Porcine adenovirus serotype 3 internalization is independent of CAR and alphavbeta3 or alphavbeta5 integrin

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus-adenovirus receptor (CAR) serves as a primary receptor and alphavbeta3 or alphavbeta5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, alphavbeta3 or alphavbeta5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-alphavbeta3 or anti-alphavbeta5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as alphavbeta3 or alphavbeta5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and alphavbeta3 or alphavbeta5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.     

Beta4 integrin subunit gene expression correlates with tumor size and nuclear grade in early breast cancer.

In vitro data support a role for the alpha6beta4 integrin in tumor cell migration and invasion, particularly in breast carcinoma cells, but clinical data on this potentially important integrin are limited. The beta4 integrin subunit has been shown to cluster with genes characteristic of basal/myoepithelial cells in cDNA microarray analyses of breast cancer, and the subset of breast cancers with increased expression of genes characteristic of basal/myoepithelial cells appears to be particularly aggressive. The purpose of this study was to determine whether alpha6beta4 integrin expression correlates with aggressive clinicopathologic features of breast cancer and whether expression of this integrin has prognostic significance in early breast cancer. We evaluated tumor expression of the beta4 integrin subunit gene in a cohort of patients with early invasive breast carcinoma by in situ hybridization and correlated expression levels with multiple clinicopathologic characteristics. We also evaluated expression of laminin-5 protein, the principal ligand of alpha6beta4, in this patient cohort. Although we observed a slight trend towards decreased disease-free survival for patients whose tumors had high beta4 gene expression and coexpression of laminin-5, this did not reach statistical significance (P=0.11). However, we did observe a correlation between beta4 mRNA expression and both tumor size (P=0.01) and tumor nuclear grade (P<0.01). These results do not demonstrate prognostic significance for beta4 gene expression and/or laminin-5 protein expression in early breast cancer, but increased beta4 gene expression in larger tumors and in higher grade tumors does support a potential role for the alpha6beta4 integrin in tumor progression.     

Zoledronate inhibits alphavbeta3 and alphavbeta5 integrin cell surface expression in endothelial cells.

Zoledronate exhibits antiangiogenic properties in vitro and in vivo. Integrins alphavbeta3 and alphavbeta5 are involved in angiogenesis. Because zoledronate inhibits endothelial cell adhesion, the authors explored the hypothesis that it could alter these integrins recruitment to focal adhesion sites. Human umbilical vein endothelial cells (HUVECs) were treated with zoledronate or with mevalonate pathway intermediates geranylgeraniol (GGOH) and farnesol (FOH). Zoledronate generated a significant decrease in alphavbeta3 and alphavbeta5 expression at HUVEC cell surface using flow cytometry and immunofluorescence. This inhibition was reversed by GGOH but not by FOH. Cells cotreated with zoledronate and GGOH were able to attach to vitronectin through alphavbeta3 and alphavbeta5, as confirmed by the use of specific function-blocking antibodies. The authors showed that zoledronate alters endothelial cell integrin-mediated adhesion. This effect is likely to contribute to the previously demonstrated antiangiogenic effect of zoledronate. Whether this mechanism of action also applies to metastatic tumor cells is under investigation.     

Expression of the hyaluronan receptor LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vessels

Expression of the hyaluronan receptor LYVE-1 is one of few available criteria used to discriminate lymphatic vessels from blood vessels. Until now, endothelial LYVE-1 expression was reported to be restricted to lymphatic vessels and to lymph node, liver, and spleen sinuses. Here, we provide the first evidence that LYVE-1 is expressed on blood vessels of the yolk sac during mouse embryogenesis. LYVE-1 is ubiquitously expressed in the yolk sac capillary plexus at E9.5, then becomes progressively down-regulated on arterial endothelium during vascular remodelling. LYVE-1 is also expressed on intra-embryonic arterial and venous endothelium at early embryonic stages and on endothelial cells of the lung and endocardium throughout embryogenesis. These findings have important implications for the use of LYVE-1 as a specific marker of the lymphatic vasculature during embryogenesis and neo-lymphangiogenesis. Our data are also the first demonstration, to our knowledge, that the mouse yolk sac is devoid of lymphatic vessels.     

