Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that α6β1 but not α6β4 functions as a major receptor for fragment E8 (original) (raw)
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Tumor type-specific differences in cell-substrate adhesion among human tumor cell lines
International Journal of Cancer, 1987
Cell lines derived from human tumors of 4 different histo-described (Carey et al., 1976;; logical types (SWamous carcinoma, melanomas, gliomas and McKeever et al., 1986; Varani et al., 1986). All of the lines a fibrosarcoma) were examined for cell-substrate adhesion on were grown in monolayer culture using minimal essential plastic culture dishes and dishes coated with 50 pg of type-IV medium of ~~~l~ (MEM) with ~~~l~' salts, non-essential collagen. In the absence of exogenous adhesion factors the squamous carcinoma cells attached and spread amino acids, L-glutamine and 15% fetal bovine serum as the than the other cells on both substrates. Once attached, the "lture medium* The were grown at 370c and 5% c02 squamous carcinoma cells were also more difficult than the and subcultured by tVPsiniZatiOn as required. other cells to remove with proteolytic enzymes/EDTA. While R~~~~~~~ the cell lines derived from melanomas, gliomas and the fibrosarcoma were less adhesive than the squamous carcinoma Laminin prepared in our laboratory or purchased from lines in the absence of exogenous adhesion factors, these cells GIBCO (Grand Island, NY) was isolated from the murine were highly responsive to laminin. In contrast, laminin only Englebreth-Holm-Swam (EHS) tumor by the method of Timpl Slightly enhanced the attachment and spreading of squamous et al. (1979). The purity of each lot of laminin was confirmed carcinoma cells on the plastic dishes and actually inhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis characteristic of laminin A (M, = 400,000) and B (M, = results indicate that there are tumor-type-specific differences in adhesiveness among human tumor cell lines and that cells from different tumor types may have distinct mechanisms for 2~,~) subunits, Were Seen on the gels after staining with carrying out one of the functions critical to invasion.
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.
A Novel Recognition Site on Laminin for the α3β1 Integrin
Experimental Cell Research, 1996
hesion, migration, protease secretion, and angiogenesis Synthetic peptide GD-2 is a sequence of amino acids . Proteolytic fragments of laminin and synthetic derived from the carboxy-terminal long arm of the A peptides corresponding to amino acid sequences within chain of laminin. Previous studies have shown that laminin have been utilized to identify specific domains peptide GD-2 promotes the adhesion of human squaof laminin that mediate specific activities involved in mous cell carcinoma (SCC) cells as well as a variety of the metastatic process . At present, three synthetic other cell lines. In this study, we attempted to identify peptides derived from laminin, YIGSR, PDSGR, and the receptor that SCC cells use to adhere to peptide SIKVAV, have been reported to alter metastasis of tu-GD-2. Monoclonal antibodies (mAbs) against a human mor cells in mice [4 -6]. Another laminin peptide, GD-SCC cell line were generated. One of these mAbs, ASC-2, has been shown to promote the adhesion of a variety 1, bound to the surface of SCC cells as determined by of cell types, including human squamous cell carcinoma flow cytometry. This mAb inhibited SCC cell adhesion (SCC) cell lines and HT-1080 human fibrosarcoma cells to peptide GD-2 and laminin, but not fibronectin or [7,. In addition, peptide GD-2 has been shown to type IV collagen, suggesting that mAb ASC-1 binds to promote the outgrowth of neurites from chick spinal the SCC receptor for the peptide GD-2 sequence of lamcord and dorsal root ganglia neurons . The identifiinin. MAb ASC-1 immunoprecipitated a complex comcation of sites on laminin that promote cell adhesion posed of two components of 135 and 116 kDa. Immuand the identification of receptors on cell surfaces that noadsorption of ASC-1-binding material from the SCC bind laminin are important in understanding the role cell extract by incubation with mAb ASC-1 resulted of laminin in cell adhesion and the metastatic process in the removal of the a3b1 integrin from the extract. of tumor cells. Immunohistochemical staining of tissue from a normal Integrins are a family of transmembrane heterodihuman tongue and from a patient with SCC of the meric glycoproteins composed of a and b subunits that tongue revealed that mAb ASC-1 stained the surface of function in cell -matrix and cell-cell adhesion [9 -11].
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.
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...
Journal of cell science, 2000
Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 beta 1 and alpha 6 beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this po...
Cell Biology International, 2001
A comparison of the adhesion of human primary keratinocytes to laminin-1 from murine EHS sarcoma and laminin-2/4 from human placenta was carried out using two methods, cell adhesion to substrates covered with the laminin isoforms, and interaction of keratinocytes from suspension with latex beads coated with the proteins. Laminin-2/4 was considerably more potent as a promoter of attachment of primary human keratinocytes than laminin-1 (and fibronectin), with increased attachment of cells correlating well with the number of latex bead binding sites. Only small cells of diameter of less than 20 m bound more than 5 beads. Staining of keratinocytes with involucrin antibodies confirmed the existence of an inverse relationship between laminin-2/4-coated bead binding and differentiation.
Cancer Research, 1991
We previously reported that altered expression of the A9 antigen (defined by monoclonal antibody UM-A9) is a predictive marker of early recurrence and progression of squamous cell carcinoma (SCC). In normal squamous cells A9 expression is limited to the site of contact with the basement membrane in vivo and the culture surface in vitro, whereas aggressive SCCs exhibit loss of polarity and increased intensity of A9 expression. The potential relationship of the A9 antigen to structures known to be involved in cell adhesion was analyzed by immunobiochemical and cell adhesion assays. UM-A9 precipitates a complex of protein chains reminiscent of the a and /ÃOE heterodimer glycoproteins that char acterize the integrin family of extracellular matrix receptors. Proteins were isolated from A9-positive cells using UM-A9 and well-defined antibodies specific for integrin a and 0 chains. UM-A9, anti-a6, and anti-0t monoclonal antibodies (mAbs) all precipitated proteins with com parable electrophoretic mobilities. Furthermore, UM-A9 mAb precleared the SCC n'lh integrin complex isolated with anti-a6 or ;inii-/l, mAbs but not that isolated by anti-/?, mAh. The isoelectric points of the A9 complex chains were consistent with those reported for a6 and ßt. Three of the polypeptide chains (140, 175, and 205 kDa) precipitated by UM-A9 showed peptide homology to one another and to the 04 chain precipitated by mAh 439-9B. The A9/a6 subunit is composed of 125-and 30-kDa chains and was distinguished from 04 and ß\ chains by its peptide map and isoelectric point. UM-A9 binds to an epitope common to the ¡i., subunits since in pulse-chase analysis the A species are precipitated at an early time point, whereas detection of «-subiinil synthesis is detected during assembly of the mature complex. Immunoprecipitation and pre clearing experiments demonstrated that in SCC the a6 subunit is associ ated primarily with the /ii species and not with the 130-kDa ,1, subunit. In cell adhesion assays on extracellular matrix proteins, the «'-specific GoH3 mAb inhibited binding of SCC to laminili, suggesting that «"/ij may function as a laminin receptor in SCC. These data and our prior observations showing an association between altered A9 expression and early recurrence in SCC provide the first evidence that altered expression of a604 integrin is associated with the clinical behavior of human squa mous cell carcinomas.