Microenvironments surrounded with various extracellular matrix (ECM) components can decide specifically the fate of spermatogonial stem cells (SSCs) and integrin heterodimers recognizing directly ECM proteins play an important role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrin heterodimers expressed functionally on the undifferentiated SSCs derived from mouse with hybrid strain remain unclear. Therefore, we tried to investigate systematically what kind of integrin heterodimers are expressed transcriptionally, translationally and functionally in the SSCs derived from testis of hybrid (B6CBAF1) mouse. For these, magnetic activated cell sorting (MACS) using Thy1 antibody was used for isolating SSCs from testis, and real-time PCR or fluorescence immunoassay was conducted for measuring transcriptional or translational level of integrin α and β subunits in the isolated SSCs. Subsequently, antibody inhibition assay was conducted for confirming functionality of presumed integrin heterodimers. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, 7 integrin α (α4, α6, α7, α9, αV, αL and αE) and 2 integrin β (β1 and β5) subunit genes showed significantly increased transcriptional up-regulation, compared to the other integrin subunit genes. In contrast, integrin α3, α5, α10 and α11, and integrin β2, β3, β4 and β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV, αL and αE) were measured, significantly strong translational up-regulation of integrin α6, α7, α9, αV and αL subunit genes were detected, whereas integrin α4 and αE subunit genes were weakly. In case of integrin β subunit, β1 evaluated more expression than β5. Based on these results, we speculated that the undifferentiated SSCs derived from B6CBAF1 mouse might express integrin α4β 1, α6β1, α7β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Subsequently, the hybrid strain SSCs showed significantly increased adhesion to fibronectin, laminin, tenascine-C and vitronectin and functional blocking of integrin α4β1, α6β1, α9β1, and αVβ1 or αVβ5 in SSCs significantly inhibited attachment to fibronectin, laminin, tenascin-C and vitronectin, respectively. Accordingly, we could identify that the hybrid (B6CBAF1) mouse-derived SSCs had integrin α4β1, α6β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.

Generally, fate of spematogonial stem cells (SSCs) can be determined specifically by microenvironments enclosed with various extracellular matrix (ECM) components and integrins recognizing directly ECM proteins play an pivotal role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrins expressed on the undifferentiated SSCs remain unclear. Therefore, we tried to investigate systematically what kind of integrin subunits are expressed transcriptionally or translationally in the SSCs derived from testis of hybrid B6CBAF1 mouse. For these, isolation of SSCs from testis were conducted by magnetic activated cell sorting (MACS) using Thy1 antibody. Subsequently, transcriptional and translational level of integrin α and β subunits in the isolated SSCs were measured by real-time PCR and fluorescene immunoassay, respectively. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, and integrin α4, α6, α7, α9, αV, αL and αE and integrin β1, β5 showed higher expression levels than other subunits. By contrast, integrin α3, α5, α 10 and α11 and integrin β2, β3, β4, β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV αL, and αE) were measured, integrin α6, α7, α9, αV and αL were higher than integrin α4 and αE. In case of integrin β subunit, β1 evaluated more expression than β5. From these results, we speculate that the undifferentiated SSCs derived from hybrid B6CBAF1 mouse may express integrin α4β1, α6β1, α7β1, α9β1, αVβ1 and/or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.

Integrin is a heterodimer protein that locates on cell membrane to interact with neighboring cells or extracellular matrix. A transcriptome analysis of the brassica leaf beetle, Phaedon brassicae, midgut identified both α and β subunits of integrin. RNA interference of β subunit genes significantly impaired survival of both larvae and adults of P. brassicae. A recombinant bacteria expressing double-stranded RNA specific to β integrin of P. brassicae were constructed and showed significant oral toxicities.

Oral toxicity of double-stranded RNA (dsRNA) specific to integrin β1 subunit (SeINT) was known in a polyphagous insect pest, Spodoptera exigua. For an application of the dsRNA to control the insect pest, this study prepared a recombinant Escherichia coli expressing dsRNA specific to SeINT. The dsRNA expression was driven by T7 RNA polymerase overexpressed by an inducer in the transformed E. coli. The produced dsRNA amount was proportional to the number of the cultured bacteria. The bacteria gave a significant oral feeding mortality to S. exigua larvae with a significant reduction of the SeINT expression. The resulting insect mortality increased with the fed number of the bacteria. Pretreatment with a sonication to disrupt bacteria cell wall membrane significantly increased the insecticidal activity of the transformed bacteria. Compared to the control bacteria transformed by non-recombinant vector, the larvae fed the bacteria expressing dsRNA specific to SeINT suffered tissue damage in the midgut epithelium, which was characterized by a loose cell-cell contact and a significant cell death. The dsRNA-treated larvae were significantly more susceptible to a Cry toxin derived from Bacillus thuringinesis (Bt) than the larvae treated only with Cry toxin. This study demonstrates that a transformed bacterium expressing dsRNA specific to SeINT has a significant insecticidal activity by oral application against S. exigua and makes the target insects to be highly susceptible to Bt toxin.