Vascular endothelial growth factor expression correlates with tumour grade and vascularity in gliomas

AIMS: Tumour vascularity and vascular endothelial growth factor (VEGF) expression were studied in 41 primary brain tumours of astrocytic and oligodendroglial origin, in order to define the potential role of VEGF in the vascularization and growth of these tumours. METHODS AND RESULTS: Two commercial monoclonal antibodies to the VEGF protein (from R&D Systems and NeoMarkers), raised against different isoforms, were utilized. Each monoclonal antibody consistently detected the expression of VEGF in different cell types. The R&D Systems antibody only produced surface staining of endothelial cells in tumour capillaries, whereas staining with the Neomarkers antibody was largely confined to tumour cell cytoplasm. High levels of staining were seen with the R&D Systems and NeoMarkers antibodies in 13 and 14 of 15 glioblastomas, respectively, four and three of five oligodendrogliomas, four and seven of 10 anaplastic astrocytomas, one and three of six low-grade astrocytomas and none and none of five pilocytic astrocytomas. There was a close correlation between VEGF expression, tumour vascularity and grade. CONCLUSIONS: These findings support a role for VEGF in the angiogenesis of glioblastoma, anaplastic astrocytoma and oligodendroglioma. The distinct immunoreactivities of the two commercial monoclonal antibodies indicate either there is expression of different splice variants of VEGF or that the epitopes are differentially revealed during synthesis, secretion and receptor-binding of the growth factor. This highlights the importance of using more than one antibody in the evaluation of tissue VEGF expression.     

抗人整合素αυβ3单链抗体的构建和表达

目的：利用基因工程抗体技术构建抗人整合素αυβ3单链抗体(scFv)。方法：从分泌抗人整合素αυβ3单抗(mAb)的杂交瘤细胞E10总RNA中，用RT-PCR扩增VH和VL基因，对其核苷酸序列分析后，通过PCR在VH和VL基因间插入柔性连接子(Gly4Ser)3 组装成scFv基因，并克隆至原核表达载体pTIG—TRX中。以重组子转化大肠杆菌B121(DE3)诱导目的基因表达。结果：转化菌可表达相对分子质量(Mr)为31000的scFv。Western blot证实，具有His6标签蛋白的表达。经低剂量的IPTG诱导和较低温度培养，scFv获得了可溶性形式表达。表达产物经Ni—NTA琼脂糖层析纯化后纯度达91％以上。ELISA鉴定证实，scFv具有良好的抗原结合活性。结论：成功地构建并表达抗人整合素αυβ3的scFv，为进一步临床研究奠定了基础。     

Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease

Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1.     

Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.

Angiogenesis has been implicated in the progression of human neoplasia from benign precursor to invasive and metastatic phenotypes. The acquisition of dominant oncogenes in preneoplastic cells in vitro and in vivo has been associated with the increased ability of tumor cells to secrete angiogenic mediators and recruit blood vessels. However, in a subset of benign lesions, high levels of angiogenesis have been found before the conversion to invasive and metastatic phenotypes. In many of these benign lesions, dominant oncogenic pathways are activated first; then as malignant potential is acquired, there is a loss of nuclear tumor suppressor genes, such as p53 and p16. We studied neuroendocrine lung tumors (NLT) ranging from typical and atypical carcinoid tumors to large cell neuroendocrine and small cell carcinomas in order to determine whether angiogenesis (as assessed by mean vessel density) and proliferation rates (as assessed by MIB-1 nuclear immunohistochemical staining) correlate with tumor type. We found that increased rates of proliferation, but not angiogenesis, correlate with tumor type. The association of increased proliferation and tumor type may prove to be clinically useful and shed light on the role of sequential oncogenic alterations in NLT.     

