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At least 649 records · Page 36Linked to original sources
PubMed2005

Protein kinase D1 and the beta 1 integrin cytoplasmic domain control beta 1 integrin function via regulation of Rap1 activation.

The functional activity of integrins is dynamically regulated by T cell receptor stimulation and by protein kinase C (PKC). We report a novel function for the PKC effector protein kinase D1 (PKD1) in integrin activation. Constitutively active and kinase-inactive PKD1 mutants lacking the PKD1 pleckstrin homology (PH) domain block phorbol ester- and TCR-mediated activation and clustering of beta1 integrins. The PH domain of PKD1 mediates the association of PKD1 with the GTPase Rap1 and is central to Rap1 activation and membrane translocation in T cells. Furthermore, PKD1 and Rap1 associate with beta1 integrins in a manner that is dependent on the carboxy-terminal end of the beta1 integrin subunit cytoplasmic domain. beta1 integrin expression is required for Rap1 activation and membrane localization of the PKD1-Rap1 complex. Therefore, PKD1 promotes integrin activation in T cells by regulating Rap1 activation and membrane translocation via interactions with the beta1 integrin subunit cytoplasmic domain.

Cell Adhesion↗
PubMed2003

Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.

Adenoviridae↗
PubMed2005

[Cell differentiation during carcinogenesis of cervical epithelia].

BACKGROUND & OBJECTIVE: The disturbance of cell differentiation is an important characteristic of tumor, but, up to date, the researches on this aspect are not as deep as those on cell proliferation of tumor. This study was to detect the alterations of cell differentiation and relative regulating factors during the carcinogenesis of cervical epithelia. METHODS: The protein expression of keratin (KT) 10, 14, 19 and beta1-integrin and beta-catenin in 20 specimens of normal squamous epithelium (NSE), 19 specimens of dysplasia squamous epithelial hyperplasia (DSEH), 39 specimens of squamous carcinoma in situ (SCIS), and 20 specimens of invasive squamous epithelial carcinoma (ISEC) were detected by inmmunohistochemistry; the mRNA expression of of beta1-integrin and beta-catenin were detected by in situ hybridization. RESULTS: The strong positive rates of KT10 in NSE, DSEH, SCIS, and ISEC were 85.0%, 52.6%, 18.0% and 0, respectively, with a descending trend; it was significantly lower in SCIS and ISEC than in NSE and DSEH (Chi(2)=11.28, P<0.05; Chi(2)=8.53, P<0.05). The expression of KT14 and KT19 showed a heterogeneity in SCIS and ISEC: the proteins were negative in some cases, but overexpressed in other cases with positive cells moved upwards from the basal layer; positive cells scattered at the full epithelial layer of SCIS. The expression of beta1-integrin also showed a descending trend in NSE, DSEH, SCIS, and ISEC. The positive rate of beta1-integrin was significantly lower in ISEC than in NSE and DSEH (Chi(2)=7.62, P <0.05; value of exact probability=0.014, P < 0.05); the mRNA expression of beta1-integrin showed the same trend as its protein expression in the samples although the difference were not significant. The protein and mRNA expression of beta-catenin was located in nuclei or cytoplasm of SCIS and ISEC; increased positive cells moved upwards from the basal layer. CONCLUSIONS: Restraint of terminal differentiation of epithelial cells may relate to the abnormal expression of beta1-integrin and the dysfunction of Wnt signaling pathway on regulating cell differentiation during the carcinogenesis of cervical epithelia. The expression of KT10 and beta1-integrin in SCIS is not obviously different from that in ISEC.

Carcinoma in Situ↗
PubMed1999

beta1-chain integrins are not essential for intimin-mediated host cell attachment and enteropathogenic Escherichia coli-induced actin condensation.

Intimin is a bacterial outer membrane protein required for intimate attachment of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) to mammalian cells. beta1-chain integrins have been proposed as candidate receptors for intimin. We found that binding of mammalian cells to immobilized intimin was not detectable unless mammalian cells were preinfected with EPEC or EHEC. beta1-chain integrin antagonists or inactivation of the gene encoding the beta1-chain did not affect binding of preinfected mammalian cells to intimin or the actin condensation associated with the attachment of EPEC. The results indicate that beta1-chain integrins are not essential for intimin-mediated cell attachment or EPEC-mediated actin polymerization.

Actins↗
PubMed2000

Leukocyte integrin expression in patients undergoing cardiopulmonary bypass.

