to continue the CPR.

Echocardiographic diagnosis of extrapericardial tamponade due to dilated gastric roll following oesophagectomy.

A complication of lower thoracic oesophagectomy for oesophageal carcinoma is reported. Extrapericardial tamponade was caused by a dilated retrosternal gastric roll. Echocardiography was useful for diagnosis. Diagnosis, investigation and management of this unusual but life-threatening complication are discussed. Transthoracic echocardiography is a useful and practical investigation for the evaluation of complications of oesophagectomy.

Venous blood flow measurement by determination of change in venous hemoglobin saturation.

OBJECTIVE: To evaluate a method of measuring venous blood flow in vitro by using the Fick principle and change in saturation of venous blood and to apply the method to the clinical measurement of hepatic blood flow. DESIGN: Experimental study using an in vitro model and clinical study for critically ill patients. SETTING: Department of Anesthesiology and Intensive Care Medicine in Osaka City University Medical School. MODEL: Human blood deoxygenated by bubbling of nitrogen was circulated in a closed circuit at 10-120 mL/min by a roller pump. A fiber optic sensor was attached to the circuit for continuous monitoring of hemoglobin saturation. PATIENTS: Eight critically ill patients, aged 54.3 +/- 15.1 yrs, were studied. INTERVENTIONS: Hemoglobin saturation was changed in the in vitro study by the injection of 0.2 mL of oxygenated blood. Signals from the optical fiber were analogue-digital converted and recorded in a computer. In the clinical study, an oximetry catheter was inserted into the inferior vena cava (IVC) via the femoral vein. Arterial blood (10 mL) was drawn from the radial artery, and injected into the IVC. The changes in oxygen saturation in the venous blood were recorded. MEASUREMENTS AND MAIN RESULTS: Blood flow was calculated using the Fick principle, assuming that all the injected blood passes through the sensor. In vitro estimation of blood flow was well correlated with the actual flow (r2 = .94). IVC blood flow was measured above and below the merging point of the hepatic vein. The difference of the two values was assumed to represent hepatic blood flow. IVC blood flow was calculated by the same method as for the in vitro study. The blood flows in the IVC above and below the anastomosis with the hepatic vein were 2.82 +/-0.56 (SD) Umin and 1.96 +/- 0.61 (SD) L/min. Average estimated hepatic blood flow was 0.86 L/min (range, 0.34-1.75 Umin). CONCLUSION: We examined the accuracy and reliability of this new method in the present in vitro study. This method may be clinically useful for measuring hepatic blood flow.

Intracellular distribution of macrophage migration inhibitory factor predicts the prognosis of patients with adenocarcinoma of the lung.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is known to be a proinflammatory cytokine and glucocorticoid-induced immunomodulator as well as a regulator of tumor growth. Although positive and negative effects of MIF on tumor cell growth have been reported, to the authors' knowledge the precise role of MIF in tumorigenesis remains unclear. In the current study the authors assessed expression of MIF protein and mRNA in lung adenocarcinomas with regard to patient prognosis. METHODS: Immunohistochemical analysis was performed on tissue specimens surgically obtained from 74 patients with primary lung adenocarcinoma (American Joint Committee on Cancer pathologic Stages I, II, and IIIa). In addition, expression of MIF mRNA in the cancerous tissue was investigated using in situ hybridization. Patient prognosis was evaluated with regard to MIF expression levels and its distribution was analyzed with the Kaplan-Meier method. RESULTS: MIF mRNA and MIF protein were observed in the bronchial epithelium, alveolar epithelium, vascular smooth muscle, and alveolar macrophages in the normal lung tissue. In tumor tissue from lung adenocarcinoma specimens, both MIF mRNA and protein were observed at much higher levels than in the normal alveolar epithelium. MIF protein was observed diffusely in the cytoplasm of tumor cells in all tumor specimens examined. MIF protein also was observed in the nuclei of tumor cells from 59 patients (79.7%), whereas it was not observed in the nuclei of tumor cells from 15 patients (20.3%). The patients without nuclear MIF expression had a worse prognosis compared with those patients with MIF expression in the nuclei (P = 0.04). CONCLUSIONS: The results of the current study suggest that intracellular MIF distribution predicts patient prognosis in individuals with adenocarcinoma of the lung.

Membrane type 1-matrix metalloproteinase expression is regulated by E-cadherin through the suppression of mitogen-activated protein kinase cascade.

To elucidate the role of E-cadherin in matrix metalloproteinases (MMPs) expression, we transfected to squamous carcinoma cells with E-cadherin cDNA. HN5 cells and mock-transfected HN5-neo cells expressed proMMP-2 and active MMP-2. E-cadherin-transfected HN5-EC cells produced comparable proMMP-2 but low active MMP-2; and membrane type 1-MMP (MT1-MMP) mRNA declined. Phosphorylated ERK, a marker of mitogen-activated protein (MAP) kinase cascade, also declined in HN5-EC cells. The addition of anti-E-cadherin antibody resulted in the disappearance of these alterations in HN5-EC cells. These results suggest that E-cadherin suppresses MAP kinase cascade and down-regulates MT1-MMP.