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.

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.

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.

The 273rd codon mutants of p53 show growth modulation activities not correlated with p53-specific transactivation activity.

Cancer-related mutations of the p53 tumor suppressor gene are clustered in the four so-called 'hot spots', codons 175, 248, 273 and 281/282. By using recombination PCR in vitro mutagenesis, we introduced point mutations into the codon 273 of wild-type (wt) p53 (pC53-SN3) from Arg to His (pC53-273H [273H]), Asp (273D), Pro (273P), Lys (273K), Leu (273L) or Thr (273T), and compared their biological and biochemical activities with wt p53 and cancer-derived 175H, 248W and 273H/309S. Among them, 273H/309S, 273H and 273D as well as wt p53 transactivated the chloramphenicol acetyltransferase (CAT) gene placed downstream of the p53 binding consensus, while none of the other mutants including 273L did. Transcriptions from human c-fos and rat PCNA promoters were suppressed by wt p53 and 273D, while they were enhanced variously by all other mutants in Saos-2 and/or NIH3T3 cells. On the other hand, growth of human squamous carcinoma cell lines measured by the plating efficiency of G418-resistant colonies was enhanced by transfection of 175H, 248W, 273H/309S and 273P, while suppressed by not only wt p53, 273D and 273H but also 273L. Thus, 273H/309S enhanced cell growth in spite of its p53-specific transactivation activity, while 273L suppressed cell growth in spite of its complete loss of the p53-specific transactivation. We concluded that the sequence-specific transactivation of p53 is not always correlated with its growth inhibitory activity.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay.

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT-PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR-SSCP) of exon 5-8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR-SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity.

The p53 gene has been indicated to be a tumour suppressor gene that is found in mutated form in common human cancers. Human papillomavirus (HPV) has oncogenic activity in cervical and oral squamous cell carcinomas (SCCs). The E6 protein of HPV is known to bind with p53 protein and inactive the tumor suppressor activity by promoting p53 degradation. Because of this background, we examined 38 primary, resected specimens of oral SCCs for detection of p53 mutations and HPV DNAs. Exons 5 through 8 of the p53 Mutations were observed in nine cases (24%). HPV-DNA detection and typing were performed using PCR with ¿high risk group' HPV-specified primers. HPV DNA sequences were detected in eight cases (21%). The AvaII digestion pattern of PCR-amplified HPV DNA showed that HPV-16 was present in all eight cases. Seven cases were p53 mutation-positive/HPV-negative, six cases were p53 mutation-negative/HPV-positive, and two intraosseus SCC cases were p53 mutation-positive/ HPV-positive. Thus, 15/38 (40%) cases had inactivation of the p53 protein. Interestingly, p53 mutation-negative/ HPV-negative cases had a poorer prognosis than p53 mutation positive or HPV-positive cases (P < 0.01). We conclude that (1) mutation in the p53 gene and/or HPV infection are frequent (40%) in oral SCC; (2) inactivation of p53 function by mutation and HPV infection are important genetic events in the development of 40% integral of oral SCCs; (3) p53 mutation and HPV infection are not mutually exclusive events and (4) other oncogenes or tumor suppressor genes may be crucial in the development of oral SCC if the prognosis is poor.

Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis.

To investigate the clinical significance of vascular endothelial growth factor (VEGF) in osteosarcoma, we immunohistochemically stained biopsy specimens of 27 primary osteosarcomas using an antibody against VEGF and evaluated the correlation between the expression of VEGF and local density of CD34-positive microvessels, clinicopathological variables, and survival of patients. VEGF staining was positive in 17 tumors (63.0%) in which the density of CD34-positive microvessels was significantly higher than that in VEGF-negative 10 tumors (P < 0.05). In terms of clinicopathological variables, there was no correlation between the expression of VEGF and histological subtype, stage, or response to neoadjuvant chemotherapy, or, strikingly, to the development of pulmonary metastasis (89% of VEGF-positive tumors versus 10% of VEGF-negative tumors; P < 0.0003). Moreover, patients with a VEGF-positive tumor were poorer in both disease-free survival (P < 0.001) and overall survival (P < 0.03) compared to those with a VEGF-negative tumor. These findings strongly suggest that VEGF expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis in patients who underwent aggressive therapy and also provide the basis for a therapeutic strategy targeting angiogeneic property of osteosarcoma.

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.

High-risk HPV-positive human cancer cell lines show different sensitivity to cisplatin-induced apoptosis correlated with the p21Waf1/Cip1 level.

