Identification of laminin binding proteins in cell membranes of a human colon adenocarcinoma cell line (original) (raw)
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Expression of 32/67-kDa laminin receptor in laminin adhesion-selected human colon cancer cell lines
British Journal of Cancer, 1998
Laminin promotes the malignant phenotype, and the expression of certain laminin receptors is increased in malignancy. Previously, we demonstrated that a laminin-adhesive subolone of a human colon cancer cell line showed increased tumorigenicity in nude mice and increased affinity of the P, integrin for laminin relative to the laminin-non-adhesive subolone. The total amount of either 1 integrin protein or mRNA did not increase. As levels of the 32/67-kDa laminin receptor (67LR) correlate with malignancy, we examined 67LR expression in the laminin adhesion-selected human colon cancer cells. The laminin-adhesive subolone, which was more tumorigenic in both heterotopic and orthotopic locations than in a laminin-non-adhesive subolone, showed cell-surface membrane staining of 67LR, whereas the laminin-non-adhesive subolone showed cytoplasmic staining of 67LR. No difference in either the amount of 67LR mRNA or the amount of protein was observed in the parental cells than in the laminin-adhesive and non-adhesive subolones. When assayed on a laminin affinity column, more 67LR molecules bound to the column with cell extracts from the laminin-adhesive subolone than was observed with the nonadhesive subclone. These findings suggest that the increased tumorigenicity of laminin adhesion-selected tumour cells might be due to an alteration in the distribution and/or adhesiveness of multiple receptors including 67LR and P1 integrin.
Journal of Biological Chemistry, 1996
We investigated the effect of peptide G, a synthetic peptide derived from the sequence of the 37-kDa laminin receptor precursor, on the interaction of laminin in two tumor cell lines one of which produces laminin and one of which does not. Addition of peptide G to the culture medium induced a significant increase in the amount of endogenous laminin detectable on the cell membrane of both cell lines. Moreover, pretreatment of exogenous laminin with peptide G dramatically increased laminin binding on both cell lines. Kinetics analysis of membrane-bound labeled laminin revealed a 3-fold decrease in the k d of peptide G-treated laminin compared with untreated or unrelated or scrambled peptide-treated laminin. Moreover, the affinity constant of peptide G-treated laminin increased 2-fold, with a doubling of the number of laminin binding sites, as determined by Scatchard analysis. Expression of the VLA6 integrin receptor on the cell membrane increased after incubation with peptide G-treated laminin. However, the lower binding inhibition of peptide G-treated laminin after anti-VLA6 antibody or cation chelation treatment indicates that membrane molecules in addition to integrin receptors are involved in the recognition of peptide G-modified laminin. These "new" laminin-binding proteins also mediated cell adhesion to laminin, the first step in tumor invasion. Together, the data suggest that peptide G increases and stabilizes laminin binding on tumor cells, involving surface receptors that normally do not take part in this interaction. This might explain the abundant clinical and experimental data suggesting a key role for the 67-kDa laminin receptor in the interaction between cancer cells and the basement membrane glycoprotein laminin during tumor invasion and metastasis.
