Biomedical subjects

R Falcioni

Publications and source records attributed to R Falcioni.

At least 19 recordsLinked to original sources

PubMed2000

Cooperative signaling between alpha(6)beta(4) integrin and ErbB-2 receptor is required to promote phosphatidylinositol 3-kinase-dependent invasion.

We previously demonstrated that beta(4) integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large beta(4) cytoplasmic domain that are involved in the interaction of alpha(6)beta(4) with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of beta(4) that lacked either all or portions of the beta(4) cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of beta(4) was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of alpha(6)beta(4) to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the beta(4) cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of alpha(6)beta(4) integrin to increase invasion. Interestingly, the extracellular domain of beta(4) is not necessary for alpha(6)beta(4) to stimulate invasion. The association of alpha(6)beta(4) with ErbB-2 is dependent upon the beta(4) cytoplasmic domain and can occur in the absence of alpha(6)beta(4) heterodimerization. Finally, we observed strong activation of PI3K with beta(4) wild type and with those beta(4) deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between alpha(6)beta(4) and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the beta(4) cytoplasmic domain (amino acids 854-1183) in this event.

3T3 Cells↗

PubMed1999

p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB.

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

Antigens, Neoplasm↗

PubMed1999

Activation of p53 function in carcinoma cells by the alpha6beta4 integrin.

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the alpha6beta4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of alpha6beta4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that alpha6beta4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these alpha6beta4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of alpha6beta4 with a beta4 integrin-specific antibody promotes p53-dependent apoptosis in cells that express both alpha6beta4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of alpha6beta4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949-960), these studies suggest that the ability of alpha6beta4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.

Antigens, Surface↗

PubMed1998

N-methylformamide induces changes on adhesive properties and lung-colonizing potential of M14 melanoma cells.

We have studied whether N-methylformamide can affect the expression pattern of adhesion molecules and the attachment behaviour of M14 human melanoma cells. The role of N-methylformamide on experimental and spontaneous pulmonary metastases from M14 cells in nude mice was also investigated. We demonstrate that N-methylformamide in vitro pretreatment of M14 cells, although inducing a significant increase in the expression of alpha2beta1, alpha6beta1 and alpha(v)beta3 integrin receptors, slightly modifies alpha5beta1 heterodimer and beta1 subunit expression. After this modulation, enhancement of cell adhesion to laminin, collagen I, vitronectin and fibrinogen, which is blocked by specific anti-integrin antibodies, also occurs. No changes in binding to fibronectin are observed. In vitro N-methylformamide pretreatment also results in an increased number of experimental nodules and in a decrease in spontaneous metastases. Moreover, in vivo treatment with N-methylformamide significantly reduces the number of spontaneous metastases. Collectively, these data show that N-methylformamide modulates the expression of some adhesion receptors, cell adhesion to laminin, collagen I, vitronectin and fibrinogen as well as the metastatic behaviour of M14 cells. Our data also suggest that the effect of N-methylformamide might be evaluated in combination with antineoplastic agents for the treatment of human melanoma.

Animals↗

PubMed1997

Alpha 6 beta 4 and alpha 6 beta 1 integrins associate with ErbB-2 in human carcinoma cell lines.

Growth factors modulate integrin-mediated cell adhesion and motility, and their receptors are thought to share proteins that mediate intracellular signaling with integrin receptors. The crosstalk between these receptors is thought to play a relevant role in transformation and tumor progression. To highlight possible interactions between growth factors and cell adhesion receptors we investigated whether integrins associate with tyrosine kinase receptors in tumor cells. By affinity chromatography and Western blot analyses of purified immune complexes, we studied the association of laminin receptors (alpha 6 beta 1 and alpha 6 beta 4) with ErbB-2 tyrosine kinase in human carcinoma cell lines. We demonstrated that the alpha 6 beta 4 and alpha 6 beta 1 integrins coprecipitated with ErbB-2 in lysates from carcinoma or NIH3T3 cells overexpressing ErbB-2. Integrin-mediated activation of ErbB-2 receptors suggested that this association is functionally meaningful. Indeed, carcinoma cells treated with a monoclonal antibody to the alpha 6 integrin subunit showed a ligand-dependent increase of ErbB-2-phosphorylated molecules coprecipitated with integrins and an increased DNA synthesis. The interaction between growth factor receptors and integrins was also studied in NIH3T3 cells overexpressing alpha 6 beta 4 receptors and ErbB-2 protein. We report that cells overexpressing both receptors, but not those overexpressing a crippled ErbB-2, showed enhanced proliferation rates and invasiveness, further suggesting that alpha 6 beta 4 integrin and ErbB-2 receptor interaction might contribute to generate a more malignant phenotype in carcinoma cells.