Double-stranded RNA(dsRNA) had been used to specitically suppress target gene expression at post-tanscription level. Injection of dsRNA to hemocoel is the most efficient to knockdown target mRNA. However, some insects have shown to be susceptible to feeding dsRNA. Spodoptera exigua was susceptible to dsRNA at oral treatment. Especially dsRNA specific to β-integrin was potent to survival of S.exigua larvae. This study advanced our dsRNA application technology by generating recombinant E.coli expressing dsRNA specific the β-integrin. A recombinant vector L4440 was constructed with a partial β-integrin gene under T7 RNA polymerase promoter. The recombinant vector was used to transform HT115 competent cells of E.coli. The transformed E.coli expressed the dsRNA. The production of dsRNA was proportional to the bacterial number. By feeding the recombinant E.coli, S.exigua underwent significant mortality. By adding E.coli expressing Cry1Ca Bt toxin to E.coli expressing dsRNA, S.exigua exhibited highly enhanced mortality. This study suggests a possibility to use a recombinant E.coli expressing dsRNA to control S.exigua.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.

Human pituitary tumor transforming gene 1 (PTTG1), which is a newly identified proto-oncogene, is highly expressed in normal pituitary tissues containing proliferating cells and in several cancers. Also, PTTG1 has been known as a securin to involve in the regulation of c ell-cycle and in t he p rogression o f tumor. B u t the effect o f PTTG1 in o ral squamous cell carcinoma (oral SCC) h as not b een studied yet. The objective of this study is to analyze the expression of PTTG1 in oral SCC cell lines (YD-10B and YD-15) and to evaluate the effect of PTTG1 on oral SCC cell lines for the migration effect by PTTG1 siRNA treatment. Western blot, migration assay, and zymography were performed to evaluate the effects of PTTG1 on the expression of MMP-2/-9 and migration activity after PTTG1 siRNA treatment. PTTG1 was expressed in oral SCC cells lines, otherwise, significantly decreased after PTTG1 siRNA treatment. There is no difference in expression of MMP-2 regardless of PTTG1 siRNA treatment. However, the enzyme activity of MMP-9 was significantly decreased. In addition, the migration activities of oral SCC cells were significantly decreased after PTTG1 siRNA treatment (p<0.001). These results suggested that the down-regulated PTTG1 could inhibit the migration of human oral SCC cells through the low MMP-9 expressions. Therefore, these findings provide a useful guideline for the migration mechanism of oral SCC depend on PTTG1 expression.

Cancer cells are often found in an ischemic condition due to the rapid outgrowth of their vascular supply, and these cells are expected to develop an increased potential for local invasive growth. Since the first steps are characterized by increased motility and invasiveness, expression of molecules involved in cellular adhesion to extracellular matrix (ECM) is increased in the process of cancer cell invasion and metastasis. In this work we explored the molecular characteristics and its regulatory mechanism of hypoxic oral squamous cell carcinoma (OSCC) cells. Our experiment identified that hypoxia increases α5 integrin protein levels through phosphoinositide 3-kinase (PI3K)/Akt pathway in OSCC cells.

The purpose of this study was to evaluate the role of integrin α3 and integrin β1 in the ameloblastomas. For this study, 10 specimens diagnosed as amoblastomas referred to the Department of Oral Pathology, School of Dentistry, Kyung Hee University, and 5 specimens of normal oral mucosa without any inflammatory changes were used as experimental and control groups, respectively. The ameloblastomas devided into follicular type, plexiform type, acanthomatous type, and granular cell type. All specimens; experimental and control group were fixed in 10% neutral formalin solution and embedded in paraffin, and then the serial tissue sections were made 5㎛ in thickness and processed for immunohistochemical observation. The specimens were incubated with primary antibody against integrin α3 or integrin β1, each was diluted at 1 : 100, followed by the Supersensitive non-biotin horse radish peroxidase detection system with DAB as chromogen. After counterstaining with Gill's hematoxylin stain method and mounted, and examined under the light microscope. Based on the intensity of the immunoreactivity, intensity of the immunity was scored no epithelial stain, weak or focal epithelial stain, moderate or focal intensive epithelial stain, intense generalized epithelial staining for the epithelial, and connective tissue component in ameloblastomas, and normal oral mucosa on each. Attained results as follows. Expression of integrin α3 in the oral mucosa, weak reaction was noted on the all layers of epithelium, and submucosa. Expression of integrin β1 in the oral mucosa, intense reaction on the superficial layer, moderate reaction in basal layer were shown. Expression of integrin α3 in ameloblastomas, it was noted that weak reaction on the ameloblast like cells in the all types and rarely in basement membrane. Expression of integrin β1 in ameloblastomas, intense reaction on the tumor cell ,and partly in the nuclei in follicular type was noted, And moderate reaction on the tumor cell in plexiform , acathomatous types, but weak reaction in granular cell type was shown. This results result suggest that integrin α3 may influenced negligibly, but the integrin β1 influenced significantly the development of the ameloblastomas considering the response is increased on the region with highly cellular activities