The Journal of Pathology, 2003
In human tissues, the laminin (Ln) α1 chain shows a restricted and developmentally regulated distribution in basement membranes (BMs) of a subset of epithelial tissues, including those of renal proximal convoluted tubules. The present study investigated the distribution of the Ln α1 chain in renal cell carcinomas (RCCs) and oncocytomas as well as in xenografted tumours induced in nude mice with four characterized RCC cell lines. These cell lines were also used in cell adhesion studies with purified laminins. By immunohistochemistry it was found that the Ln α1 chain is widely present in the BMs of RCCs, all of the specimens presenting immunoreactivity. High-grade RCCs tended to contain more BM-confined and stromal immunoreactivity than low-grade tumours, none of the grade 3 (G3) carcinomas being negative and all of the metastatic specimens showing partial or overall BM immunoreactivity. Double immunolabelling experiments showed that in RCC BMs but not in vessel walls, the Ln α1 chain was co-distributed with Ln α5, β1, and β2 chains, implying the presence of Ln-1/Ln-3 and Ln-10/Ln-11. In papillary RCCs, the Ln α1 chain co-localized with Ln-5. The oncocytomas lacked immunoreactivity for the Ln α1 chain. Xenografted tumours induced in nude mice showed BM-like deposition of the Ln α1 chain. In cell adhesion studies, mouse and human Ln-1 were equally effective in promoting cell adhesion of all RCC cell lines. For each cell line, Ln-10 and Ln-10/11 were equally effective adhesive substrates, all cell lines adhering more avidly to these laminins than to mouse or human Ln-1. As judged by inhibition assays employing specific integrin antibodies, adhesion of normal human renal proximal tubular epithelial (RPTE) cells and RCC cells from a G1 tumour to human Ln-1 was mediated mainly by α 6 β 1 integrin, while only the G1 RCC cells adhered to mouse Ln-1 by using α 6 β 1 integrin. For adhesion to Ln-10, RPTE cells and RCC cells from a G1 tumour used an unidentified β 1 integrin. Cells from G3 tumours mainly used an α 3 β 1 integrin complex for adhesion to mouse Ln-1 and to human Ln-1 and Ln-10. For all cells, adhesion to the Ln-10/11 mixture was mediated by an unidentified integrin complex or by other adhesion molecules. These results show that laminin trimers containing the α1 chain are, in contrast to oncocytomas, abundant in the BMs of RCCs. This is in keeping with their suggested origin from renal proximal tubular epithelium known for its capacity to produce the Ln α1 chain. The results also show that RCC cells utilize complex, mainly integrin α 3 β 1 -and integrin α 6 β 1 -mediated, mechanisms for adhesion to laminins. The adhesion to Ln-1 changes from integrin α 6 β 1 to integrin α 3 β 1 upon increasing malignancy and, especially for Ln-10 and Ln-10/11, other adhesion molecules of non-integrin type may contribute to the adhesion.
Role of laminin receptor in tumor cell migration
Cancer Research, 1987
Polyclonal antisera were made against biochemically purified laminili receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U. M. Wewer et al., Proc. Nati. Acad. Sci. USA, 83: 7137-7141, 1986). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A20S8 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized mela noma and carcinoma cells. The anti-laminin receptor antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A20S8 mela noma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on lam inin but not on fibronectin. Synthetic peptide GRGDS corresponding to the fibronectin cell-binding domain inhibited haptotaxis on fibronectin but not on laminin. Both types of anti-laminin receptor antisera inhibited haptotaxis on laminin but not on fibronectin. Using immunohistochemis-Iry, invading human carcinoma cells in vivo exhibited a marked cytoplasmic ¡mmunoreactivityfor the receptor antigen. Together these find ings indicate a specific role for the laminin receptor in laminin-mediated migration and that the ligand binding of the laminin receptor is encom passed in the COOH-terminal end of the protein.
European Journal of Biochemistry, 1989
The laminin proteolytic fragments 1 (derived from the intersection of the short arms of the cruciform laminin molecule) and 8 (derived from the laminin long arm) bind to distinct receptors on HT-1080 human fibrosarcoma cells; both fragments are shown here to inhibit the high-affinity binding of laminin to these cells. Inhibition of binding between fragment 8 and laminin was competitive, whereas that between fragment 1 and laminin was noncompetitive. This indicates that laminin and fragment 8 most probably share the same cellular receptors, whereas laminin and fragment 1 bind to distinct receptors, inhibition being due to steric hindrance. Surprisingly, fragment 1 -4 (corresponding to the complete short arms of laminin) neither bound to HT-1080 cells nor inhibited the binding of laminin or fragment 1. After treatment of fragment 1-4 with pepsin, however, the smaller subfragment 1 was liberated, which could then bind to the cells, and so was shown to block the binding of laminin and fragment 1. We conclude that native laminin bound to HT-1080 cells via the fragment-%binding site near the end of its long arm. Although these cells also have distinct receptors for the short arm fragment 1, this receptorbinding site was not used as it appeared to be latent within the native laminin molecule.