We investigated the sensitivity and cell-cycle inhibitory gene expression of human papillomavirus (HPV) 16- and 18-positive human cancer cell lines after DNA damage induced by treatment with the anti-cancer drug cisplatin. Four HPV-positive cell lines (Caski, SiHa, HeLa and KB) were treated with cisplatin at various concentrations. Apoptotic cell death was observed in a dose-dependent manner in all cell lines treated with cisplatin; however, colony assay for chemosensitivity revealed that HeLa and KB cells (HPV 18-positive cell lines) were more sensitive than SiHa and Caski cells (HPV 16-positive cell lines). Northern blot analyses showed that p53 and p21Waf1/Cip1 mRNA were detectable in all untreated cells, and increasing amounts of these transcripts were identified in all cell lines treated with cisplatin. However, signals were more prominent in HeLa and KB, HPV 18-positive-cells. Immunohistochemical detection of p21Waf1/Cip1 protein showed that the p21-positive cells with apoptotic features were more distinct in KB and HeLa cells (HPV 18-positive) than in SiHa and Caski cells (HPV 16-positive). Our results show that there were differences in sensitivity to cisplatin among four types of high risk HPV-positive cells, possibly due to different levels of p21Waf1/Cip1 up-regulation by functional p53.

Identification of human papilloma virus DNA sequence in the hyperplastic epithelium of an oral denture fibroma.

The human papilloma virus (HPV) associated with hyperplastic epithelium in an oral denture fibroma was examined by southern blot hybridization. Extracted DNA was hybridized with full length linear HPV type 2a, 6b, 11, 16, 18, 31 and 33 DNAs as a mixed probe only under low stringent conditions. The hybridized bands digested with Bam HI and Eco RI were approximately 8.8 kbp and 15 kbp, respectively. Thus the lesional HPV DNA was different from HPV types used as probes and was probably integrated into host cell chromosomal DNA judging by the off-size high molecular weight bands. Considering the contaminating mesenchymal region and uninfected epithelial cells as well as the evidently limited homology with probe HPV DNAs, the virus copy number in infected cells was poorly defined. In situ antigen staining signals were widely detected in the hyperplastic epithelial layer.

Abnormal p53 expression in human lung cancer is associated with histologic subtypes and patient smoking history.

Among the most common mutations in human lung cancer are those affecting the p53 gene. The expression of p53 in the nucleus is considered an immunohistochemical reflection of the nuclear accumulation of mutant p53 protein, which is coded by the p53 gene with missense mutation and has a prolonged half-life. In the present study, p53 expression detected by means of immunohistochemistry occurred frequently in human lung cancer and was associated with histologic subtypes. The alteration in the p53 gene was found to be a relatively early genetic event in the development and progression of lung cancer and to be maintained in the process of metastasis: abnormal p53 expression was found in both the early and late clinical stages, and identical p53 expression was detected consistently among primary and metastatic lesions from the same patients. Furthermore, an observed association between abnormal p53 expression and the patients' smoking history suggests that the p53 gene could be a common target of tobacco-associated carcinogenesis in lung cancer.

The enhancing effect of excess retinol palmitate on induction of odontogenic tumors and inhibitory effect on squamous cell carcinoma of the gingiva in hamsters treated with N-methylnitrosourea.

The influence of excess retinol palmitate on induction of tumors in the oral region was examined histopathologically. Sixty-three weanling Syrian golden hamsters were divided into five groups and received either 0.2% N-methylnitrosourea (MNU) (1 mg/100 g body weight) or retinol palmitate (RP) (25,000 IU/100 g body weight) twice a week for 16 weeks, singly or in combination. Animals received RP intraperitoneally or intragastrically and then, 6 hours later, the animals received intragastric administration of MNU. To accelerate the cell activity of the incisal tooth buds, intentional disocclusion of the left upper and lower incisor of all hamsters was carried out by repeated cutting with cooled diamond disks to a level just above the inter-dental papilla twice a week for 12 weeks. The right incisors were left in occlusion. In all animals exposed to RP + MNU, while the induction of squamous cell carcinomas of the gingiva and forestomach were prevented, the notable findings were a significantly increased incidence of odontogenic tumors in cut incisal regions of the animals with intragastric administration of RP + MNU and an induction of maxillary neurogenic tumors. The incidence of MNU-induced disturbances in odontogenesis in the incisors was reduced but marked disturbances were increased. RP seemed to have opposite effects of prevention and enhancement for development of neoplastic changes in the oral region.

Predominant expression of the src homology 2-containing tyrosine phosphatase protein SHP2 in vascular smooth muscle cells.

src homology 2 (SH2)-containing protein-tyrosine phosphatase SHP2 is known to transduce positive signals from activated receptor protein-tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) beta and insulin receptor. Here, we demonstrate the physiological expression of SHP2 in rats. In northern and western blot analyses, SHP2 expressions were recognized in all tissues, but their expression levels varied significantly among tissues: it is lowest in the liver and kidney. Immunohistochemical staining and in situ hybridization showed SHP2 was expressed ubiquitously but predominantly in vascular smooth muscle cells (SMC). During the development of granulations. SHP2 was expressed predominantly in vascular SMC and also highly expressed in capillary cells. The functional associations of SHP2 with PDGFR beta, which transduces major growth signals in vascular SMC, identify a crucial function of SHP2 in blood vessels in consert with PDGFR beta.

Prevention of carcinoma in situ of human papillomavirus type 16-immortalized human endocervical cells by retinoic acid in organotypic raft culture.