BACKGROUND: The recruitment of leukocytes to vascular endothelium is controlled by adhesion events mediated through the beta2 integrins, whereas the response of extravasated leukocytes within the tissues is controlled through the beta1 integrins. Although cardiopulmonary bypass (CPB) has been shown to be associated with a systemic inflammatory response and elevated levels of beta2 integrins on leukocytes, its effect on the beta1 integrins is not known. This study investigated the effect of the protease inhibitor aprotinin on the expression of the beta1 and beta2 integrins on circulating leukocytes in patients undergoing CPB. METHODS: Patients undergoing primary elective coronary artery bypass grafting were randomized into full-dose aprotinin or placebo groups. Blood samples were obtained at nine time points preoperatively, intraoperatively, and up to 6 days postoperatively. The surface expression of the beta1 integrins VLA-1, -3, -4, -5, and -6 and of the beta2 integrins CD11a/CD18, CD11b/CD18, and CD11c/CD18 was measured by flow cytometry on gated neutrophil and monocyte subpopulations in whole blood. RESULTS: Expression of the beta1 integrins was not significantly altered during the study period and, therefore, aprotinin had no effect on the expression of these molecules. Of the beta2 integrins, CD11b/CD18 expression was significantly increased on neutrophils at 15 minutes after onset of CPB in the placebo group (p < 0.01) but not in the aprotinin group. CONCLUSIONS: This study showed that expression of the beta1 integrins on neutrophils and monocytes did not alter during the first 6 days after CPB. Expression of the beta2 integrin CD11b/CD18 increased significantly on neutrophils during CPB in control patients but not in patients treated with full-dose aprotinin.

Aprotinin↗
PubMed1998

Prolidase in human breast cancer MCF-7 cells.

Prolidase (EC 3.4.13.9) is an ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. We have shown previously that prolidase activity in normal human skin fibroblasts is regulated by the interaction of type I collagen with beta1 integrin receptor. In the present study, we investigate prolidase activity in MCF-7 cells and find it is only one-third of that in normal human skin fibroblasts. The relative difference in prolidase activity is corroborated by enzyme protein with Western immunoblot analysis. We propose that the decrease in prolidase activity is due to derangement of regulation by the collagen-beta1 integrin receptor axis. Supporting evidence comes from the following observations: (1) relative collagen content elaborated by MCF-7 cells as compared to fibroblasts is lower by 30% in sparse cells and by 80% at confluence; (2) collagenase treatment of both cell types results in decreased enzyme activity; (3) in contrast to fibroblasts, prolidase activity in MCF-7 cells is not stimulated by the addition of type I collagen or beta1 integrin antibodies (agonist for beta1 integrin receptor); and (4) in contrast to fibroblasts, MCF-7 cells express only trace amounts of beta1 integrin receptor as shown by Western immunoblot analysis. Thus, we conclude that depressed prolidase activity in MCF-7 cells may be a result of disturbances in signaling mediated by beta1 integrin-collagen interaction.

Antibodies↗
PubMed1998

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion.

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.

Antibodies, Monoclonal↗
PubMed2001

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

Acetylcholine↗
PubMed2005

Matrix-protein-specific regulation of Cx43 expression in cardiac myocytes subjected to mechanical load.

To elucidate mechanisms responsible for mechanotransduction in the heart and define the effects of remodeling of the extracellular matrix, we cultured neonatal rat ventricular myocytes on native type I collagen, fibronectin, or denatured collagen and subjected them to uniaxial, pulsatile stretch. Changes in expression of the cardiac gap junction protein, Cx43, were measured by confocal microscopy and immunoblotting. Cells grown on fibronectin or denatured collagen exhibited significantly greater Cx43 expression than cells grown on native collagen. Stretch induced a approximately 2-fold increase in Cx43 expression in cells grown on native collagen but no increase in cells grown on fibronectin or denatured collagen. Incubation of cells on native collagen with a peptide containing the arginine-glycine-aspartate (RGD) motif upregulated Cx43 expression equivalent to that induced by stretch. Nonselective activation of integrin signaling with MnCl2 also upregulated Cx43 expression in cells grown on native collagen. This effect was blocked completely by pretreatment with anti-beta1 integrin antibody but not by anti-beta3 integrin antibody. Stretch led to a marked increase in beta1 integrin immunofluorescent signal in cells grown on native collagen but not in cells grown on fibronectin or denatured collagen. Stretch-induced upregulation of Cx43 was also blocked by anti-beta1 integrin antibody. Thus, matrix protein-myocyte interactions regulate Cx43 expression via beta1 integrin signaling initiated by mechanical stimulation in cells grown on native type I collagen, or by RGD-integrin signaling independent of mechanical stress in cells grown on fibronectin or denatured collagen. Changes in the composition of the extracellular matrix may affect electrical coupling in cardiac myocytes.