Differential expression of a laminin-Like substance by high- and low-Metastatic tumor cells
American Journal Of Pathology
High-metastatic murine fibrosarcoma cells readily attached to Type IV (basement membrane) collagen, whereas low-metastatic cells isolated from the same tumor did not. The addition of laminin--a glycoprotein that facilitates the adherence of epithelial cells to their basement membranes--enhanced the attachment of the low-metastatic cells, but not the high-metastatic cells. Using anti-laminin antibodies and a laminin-binding lectin as probes, the authors were able to identify by immunofluorescence a moiety associated with the high-metastatic cells, but not the low-metastatic cells, which cross-reacted with murine laminin purified from the EHS sarcoma. When extracts from the high-metastatic cells were separated by affinity chromatography, with the laminin-binding lectin as the affinity substrate, a substance was isolated that had an apparent molecular weight of 56,000 daltons. The affinity-purified material reacted strongly with anti-laminin antibodies by enzyme-linked immunosorbent as...
International Journal of Cancer, 2009
Laminin-332 (LN-332), which is essential for epithelial cell adhesion and migration, is up-regulated in most invasive carcinomas. Association between LN-332 and carcinoma cell integrins and stroma collagen is thought to be important for tumor growth and metastasis. Here, we show that function blocking LN-332 antibodies interfering with cellular adhesion and migration in vitro evoke apoptotic pathways. The antibodies also target epithelial tumors in vivo. Antibodies against the cell binding domain of the a3 chain of LN-332 inhibited tumor growth by up to 68%, and antibodies against the matrix binding domains of the b3 and c2 chains significantly decreased lung metastases. The LN-332 antibodies appear to induce tumor cell anoikis and subsequent programmed cell death and reduce migration by interfering with tumor cell matrix interactions. ' 2009 UICC Laminins are trimeric basement membrane (BM) glycoproteins with roles in cell adhesion, proliferation, migration and differentiation. In mammals, 5 genetically distinct a, 3b and 3g chains can form at least 14 different combinations of these chains. 1,2 Laminin-332 (LN-332, previously termed laminin-5) that has chain composition a3:b3:g2 (Supplementary ) is essential for anchorage of epithelial cells and specifically found in epithelial BMs. 3-5 LN-332 defects lead to detachment of epithelia and the fatal skin blistering disease junctional epidermolysis bullosa. 6-8 LN-332 also has a role in proliferation and locomotion of epithelial cells, such as in keratinocytes of healing wounds. 9,10 The globular domain (Gdomain) of the a3 chain binds to the cell surface through integrin receptors a6b4 and a3b1 11 and evokes anti-apoptotic signals through focal adhesion kinase, 12,13 while the short arms of the b3 and g2 chains bind to Type VII collagen in the stroma. 14 LN-332 is up-regulated in various epithelial cancers, including colon, gastric, mammary duct and squamous cell carcinomas, as well as melanomas, 15-18 but not in mesenchymal cancers. 15,16 High expression of the g2 chain of LN-332 has been found to correlate with poor prognosis of cervical squamous cell carcinomas. 19 LN-332 is also a major scattering factor stimulating invasive and metastatic capacity of several tumor cell lines in vitro. 20,21 In the cancer tissue, the protein is primarily expressed at the invasive front, as well as in micro-metastases. Down-regulation of LN-332 has been reported in epithelial prostate cancer 22 and also in breast cancers. LN-332 expression has been associated with tumorigenesis. Thus, when HT1080 tumor cells constitutively expressing laminin b3 and g2 chains but not a3 were transfected with laminin a3 cDNA, the cells grew significantly larger tumors in nude mice than untransformed cells. 24 Moreover, LN-332 negative (as well as a4 integrin negative) keratinocytes did not become tumorigenic upon transfection with Ras-IjBa in contrast to normal keratinocytes. Since most cancers are of epithelial origin and positive for LN-332 expression, the question arises if this protein can have a general role for the adhesion and migration process of invading carcinoma cells, and if interference with those functions might influence tumor growth and spread. To address these questions, we have studied the role of LN-332 for carcinoma cell adhesion and migration in vitro and shown that interference with the binding of this protein to the cells inhibits these functions and induces apoptosis. Furthermore, we show that antibodies against the cell and matrix binding domains of LN-332 target to several types of carcinomas growing in vivo and effectively inhibit tumor growth and metastasis in mice. We hypothesize that the LN-332 antibodies induce tumor cell anoikis and decrease metastasis by dissociating the cells from the extracellular matrix.
Journal of Cell Science, 1998
In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observat...