3T3 Cells↗

PubMed1997

Beta 4 integrin subunit expression is downregulated in low metastatic carcinoma variants.

Organization and expression of the gene coding for beta 4 integrin subunit were investigated in murine carcinoma lines endowed with low and high metastatic potential maintained both in vivo and in vitro. Probes corresponding to either the extracellular or the cytoplasmic domains of the protein were generated and employed for Southern and Northern blot hybridization experiments. Southern blot analysis demonstrates a restriction fragment-length polymorphism between BALB/c and C57BI/6J DNAs. The different genomic organization of the beta 4 gene apparently does not generate variation in mRNA length. Northern blot analysis reveals a 7-kb transcript encoding full-length beta 4 protein and shorter transcripts that apparently correspond to nonfunctional mRNAs. In carcinoma cells endowed with low metastatic capacity, the 7-kb mRNA cannot be revealed in spite of the presence of shorter transcripts. In contrast, the 7-kb transcript is always present in tumor cells endowed with high metastatic capacity. Accordingly, the beta 4 protein is detectable, either by immunofluorescence or by Western blot analyses, only in highly metastatic carcinoma cells. In conclusion, the data presented show that in these murine carcinoma cell lines (i) the 7-kb mRNA is associated with a more invasive phenotype, and (ii) missing expression of the beta 4 integrin subunit in low metastatic carcinoma cells depends, at least in part, on mechanisms acting at a post-transcriptional level. The possibility that beta 4 subunit expression is a consequence of specific differentiation stages of lung carcinoma cells is discussed.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed1996

The beta 4 integrin subunit is expressed in mouse fibroblasts and modulated by transforming growth factor-beta 1.

Integrin beta 4 subunit is present in association with alpha 6 chain on both normal and transformed epithelial cells. Recently alpha 6 beta 4 heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that beta 4 subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of alpha 6 beta 4 has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor beta 1 (TGF-beta 1) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether alpha 6 beta 4 expression is modulated by TGF-beta 1, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-beta 1 cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of beta 4 subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-beta 1.

3T3 Cells↗

PubMed1994

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human lung carcinoma cells of different histotypes.

Structural and functional analyses of several integrin heterodimers were performed in non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines. The expression of beta 1, beta 3, beta 4, and beta 5 heterodimers was evaluated at protein and mRNA levels. By flow cytometry and immunoprecipitation experiments we demonstrate that NSCLC cells (A549 adenocarcinoma and DG 3 large cell carcinoma) coexpress integrin heterodimers composed of beta 1, beta 3, beta 4, and beta 5 subunits, whereas SCLC cells (AE2 and H69) express only beta 1 integrin heterodimers. Northern blot experiments confirmed immunochemical analysis: SCLC cells in contrast to NSCLC cells express only the mRNA coding for the beta 1 subunit. These data indicate that in lung carcinoma cells the diversity in the integrin repertoire depends upon differential gene expression. The functionality of integrin receptors has been studied using antibody blocking experiments. Data reported demonstrate that the alpha 6 beta 1 integrin is a laminin receptor in either SCLC or NSCLC cells. An antibody to the beta 4 subunit partially inhibits the adhesion of adenocarcinoma cells to lamin but does not block lamin adhesion of large cell carcinoma cells, even though alpha 6 beta 4 complexes are expressed on both cell types. Two antisera to vitronectin receptors inhibit the adhesion of NSCLC cels to both vitronectin and fibronectin. The same antisera inhibit the adhesion of SCLC cells only to laminin, indicating that the alpha v beta 1 integrin might function in these cells as laminin receptor.

Carcinoma, Non-Small-Cell Lung↗

PubMed1994

Retinoic acid negatively regulates beta 4 integrin expression and suppresses the malignant phenotype in a Lewis lung carcinoma cell line.

Retinoic acid (RA) is a potent inhibitor of the malignant phenotype and of tumour cell growth. We observed that in vitro RA treatment of a highly metastatic lung carcinoma cell line (C87) induced a marked reduction in the amount of the beta 4 integrin subunit. The downregulation of this adhesion molecule was assessed by immunofluorescence, immunoprecipitation, and northern analysis. In order to investigate the effects of RA on the malignant phenotype in C87 cells we performed morphological and functional analysis after RA treatment. We found that RA was able to produce marked changes in C87 cell shape, increasing the number of flat cells (90% of the total cell population), and significantly inhibiting the malignant and invasive phenotype of C87 cells. RA treatment suppressed their clonogenic potential in soft agar (control, 20 +/- 5; RA, 0), and strongly reduced their chemotactic and chemoinvasive capacity (chemotaxis: control, 231 +/- 5; RA, 28 +/- 0; chemoinvasion: control, 132 +/- 11; RA = 2 +/- 1). FACS analysis and cell count, however, indicated that RA reduced the growth of C87 cells only partially. After 72 h of treatment we observed only a 10% reduction in the S phase fraction of the cell population. Finally, the reduced lung colony-forming ability, observed after i.v. injection of RA-treated cells (lung foci/animal: RA-treated cells, 1 +/- 0.1; untreated, 8.5 +/- 0.8), further supports the conclusion that in this murine lung carcinoma cell line a marked reduction in the expression of the beta 4 integrin subunit is associated with a marked inhibition of the malignant phenotype.