Animals↗
PubMed2004

In vitro exposure to paclitaxel modulates integrin expression by human T lymphocytes and inhibits T cell adhesion to breast carcinoma cells.

Paclitaxel (Taxol) is a drug that is commonly used in the treatment of metastatic breast cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel on the subsequent ability of human Jurkat T lymphocytes to adhere to monolayers of MDA-MB-435 human breast carcinoma cells. Jurkat T cells that survived culture for 24 h in the presence of paclitaxel (0.1 or 3 micro g/ml) exhibited a diminished ability to adhere to MDA-MB-435 breast cancer cell monolayers. Flow cytometric analysis of paclitaxel-treated Jurkat T cells revealed reduced surface expression of alphaL, alpha4, alpha5, and beta7 integrins, but normal beta1 integrin expression. These data suggest that paclitaxel interferes with the expression of alphaLbeta2 (LFA-1), alpha4beta1 (VLA-4), alpha5beta1 (VLA-5), and alpha4beta7 (LPAM-1) adhesion molecules by surviving T cells. Blocking experiments with antibodies against alphaL, alpha4, alpha5, beta1, and beta7 integrins revealed that adhesion of Jurkat T cells to MDA-MB-435 breast cancer cell monolayers was dependent upon alphaL, alpha4, and beta7 but not alpha5 or beta1 integrins. We conclude that prior exposure to paclitaxel caused a reduction in Jurkat T cell adhesion to MDA-MB-435 breast carcinoma cells by preventing optimal alphaLbeta2 and alpha4beta7 interactions with their corresponding ligands on tumor cells. A similar effect by paclitaxel on T lymphocytes of breast cancer patients may compromise cell-mediated anti-tumor immune responses.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes.

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in beta-adrenergic receptor (beta-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). beta-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed beta-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited beta-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with beta1-integrins after beta-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of beta1-integrin signaling using laminin inhibited the increased association of MMP-2 with beta1-integrins. beta-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) beta-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits beta-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with beta1 integrins and poly-ADP-ribose-polymerase cleavage.

Adrenergic beta-Agonists↗
PubMed2000

Alpha4beta1-integrin activation is necessary for high-efficiency T-cell subset interactions with VCAM-1 under flow.

OBJECTIVE: The purpose of this study was to examine the relationship between alpha4beta1-integrin state of activation on CD4+ T-cell subsets and their adhesive interaction to VCAM-1 under flow. METHODS: Human CD4+ memory and naive T-cells were freshly isolated and effector-helper T-cell subsets. Th1 and Th2 cells, were differentiated in vitro from CD4+ naive T-cells. The expression of activation/ligand induced epitopes on beta1-integrins of each T-cell subset was assessed using mAb HUTS21 and mAb 15/7. T-cell subsets attachment and rolling on VCAM-1 was determined under defined flow conditions and the rates of attachment (ka), accumulation, and instantaneous rolling velocities were correlated to their beta1-integrin activation epitope expression. RESULTS: A subset of memory T-cells constitutively express activation/ligand induced epitopes on beta1-integrins recognized by mAb HUTS21 and 15/7, whereas expression levels on naive T-cells is low or not detectable. Consistent with an activated phenotype, memory T-cells exhibit significantly higher rates of attachment and accumulation on VCAM-1 under flow as compared to naive T-cells. Interestingly, the expression of activation/ligand induced epitopes on beta1-integrins on Th2 cells and the ability of these cells to interact with VCAM-1 are comparable to memory T-cells. In contrast, Th1 cells did not interact as efficiently with VCAM-1, which correlated with lower expression of activation/ligand induced epitopes on these cells. VCAM-1 interactions are inhibited completely by pretreatment of the T-cells with blocking mAb to alpha4-integrins or beta1-integrins, indicating that alpha4beta1 is the predominant T-cell integrin involved. CONCLUSIONS: Memory T-cells express constitutively active alpha4beta1-integrins, as compared to naive T-cells, which mediate high rates of initial attachment and sustained high-affinity adhesive interactions with VCAM-1 under flow conditions in vitro. Similarly, in vitro differentiated Th2 cells but not Th1 cells, which also express elevated levels of activated alpha4beta1-integrins, are capable of sustaining high-affinity adhesive interactions with VCAM-1. The differences observed in beta1-integrin activation on T-cell subsets may underlie selective recruitment patterns of T-cell subsets in vivo.