VEGFR-3 expression in breast cancer tissue is not restricted to lymphatic vessels

We examined the immunohistochemical reactivity for vascular endothelial growth factor receptor 3 (VEGFR-3), a protein playing an important role in lymphangiogenesis, in breast cancer. A retrospective series of 77 invasive ductal breast carcinomas was investigated. The relationship between VEGFR-3 expression and clinicopathologic parameters was examined for statistical significance using Pearson's chi-square (chi2) test and Fisher's exact test (when n<5). Threshold for significance was p<0.05. Patient age ranged from 31 to 77 years (mean: 55 years). The VEGFR-3 immunoreactivity was as follows: 5 cases were negative (6.5%), 35 + (45.4%), 27+ + (35.1%), and 10+ + + (13.0%). Reactions were positive for both lymphatic and blood vessels in several cases. VEGFR-3-positive reactions were more frequent in the tumor periphery than within the tumor. Immunoreactivity was also observed in myoepithelial cells surrounding both normal ducts and ducts with ductal carcinoma in situ. Statistical analysis of VEGFR-3 reactions was not significantly related to node status, microvessel density, and tumor grade. Ploidy showed a tendency towards significance (p=0.063); however, owing to the limited number of cases, statistical significance was not reached. VEGFR-3 lacks lymphatic vessel specificity and is also expressed in blood vessels, myoepithelial cells, and neoplastic cells.     

Expression of integrin subunits in the human infant breast correlates with morphogenesis and differentiation

Integrins are widely expressed on normal tissues and their function is considered critical directly or indirectly with the control of cell growth and differentiation. Also, they are likely to play a crucial role in cell–matrix interactions during development. As the human breast develops after birth, it provides a rare opportunity in which to study human organogenesis. We have examined the distribution of integrins in the human infant breast with the aim of elucidating the possible role of these molecules in morphogenesis and differentiation. Necropsy breast specimens from six male and eight female infants, ranging in age from 1 day to 9 months, were used in this study. Cryostat sections were stained by the avidin-biotin complex technique, using a panel of monoclonal antibodies (MAbs) which recognize β₁, α₂, α₆, β₄, α_v, and α_vβ₃ integrin chains, which are candidate molecules for a role in mammory morphogenesis. MAbs to β₁ (DH12) and α₂ (HAS3) showed positive membrane and cytoplasmic staining of basal cells and luminal epithelial cells. In addition, positive staining for the β₁ integrin chain was found on fibroblasts. A MAb which recognizes the α₆ chain (MP4F10) showed positive staining of the basal cells and heterogeneous staining of the luminal epithelial cells, whilst β₄ chain (439-9B) showed positive staining in the basement membrane domain of the basal cells with no staining of the luminal epithelial cells. There was a positive correlation between the intensity of expression and the structural development of the ductal system, with integrin expression reduced or absent in the end buds and lateral buds. These data provide evidence that some integrin molecules are expressed in a pattern that correlates with the morphological and functional differentiation of the normal mammary gland. Changes in the expression and function of integrins may have an inductive role in the development of the normal mammary gland.     

Expression of caveolin-1 and caveolin-2 in urothelial carcinoma of the urinary bladder correlates with tumor grade and squamous differentiation

We immunohistochemically evaluated 94 cases of urothelial carcinoma (UC) of the urinary bladder for the expression of caveolin (Cav)-1 and Cav-2. Neither benign urothelium present in 22 cases nor flat carcinoma in situ present in 10 cases stained for Cav-1 or Cav-2. Thirty-five (37%) of 94 cases and 45 (51%) of 89 cases of UC stained positively for Cav-1 and Cav-2, respectively. The percentages of positive cases for Cav-1 in grades 1, 2, and 3 tumors were 0% (0/6), 0% (0/25), and 56% (35/63), respectively (P < .001), and for Cav-2, 0% (0/6), 13% (3/23), and 70% (42/60), respectively (P < .001). Multivariate analysis showed no significant correlation between tumor stage and Cav-1 or Cav-2 expression after correction for tumor grade. Eighty-two percent (14/17) of cases with squamous differentiation were positive for Cav-1 compared with 43% (20/46) of grade 3 tumors without squamous differentiation (P < .001). These results indicate a positive correlation of the expression of Cav-1 and Cav-2 with tumor grade and squamous features of UC and suggest that Cav-1 and Cav-2 be studied further for a possible role in tumor progression and squamous differentiation.     