Animals↗

PubMed1993

Integrin beta 4 expression in the neoplastic progression of cervical epithelium.

Integrin alpha 6 beta 4, a putative laminin receptor, is expressed in normal squamous epithelium and in squamous cell carcinomas. We evaluated by immunohistochemistry the topography of the beta 4 subunit in 35 biopsy samples representative of the sequence from normal exocervical epithelium to invasive squamous cell carcinoma. In normal and metaplastic squamous cervical epithelium, in condylomas, and in squamous epithelium with low-grade dysplasia (CIN 1), beta 4 was detected in basal and parabasal cells. In high-grade CIN (2-3) there was enhanced beta 4 expression and upward shift of beta 4 topography to the whole epithelial thickness. The beta 4 chain was diffusely expressed in most invasive squamous cell carcinomas. These observations suggest that profound alterations in the expression of the alpha 6 beta 4 adhesion glycoprotein occur in the intraepithelial phase of cervical carcinogenesis.

Antigens, Neoplasm↗

PubMed1991

Microdistribution of specific rat monoclonal antibodies to mouse tissues and human tumor xenografts.

Detailed evaluations of the microdistribution of 125I-labeled monoclonal antibodies (MoAbs) to normal tissue antigens were conducted in BALB/c mice. MoAb 273-34A, which binds to a target molecule on the lumenal surface of lung endothelial cells, localizes quickly and efficiently throughout the lung vasculature. MoAb 133-13A, which binds to an antigen on macrophage-like cells expressed in nearly equal amounts in lung, liver, and spleen, localizes most efficiently to spleen and less well to liver and lung. The microdistribution of MoAb 133-13A in liver and spleen is consistent with the antigen distribution in these organs, but in the lung a more diffuse microdistribution is observed, indicating poor access of MoAb to the antigen-positive alveolar macrophages. These findings are consistent with the hypothesis that tight endothelium (lung) represents a significant barrier to extravasation of MoAb into tissue while fenestrated (spleen) and sinusoidal (liver) endothelium are more easily penetrated. In human tumor bearing nu/nu mice, the microdistribution of MoAb to the beta 4 and alpha 6 subunits of integrin was studied. These MoAbs do not cross-react with murine integrins and thus are tumor-specific in the nu/nu mouse model. Localization of 125I-labeled MoAb 450-11A, which reacts with an intercellular domain of beta 4 integrin, is very weak and diffuse. All MoAbs to extracellular domains (mouse 450-9D, 450-30A1, and rat 439-9B) localize well to the tumor. Microdistribution of these MoAbs in the 3 different tumors is nonuniform with heavy distribution near the blood vessels, whereas antigen distribution as determined by immunoperoxidase shows a much more uniform pattern throughout the tumors. In experiments with 125I-labeled MoAb 439-9B F(ab')2, the nonuniform pattern of distribution was not changed. Gross and microdistribution of different doses of 125I-labeled MoAb 439-9B were studied. The percent of injected dose per g of MoAb in the tumor at 48 h did not vary significantly (P greater than 0.1) up to a dose of 500 micrograms/mouse, and active MoAb was recovered in comparable amounts in the serum from animals in all doses. In contrast, the microdistribution of MoAb at the high dose was different than that at low doses. At doses up to 100 micrograms/mouse, a perivascular pattern was obtained, whereas at 500 micrograms/mouse the 125I-labeled MoAb was distributed nearly evenly throughout the tumor. These data indicate that high doses of MoAb penetrate deeply into portions of the tumor that are distant from blood vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.

Animals↗

PubMed1990

Integrin (alpha 6/beta 4) expression in human lung cancer as monitored by specific monoclonal antibodies.