The purpose of this study was to evaJ uate the role of integrin a 3 and integrin ß 1 expression in the saivary gJand tumors. For this study, 11 specimens diagnosed as pleomorpic adenoma, adenoid cystic carcinoma, adenocarcinoma, mucoe pidermoid carcimoma referred to the Dept. of Oral Pathology‘ School of Dentistry, Kyung Hee University, 2 specimens 01' normaJ submandibular gland tissues were used as experimental, control groups respectively, All the tissues experimental and control group wel'e fixed in neutral formaJin solution and embedded in paraffin, seriaJ tissue section were made 511m in thickness and processed in the standard way for immunohistochemical method, using primary antibody against integrin a 3, and integrin ß 1 each was diluted at 1;100 followed by the poly- horse radish peroxidase detection system with DAB as chormogen counterstained with Mayel ’s hematoxylin stain method and mounted And examined unde1' the biologic micro scope with the criteria of no epitheliaJ stain, weak 01' focal epithelial stain, moderate 01' focal intensive epithelial s tain. intense generalized epithelial staining for the epithelial, and connective tissue components in no1'mal salivary gland, and saivary g land tumors : pleomorphic adenoma‘ adenoid cystic carcinoma, adenoca1'cinoma, mucoepide1'moid ca1'cinoma on each On the integ1'in α 3 reaction, negative to minimal posit ive reaction was noted on the salivary gland twnors and nor mal subma ndibular gland tlssues On the integrin ß 1 reactions, intense 1'eaction is shown on the serous demilune and ductal cells , and partly on the serous acini in submandibula1' gland tlssues On the integrin ß 1 reactions to pleomorphic adenoma tissues, moderate reactions were noted on the ductal celJs and myoepithelial cells. On the integrin ß 1 reactions to adenoid cystic ca rci noma‘ adenocarcinoma, mucoepidermoid ca1'cinoma tissues, intense reactions were shown on the neo plastic cell s , This resuJt suggest that integrin a 3. integrin ß 1 could be a 1'ole inducing the tumorigenesis.

The purpose of this study was to evaluate the role 0 1' integrin a 3 and integrin ß 1 in the oral squamous cell ca rcinomas. For this study‘ 10 specimens diagnosed as squamous cell carcinoma referred to the Dept. of Oral Pathology. School of Dentis try, Kyung Hee Univers ity, and 5 specimens of normal oral mucosa without any inflammatory cha nges were used as experimenta l and co nt rol groups, respectively. AlI s pecimens; experirnental and control group were f ixed in neutral f ormalin so lu tion and embedded in paraffin, and then the serial tissue section were rnade 5i1m in thickness and processed for imrnunohi stochemical observatlon The specimens were incubated with prirnary antibody against integrin a 3 r integrin ß 1‘ each was diluted at 1;100, followed by the super sensit ive non- biotin horse r adish peroxidase detection sys tem with DAB as chromogen‘ After counters ta ining with Gill ’s hematoxylin stain method and mounted and examined under the light microscope. Based on the intens ity of the immunoreactivity, intensity of the immunity was scored no ep ithelial stain, weak 0 1' focal epitheli al sta in, modera te 0 1' focal intensive epithelial stain, intense genera lized epitheli al s taining for the e pithelia l, and co nnective ti ssue component in squamous cell carcinomas, and normal oral mucosa on each Expression of integrin a 3 in t he oral mucosa was negli gible. Expression 0 1' integrin a 3 in expression in the or al s mnus cell ca rcinoma was ve ry wea k, but the express ion was increased in poorly differ entiat ed type of the oral squamous cell carcinomas ln the oral mucosa , expression of in tegr in ß 1 ra nged from weak to moderate in the cytoplasm and the cell membra nes of the kera tini zed and basal cell layer. Nuclei were mainly integrin ß 1 negative‘ but rarely revealed weak expression. ln sq uamous cell carcinoma, expression of integrin ß 1 was ntense notably in the cytoplasm, cell membrane a nd nuclear membra ne Nuclei of several tumor cells revealed moderate expression of integrin ß 1. Expression of integrin ß 1 was increased the poorly diffe rentiated type of in squamous cell carcinoma compare to that in moderate or well diffe rentiated type of oral squamous cell carCllìoma These results suggest integrin a 3 and integrin ß 1 may be influ enced the development and growth of the squamous cell carcima .