OBJECTIVE: To determine the effect of retinoic acid on the development of severe dysplasia or carcinoma in situ from endocervical cells containing human papillomavirus (HPV) type 16. METHODS: Two independent lines of HPV 16-immortalized endocervical cells were reconstructed into two squamous epithelial tissues using the organotypic raft culture system to examine the differentiated phenotype. The effect of retinoic acid on dysplastic morphology of differentiation of the epithelia was examined by light microscopy of stained sections and electron microscopy. The endocervical cell type cytokeratin expression pattern was determined by indirect immunofluorescence using specific monoclonal antibodies. Ribonucleic acid expression of the HPV 16 E7 oncogene was examined by in situ hybridization. RESULTS: Untreated HPV 16-immortalized endocervical cells were reconstructed into squamous dysplastic lesions resembling carcinoma in situ observed in women. Retinoic acid-treated rafts formed epithelia composed of two to three cell layers of columnar-like cells resembling simple epithelium of the endocervix. Electron microscopy and cytokeratin expression patterns confirmed the histology of a differentiated endocervical phenotype after treatment with retinoic acid. Expression of HPV 16 E7 was modestly lower in treated epithelia, preferentially in basal cells. CONCLUSION: Retinoic acid prevents the histology and cytokeratin differentiation markers of carcinoma in situ of HPV 16-immortalized endocervical cells. Because the epithelia closely mimic HPV 16-containing severe dysplasias and native endocervical epithelium in women, this immortalized endocervical cell-raft system may be useful as a model to assess the efficacy of agents such as retinoic acid for preventing progression of these lesions to malignant cervical carcinoma.

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.

Expression of E1AF, an ets-family transcription factor, is correlated with the invasive phenotype of oral squamous cell carcinoma.

E1AF is a newly identified ets-oncogene family transcription factor. Previous reports have noted that E1AF can upregulate promoter activities of several matrix metalloproteinase (MMP) genes and showed that invasive potentials of oral squamous cell carcinoma-derived cell lines are correlated with expression of E1AF and MMPs. The invasive phenotype is restrained by transfection with an antisense E1AF expression vector. Thus, E1AF is thought to be highly correlated with malignant potentials of cancer cells. However, little is known about E1AF expression and cancer cell malignancies in in vivo tumours. In the present study, 27 oral squamous cell carcinoma (SCC) specimens were examined using RT-PCR, Southern blot hybridisation and in situ hybridisation (ISH) and compared to the clinicopathological parameters. Among the 27 patients, E1AF was detected in 15 cases. E1AF mRNA was detected in 13 of 17 invasive SCCs, whereas the majority of SCCs not expressing E1AF showed an expansive growth pattern. Increased prevalence of E1AF-positive oral SCC was observed in cases with nodal metastasis. These results indicate that E1AF may be involved in cancer cell malignancies through its ability to promote invasive potential.

BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma.

BAG-1 is a Bcl-2-binding protein that functions as an anti-apoptotic molecule. In this report we show a possible correlation between BAG-1 expression levels and the probability of oral squamous cell carcinoma (SCC) progression. We investigated BAG-1 expression levels in 22 patients diagnosed with early lesions (T1 and T2) of oral SCCs using immunohistochemistry and western blotting. High steady-state levels of BAG-1 were detected in 13 out of 22 cases (59%). High BAG-1 expression was observed more frequently in cases with nodal metastasis (89%) than in those without nodal metastasis (38%) (P<0. 03), suggesting that BAG-1 expression levels may correlate with the pathological stage of oral SCCs. Furthermore, BAG-1 expression levels correlated with the WHO grade, i.e. 45% in grade-I cases as opposed to 72% in grade-II cases (P<0.02). These data suggest that an analysis of BAG-1 expression may be useful in establishing a prognosis for patients with oral SCCs, and especially in predicting the metastatic potential of SSCs.

Induction of adenocarcinomas in the submandibular salivary glands of female Wistar rats treated with 7,12-dimethylbenz(a)anthracene.

In 12 male and 12 female Wistar rats, 7-9 weeks of age, a solution of 0.05 ml of 1% 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in acetone was injected directly into the submandibular glands biweekly, after the gland had been injured. In some rats the carcinogen was injected 1 week after the injury. Each rat received six to seven injections. Swelling was observed in the submandibular gland region as early as 4 weeks after the last injection. The animals were killed 4-8 weeks after the last injection and glands with tumour tissues were processed for light microscopy. The control rats of the same age that received a corresponding amount of acetone only (three male, three female), carcinogen injection alone (three male, five female), and injury only (four male, four female) were killed at the same time. Histological examination revealed adenocarcinomas of the submandibular gland in 12/12 (100%) female rats, six of which were associated with fibrosarcomas. The adenocarcinomas basically consisted of ductal and glandular structures. Sometimes tubular, cystic, papillary-cystic and cribriform-like structures were also observed. Male rats mainly developed fibrosarcomas, although there was one squamous-cell carcinoma. The reasons for the sex difference are not known. One adenocarcinoma developed in the submandibular gland of a female rat (1/5) that received carcinogen injections alone. Thus direct injections of DMBA into the submandibular gland produce adenocarcinomas in female Wistar rats.