In this study, the expression of the alpha 6/beta 4 integrin complex was analyzed in human lung carcinomas both in vitro and in vivo, using two monoclonal antibodies which recognize the integrin subunits alpha 6 (Mab 135-13C) and beta 4 (Mab 439-9B). Immunoprecipitation patterns obtained from established human lung carcinoma cell lines demonstrated that the alpha 6 and the beta 4 subunits were differentially expressed in carcinomas of different types. The alpha 6 subunit was expressed in all the cell lines tested (squamous cell carcinoma A431, adenocarcinoma A549, large cell carcinoma DG3, and small cell carcinoma AE2). The beta 4 subunit was expressed in non-small cell cancer lines but was not detectable in the small cell cancer line tested. Using a quantitative two-site assay, we measured the concentration of the alpha 6/beta 4 integrin in matched biopsies from primary lung tumors and from normal lung. These studies confirmed that the complex was differentially expressed in non-small versus small cell lung cancers and that it was also detectable in lysates from normal lung at low levels. The highest levels of alpha 6/beta 4 were found in moderately differentiated squamous cell carcinomas. By immunohistochemistry, the beta 4 subunit was detectable in all the squamous cell carcinoma and adenocarcinomas tested (a total of 59), but not in 10 small cell cancers. The patterns of immunoreactivity were consistent with the expected distribution of membrane glycoproteins and, in some squamous cell carcinomas, were suggestive of the localization displayed by molecules involved in carcinoma-stroma interaction. Immunohistochemical staining indicated that beta 4 was also expressed in specific types of nonrespiratory pulmonary epithelial cells.

Adenocarcinoma↗

PubMed1990

Second generation monoclonal antibodies to the human integrin alpha 6 beta 4.

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.

Animals↗

PubMed1989

Analysis of the tumor-associated antigen TSP-180. Identity with alpha 6-beta 4 in the integrin superfamily.

The tumor-associated antigen complex, TSP-180, was previously defined in carcinoma cell lines and found to be expressed in higher amounts in tumor than in normal tissue. Here, the mouse TSP-180 complex is shown to consist of three related proteins (bands 1, 2, and 3) associated with a distinct protein (band 5) that is probably derived from a precursor protein (band 4). All of these proteins are cell surface glycoproteins, and the largest protein (band 1) can be readily labeled with 32PO4. The mouse TSP-180 complex described here strongly resembles the recently described human integrin alpha 6-beta 4 complex. This homology was confirmed using two distinct rat anti-alpha 6 monoclonal antibodies, each of which recognized both human alpha 6-beta 4 and mouse TSP-180 complexes. Furthermore, the TSP-180 band 5 protein (mouse alpha 6) had an N-terminal sequence identical to that of human alpha 6. Finally, two different monoclonal antibodies are described, 346-11A and 439-9B, which directly recognize the multiple forms of mouse and human beta 4 proteins, respectively.

Animals↗

PubMed1989

Ligand-induced phosphorylation of a murine tumor surface protein (TSP-180) associated with metastatic phenotype.

A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346-11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to Mr 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (Mr 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 32PO4, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyrosine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubilized immunocomplex, obtained from cell lysates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the Mr 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand-induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advantage of such tumor clones.

Amino Acids↗

PubMed1989

Insulin-induced phosphorylation of the beta-4 integrin subunit expressed on murine metastatic carcinoma cells.

A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.

Amino Acids↗

PubMed1988

Monoclonal antibody to human carcinoma-associated protein complex: quantitation in normal and tumor tissue.

We have identified and quantitated a tumor protein complex, TSP-180, on murine carcinomas with two monoclonal antibodies (MoAbs) (Cancer Res., 46: 707-712, 1986). One of the two MoAbs, 135-13C, recognizes a TSP-180-like protein complex on several human carcinomas in culture. MoAb 135-13C has been used to purify the human TSP-180 complex from A431 cells and the purified material used to immunize F344 rats to produce another MoAb, 439-9B, to the human TSP-180 complex. This MoAb does not precipitate the murine TSP-180 or bind to murine cells. Both MoAb 135-13C and 439-9B precipitated the same proteins from A431 cells but did not compete with each other for binding sites, indicating that they recognize different epitopes on the same protein. The two MoAbs have been used in a two-site assay to quantitate TSP-180 proteins on human cells and tissues. Carcinoma cell lines A431, SW948, and A549 all give high values (46 to 443 ng/mg of protein) while murine tumors, a human melanoma, and human fibroblasts are negative (less than 10 ng/mg of protein). Most tissues from autopsy of 2 normal individuals are negative for human TSP-180 at the levels tested (less than 10 ng/mg of protein). Some organs have intermediate range expression: spleen, 5 to 111 ng/ml of protein; colon, 24 to 111; and small intestine, 39 to 99. One primary colon and one larynx tumor were positive (144 to 372 ng/mg of protein) while 5 breast carcinomas, a stomach tumor, a metastatic melanoma, and a kidney tumor were negative. These data indicate that human TSP-180 may be preferentially expressed in certain malignant carcinomas of diverse origin. The potential for TSP-180 as a tumor marker requires further study.

Animals↗