Cell behavior of the transformed cells is known to affect by interaction with extracellular matrix (ECM) proteins and integrin. To investigate the alterations of both integrin expression and cell-matrix interaction during neoplastic conversion of human oral kerationcytes, we studied expression levels of integrin subunits by flow cytometry and cellular responses to the ECM proteins in normal human oral keratinocytes (NHOKs), HPV-immortalized HOK-16B line, and three oral cancer cell lines established from HOK-16B line, CTHOK-16B-BaP, CTHOK-16B-DMBA, and CTHOK-16B-Dexa lines. The expression levels of α and β integrin subunits were shown decreased tendency in human oral keratinocytes undergoing immortalization and tumorigenic transformation except CTHOK-16B-DMBA line tested. Although αuβ6 integrin is known to be highly expressed in squamous cell carcinomas, and the altered integrin expression is suspected to be associated with cellular carcinogenesis, αu integrin subunit and αuβ6 egrin did not express in oral cancer cell lines tested. Cell behavior to the ECM proteins in HOK-16B line was generally similar to that of exponentially proliferating NHOKs. The adhesion activity profiles of type I collagen were very similar to that of its laminin counterparts, but fibronectin showed minimal adhesion activity under our conditions compared to the BSA control. The ability of the CTHOK-16B-BaP line to spread upon type I collagen and laminin markedly decreased, but migration was notably increased on type I collagen. In contrast, CTHOK-16B-DMBA and CTHOK-16B-Dexa lines spread less but migrated more upon type I collagen than immortalized HOK-16B line. These data indicate that downregulation of integrin subunits causes the changes of cellular responses to the ECM proteins during neoplastic conversion of human oral keratinocytes, and that cellular responses to the ECM proteins in oral cancer cell lines established by exposing different carcinogens are variable according to chemical carcinogens treatment.

Alterations in cell surface receptors and adhesion molecules which regulate cell-cell and cell-matrix interactions have been 뻐plicated in tumor processes. In order to investigate the effect of integrin expression on the invasiveness of oral sqllamous cell carcinoma, integ띠1 expression in the celllines such as SCC-4, SCC-9, SCC-15, and SCC-25 was analyzed, and the comparison between cell adhesion assay towards extracellular matrix proteins and in vitro invasion assay following inhibition of the functional domain of the integrins using blocking antibodies against the specific integrins 낀 nd Arg-Gly-Asp (RGD) peptide were carried out. The expression of integrin a 2, a 3, a 6 was detected in all oral squamous cell carcinoma celllines. In contrast, the expression of a vß6 integrin is detected in SCC-4 and SCC-9, not in SCC-1 5 and SCC-25. 까1e adhesion of SCC-4 cell line to collagen 1, laminin, and fibronectin was significantly reducecl by σeatment with a 3-, a 6-, and a vß6-blocking antibody, respectively (p (0.05). 꺼.1e invasion of SCC-4 cell line throllgh Matrigel was significantly reduced by treatment with v 6-blocking antibody and RGD pepticle (p(0.05). These results sllggested that specifìc integrins play an in1portant role in the process of adhesion and invasion of oral squamous cell carcinoma cells and the expression of a vß6 integrin is believed to the enhance its invasivene잃.

Heparin-bindin epidermal growth factor (HB-EGF) is one of the EGF family to be expressed at the time of implantation in the mouse uterus. Although HB-EGF has been shown to stimulate the development of embryo and uterus in the mouse, its correlation between cell adhesion molecules remains undefined. Integrin , one of the cell adhesion molecules, is an important mediator of cell-substratum and cell-cell adhesion in implantation. In the present studies, we investigated the effects of HB-EGF on the embryonic development, initiation of implantation and expression of integrin in in vitro culture, blocking of HB-EGF, RT-PCR and immunofluores cence analysis. The results showed that HB-EGF significantly improved the developmental rate of hatched embryos (24.1%, p<0.01) and outgrowth embryos (42.5%, p<0.01). On the other hand, this growth factor showed no offset before the hatching embryonic stage. Analysis of RT-PCR showed that HB-EGF upregulated the expression level of integrina subunit genes on the preimplantation embryo and outgrowth of blastocyst (120hr and 144hr after hCG injection). Immunofluorescence analysis showed that the integrin subunits localized at the pericellular borders and cell-cell contact areas. Increase in fluorescence intensity was observed in the HB-EGF treated embryos. Intrauterine injection of an anti-HB-EGF antiserum at day 3 significantly decreased the number of implantation sites (14.4, p<0.01) and significantly increased the number of recovered embryos(6.4, p<0.05) at day 5. From these results, it imply that HB-EGF improve the embryo development and accelerated the expression of integrin in the preimplantation mouse embryos.