胶质瘤中hTFERT、VEGF表达及其与肿瘤血管形成的关系

目的 探讨胶质瘤中端粒酶逆转录酶(hTERT)、血管内皮生长因子(VEGF)的表达与肿瘤血管形成问的相互关系.方法 分别用原位杂交和免疫组化染色检测106例胶质瘤hTERT和VEGF的表达;用CD34标记瘤组织血管内皮细胞,测定微血管密度(MVD).结果 hTERT、VEGF总阳性表达率分别为53.8%(57/106)、68.0%(72/106);hTERT或VEGF阳性分别定位于肿瘤细胞核内和细胞质内.hTERT阳性或VEGF阳性的瘤组织MVD分别为71.2±18.0和74.4±20.0;而相应的阴性组分别为60.3±21.8和58.4±23.1,两组差异均有显著性(P＜0.01).hTERT、VEGF的表达和MVD均与胶质瘤组织病理分级呈正相关(P＜0.01).结论 hTERT、VEGF的表达及MVD均与胶质瘤的恶性程度有关,前两者阳性表达,其MVD高于两者阴性表达,说明hTERT、VEGF在肿瘤血管形成中可能起促进作用.     

Expression and localization of alphavbeta6 integrin in extraplacental fetal membranes: possible role in human parturition

Successful outcome of human parturition is dependent upon extensive remodelling of the extracellular matrix (ECM) of the cervix, uterus and fetal membranes, a process that involves adhesion molecules and is also common in tumour invasion and metastasis. To elucidate the role of integrins in human parturition, this study characterizes the expression of the tumour-associated alpha(v)beta(6) integrin in human placenta and extraplacental membranes. Immunohistochemical analysis of the placenta and fetal membranes from normal vaginal deliveries (NVD) (n = 10) exhibited strong intensity of staining for alpha(v)beta(6) integrin (3 = dark brown) in the epithelial layer of the amnion. Weak immunohistochemical staining of alpha(v)beta(6) integrin (1 = pale brown) was detected in the chorion and at the decidual edge. These results were consistent with the immunodetection of alpha(v)beta(6) integrin by western blot analysis that showed 4-fold enhanced expression in the amnion compared to chorion of both NVD and term elective caesarean section (CS) deliveries. Even though there was no difference in the extent of immunohistochemical staining of alpha(v)beta(6) integrin between the amnion of NVD and CS groups, significantly higher intensity of staining was observed in the NVD amniotic epithelium compared to that of CS (n = 10) (chi(2) = 10.25, P = 0.0059). Western blot analysis of the fetal membranes showed no differences in the expression of alpha(v)beta(6) integrin between the NVD and CS groups. Gelatin zymography demonstrated the presence of pro-matrix metalloprotein-9 (MMP-9) and pro-MMP-2 in the amnion and chorion of NVD, whereas in CS only the presence of pro-MMP-2 was observed. These results suggest that in term pregnancy, human fetal membranes express alpha(v)beta(6) integrin and that the expression is significantly higher in amnion compared to chorion. The fact that enhanced expression of alpha(v)beta(6) integrin in fetal membranes correlates with the expression of pro-MMP-9 in NVD is consistent with the invasive role of the integrin in cancer and suggests that the molecule may have a proteolytic role in the initiation and progression of labour.     

Expression of the gene for hematopoietic cell specific protein is not restricted to cells of hematopoietic origin

Hematopoietic cell line specific protein (HS1) is an intracellular signaling protein that has been reported as specifically expressed in hematopoietic cells. HS1 is known as a major substrate of protein-tyrosine kinases following activation by B-cell or T-cell receptor complexes. We report the first evidence that HS1 is also expressed in a variety of tissues different from hematopoietic tissues by using sensitive expression analysis including real-time quantitative RT-PCR. While former studies on HS1-expression were mostly limited to cells of hematopoietic origin, we screened a larger number of human tissues including tumor samples. Normal lung tissue showed a high degree of HS1 expression, second to the expression of hematopoietic cells. Expression of HS1 in tumor tissues was also clearly detectable. Our findings suggest that the signaling protein HS1 is involved in pathways different from the ones that have a specific role in the intracellular processes of hematopoietic cells.     