Cell Adhesion↗
PubMed1999

Molecular prognostic markers in intermediate-thickness cutaneous malignant melanoma.

BACKGROUND: The limitations of morphologic criteria alone in determining the prognosis for a patient with a particular intermediate-thickness primary melanoma have prompted efforts to identify other markers. METHODS: In this study, the authors analyzed expression of p53, beta1 integrin, and beta3 integrin in primary tumors from 111 patients with intermediate-thickness malignant melanoma. RESULTS: Eighty-nine (80%) had detectable p53 protein, 58 (52%) expressed beta1 integrin, and 71 (64%) expressed beta3 integrin. Patients with beta3 positive melanomas were more likely to die of their disease (32 of 71 patients, 45%) than those with beta3 negative tumors (3 of 40 patients, 8%) (P < 0.0001). The number of involved lymph nodes, Clark's level, beta1 integrin expression, thickness, and mitotic rate also had prognostic significance. beta3 integrin was associated with subsequent lung metastases and beta1 integrin with lymph node involvement. CONCLUSIONS: Integrin expression, along with histopathologic criteria, is a prognostic marker for intermediate-thickness malignant melanoma and may indicate the site of subsequent metastasis. These observations may have clinical utility and suggest areas for future investigation.

Antigens, CD↗
PubMed2002

The neural cell adhesion molecule L1 potentiates integrin-dependent cell migration to extracellular matrix proteins.

The L1 adhesion molecule regulates axon growth and is mutated in the X-linked mental retardation syndrome CRASH (acronym for corpus callosum agenesis, retardation, aphasia, spastic paraplegia, hydrocephalus). A novel role for L1 as a potentiator of neuronal cell migration to extracellular matrix proteins through beta1 integrins and intracellular signaling to mitogen-activated protein (MAP) kinase was identified. L1 potentiated haptotactic migration of B35 neuroblastoma cells toward fibronectin, vitronectin, and laminin through the signaling intermediates c-Src, phosphatidylinositol-3 kinase, and MAP kinase. L1 potentiated migration toward fibronectin through alpha5beta1 integrin in human embryonic kidney 293 cells and depended on determinants of L1 endocytosis: dynamin I, c-Src, and the AP2/clathrin binding site (Arg-Ser-Leu-Glu) in the neuronal splice form of L1. L1 clustering on the cell surface enhanced the internalization of activated beta1 integrins and L1 into distinct endocytic vesicles. L1-potentiated migration, enhancement of beta1 integrin endocytosis, and activation of MAP kinase were coordinately inhibited by mutation of an RGD sequence in the sixth immunoglobulin-like domain of L1. Moreover, three CRASH mutations in the L1 cytoplasmic domain (1194L, S1224L, Y1229H), two of which interfere with ankyrin association, inhibited L1-potentiated migration and MAP kinase activation. Function-blocking antibodies to L1 and beta1 integrin retarded the migration of 5-bromo-2'-deoxyuridine-labeled mouse cerebellar granule cells in slice cultures, underscoring the potential physiological relevance of these findings. These studies suggest that L1 functionally interacts with beta1 integrins to potentiate neuronal migration toward extracellular matrix proteins through endocytosis and MAP kinase signaling, and that impairment of this function by L1 cytoplasmic domain mutations may contribute to neurological deficits in CRASH.

Animals↗
PubMed2002

Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers.

Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.

Cell Adhesion↗
PubMed2005

Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation.