Allelic imbalance is not restricted to numerically abnormal chromosomes in epithelial ovarian tumours.

In this study, 23 low malignant potential (LMP) and 27 invasive epithelial ovarian tumours have been examined by microdissection and microsatellite polymerase chain reaction (PCR) for allelic imbalance (AI) at loci on the p and q arms of chromosomes 1, 11, 17, and X, and the data have been compared with interphase cytogenetics for numerical abnormalities (aneusomy) of these chromosomes. AI was uncommon in LMP tumours (5 of 23 at 9 of 146 informative loci) but was significantly more common (p<0.001) in invasive carcinomas (21 of 27 at 47 of 168 informative loci). This difference remained when LMP tumours were compared specifically with stage I carcinomas (p<0.001). A greater number of loci were involved in AI amongst serous than amongst mucinous carcinomas (p=0.015). AI was present at significantly more loci in carcinomas showing aneusomy by interphase cytogenetics than in those showing no numerical chromosome abnormalities (p<0.001). However, amongst the carcinomas showing aneusomy, AI was as frequent at loci on chromosomes with no numerical abnormality as at those with the numerical changes. These data demonstrate that aneusomy and AI are interrelated phenomena but that AI does not occur simply as a consequence of numerical chromosome changes.     

Syntenin is expressed in human gliomas and may correlate with tumor migration

Introduction

Invasion is usually recognized as the main reason for the high recurrence and death rates of gliomas. Therefore, properly understanding the molecular mechanisms of migration and invasion of human gliomas has become a focus and will be helpful for the treatment of gliomas. Syntenin has been demonstrated to be implicated in the migration, invasion and metastasis of many types of malignant tumors. Therefore, we investigated the expression of syntenin in human gliomas and its relationship with glioma migration.

Material and methods

Immunohistochemistry, Western blot and real time-polymerase chain reaction (RT-PCR) were performed to detect the expression of syntenin in human gliomas. Phosphorylated FAK in human gliomas was examined by western blot.

Results

Scattered syntenin positive glioma cells were detected by immunohistochemistry in normal tissue. Syntenin expression in grade II, III and IV gliomas increased with the degree of tumor malignancy, and no syntenin expression was detected in grade I gliomas. The level of phosphorylated FAK at the tyrosine 397 site also elevated with the degree of tumor malignancy. There was a positive correlation between the syntenin level and the pathological grade of gliomas (r_s = 0.896, p < 0.05). Phosphorylated FAK was also upregulated along with the stage of glioma progression and the increase of syntenin expression.

Conclusions

Our results indicate that the enhanced expression of syntenin and phosphorylated FAK may correlate with the increase of the malignancy of human gliomas. Syntenin may promote human glioma migration through interaction with FAK.     

Expression of coronin-3 (coronin-1C) in diffuse gliomas is related to malignancy

Coronin-3 (coronin-1C), a homotrimeric F-actin binding protein, has been shown to be important for cell migration and brain morphogenesis. Here, we present for the first time a detailed analysis of the expression pattern of coronin-3 in human brain tumours and demonstrate that coronin-3 expression correlates with malignant phenotype in diffuse gliomas. In general, the expression of coronin-3 varies in different brain tumour entities. However, in diffuse gliomas, the number of coronin-3 expressing tumour cells correlates with the degree of malignancy. High-grade gliomas, such as anaplastic astrocytomas, anaplastic oligodendrogliomas, anaplastic oligoastrocytomas and glioblastomas, show high numbers of tumour cells positive for coronin-3, while diffuse low-grade gliomas, such as diffuse astrocytomas, oligodendrogliomas and oligoastrocytomas, exhibit low numbers of coronin-3-positive tumour cells. In order to explore and verify a contribution of coronin-3 to the malignant phenotype of diffuse gliomas, we employed an efficient shRNA-mediated coronin-3 knockdown in U373 and A172 human glioblastoma cells. Coronin-3 knockdown glioblastoma cells exhibited reduced levels of cell proliferation, cell motility and invasion into extracellular matrix compared to control cells. Together, our findings demonstrate evidence for a contribution of coronin-3 expression in the malignant progression of diffuse gliomas.