BACKGROUND: Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1alpha and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion. RESULTS: In immunoblotting analysis, all three pancreatic cancer cell lines (AsPC-1, BxPC-3, and Capan-2) expressed the protein of FAK and beta1 integrin. Enhancement of FAK protein association with beta1 integrin when cells were plated on Coll IV was more increased by stimulation with IL-1alpha. Preincubation with anti-beta1 integrin antibody and FAK siRNA transfection inhibited the association of FAK with beta1 integrin of pancreatic cancer cells. FAK phosphorylation was observed by adhesion to Coll IV, furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1alpha of pancreatic cancer cells adhered to Coll IV in time-dependent manner. Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation. IL-1alpha stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in pancreatic cancer cells. Activation of Ras correlated with the phosphorylation of ERK. While not statistical affecting the apoptosis of pancreatic cancer cells, IL-1alpha-induced adhesion and invasion on Coll IV were inhibited with FAK gene silencing by siRNA, beta1 integrin blocking, and inhibition of FAK phosphorylation. PD98059, a MEK inhibitor, also inhibited IL-1alpha-induced enhancement of adhesion and invasion in pancreatic cancer cells. CONCLUSION: Our results demonstrated that activation of FAK is involved with the aggressive capability in pancreatic cancer through Ras/ERK signaling pathway. Based on our results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer.

Apoptosis↗
PubMed2001

Lysophosphatidic acid promotes matrix metalloproteinase (MMP) activation and MMP-dependent invasion in ovarian cancer cells.

Ovarian cancer is an highly metastatic disease characterized by ascites formation and diffuse i.p. adhesion, invasion, and metastasis. Levels of lysophosphatidic acid (LPA) are elevated in the plasma of patients with ovarian carcinoma, including 90% of patients with stage I disease, suggesting that LPA may promote early events in ovarian carcinoma dissemination. Expression of matrix metalloproteinases (MMPs) is also up-regulated in ovarian cancer tissues and ascites, and numerous studies have provided evidence for a direct role of MMPs in i.p. invasion and metastasis. Using three-dimensional type I collagen cultures or immobilized beta1 integrin subunit-specific antibodies, we previously demonstrated that beta1 integrin clustering promotes activation of proMMP-2 and processing of membrane type 1 MMP in ovarian cancer cells (S. M. Ellerbroek et al., Cancer Res., 59: 1635-1641, 1999). In the current study, the effect of LPA on MMP expression and invasive activity was investigated. Treatment of ovarian cancer cells with pathophysiological levels of LPA increased cellular adhesion to type I collagen and beta1 integrin expression. A significant up-regulation of MMP-dependent proMMP-2 activation was observed in LPA-treated cells, leading to enhanced pericellular MMP activity. As a result of increased MMP activity, haptotactic and chemotactic motility, in vitro wound closure, and invasion of a synthetic basement membrane were enhanced. These data indicate that LPA contributes to metastatic dissemination of ovarian cancer cells via up-regulation of MMP activity and subsequent downstream changes in MMP-dependent migratory and invasive behavior.

Cell Adhesion↗
PubMed2004

Inhibition of alpha5 integrin decreases PI3K activation and cell adhesion of human colon cancers.

BACKGROUND: Phosphatidylinositol 3-kinase (PI3K), acting through the downstream kinase AKT, regulates a number of cellular processes such as cell growth and survival. Poorly differentiated colon cancers are characterized by increased integrin-mediated extracellular matrix interactions; disruption of this integrin-mediated adhesion leads to apoptosis and involves reduced PI3K activity. The purpose of this study was to determine: (1) the role of alpha5beta1 integrin expression on PI3K activity, and (2) whether blockade of alpha5beta1 integrin decreases colon cancer cell adhesion and increases apoptosis. METHODS: Human colon cancer cells were treated with anti-alpha5 integrin antibody, anti-beta1 integrin antibody, the PI3K inhibitor LY294002 (20 micromol/L), or wortmannin (400 nmol/L). Expression and function of alpha5 were quantitated by FACS analysis and cell adhesion assay, respectively. DNA fragmentation was measured to assess apoptosis. Protein was extracted to determine phosphorylated AKT (pAKT) expression as a measure of PI3K activity. RESULTS: Increased pAKT and alpha5 integrin expression were noted in the colon cancer cells; blockade of alpha5 integrin decreased alpha5 integrin and pAKT expression and decreased cell adhesion. Moreover, PI3K inhibition resulted in decreased alpha5 integrin expression. In contrast, treatment with anti-beta1 integrin antibody produced no change in pAKT expression or cell number. CONCLUSION: Our results are the first to show that blockade of cell surface alpha5 integrin expression decreases PI3K activity, inhibits colon cancer cell attachment, and induces apoptosis. These findings suggest that agents which selectively target alpha5 integrin subunit expression may enhance the effects of standard chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.

Apoptosis↗