The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution.
; ; et al
in Molecular Biology and Evolution (1998), 15(8), 1017-25
The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related ... [more ▼]
The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related to tumor invasion and metastasis. Thus far, only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated. The finding that the cDNA for the 37LRP is virtually identical to a cDNA encoding the ribosomal protein p40 has suggested that 37LRP is actually a component of the translational machinery, with no laminin-binding activity. On the other hand, a peptide of 20 amino acids deduced from the sequence of 37LR/p40 was shown to exhibit high laminin-binding activity. The evolutionary relationship between 23 sequences of 37LRP/p40 proteins was analyzed. This phylogenetic analysis indicated that all of the protein sequences derive from orthologous genes and that the 37LRP is indeed a ribosomal protein that acquired the novel function of laminin receptor during evolution. The evolutionary analysis of the sequence identified as the laminin-binding site in the human protein suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, which appeared during evolution concomitantly with laminin. [less ▲]Detailed reference viewed: 21 (0 ULg)
Formation of the 67-kDa laminin receptor by acylation of the precursor.
; ; et al
in Journal of Cellular Biochemistry (1998), 69(3), 244-51
Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene ... [more ▼]
Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule. [less ▲]Detailed reference viewed: 17 (0 ULg)
Galectin-3 and laminin expression in neoplastic and non-neoplastic thyroid tissue.
; ; et al
in Journal of Pathology (The) (1997), 181(1), 80-6
Galectin-3 is a 31 kD beta-galactoside-binding lectin which is expressed by several types of non-neoplastic and neoplastic cells and which may be involved in cell-extracellular matrix interactions. An ... [more ▼]
Galectin-3 is a 31 kD beta-galactoside-binding lectin which is expressed by several types of non-neoplastic and neoplastic cells and which may be involved in cell-extracellular matrix interactions. An immunohistochemical study has been made of the expression of galectin-3, as well as its ligand, laminin, in a spectrum of benign and malignant thyroid neoplasms and in some non-neoplastic conditions. Immunohistochemistry with anti-human recombinant galectin-3 antibody showed consistent, intense positivity in the neoplastic cells of 18 cases of papillary carcinoma and less intense staining in the five anaplastic carcinomas studied. In addition, two out of three poorly differentiated carcinomas, three out of six medullary carcinomas, and four out of eight follicular carcinomas had less intense or focal positivity. One case of Hurthle cell carcinoma showed scattered strongly positive cells. Eight follicular adenomas, three hyperplastic nodules, five nodular goitres, and normal thyroid tissue were negative. Galectin-3 mRNA expression was also evaluated in three of the papillary carcinomas, two follicular adenomas, and one hyperplastic nodule with matched normal tissue. Northern blot analysis demonstrated mRNA overexpression in the three cases of papillary carcinomas, whereas normal and benign tissues were negative. Laminin distribution in neoplastic and non-neoplastic tissue varied with architectural patterns but did not correlate with galectin-3 immunohistochemical expression. We conclude that expression of galectin-3 is limited to inflammatory foci in normal and benign thyroid tissue and is a phenotypic feature of malignant thyroid neoplasms, especially papillary carcinomas. [less ▲]Detailed reference viewed: 5 (1 ULg)
New insights into the metastasis-associated 67 kD laminin receptor.
; Castronovo, Vincenzo ; et al
in Journal of Cellular Biochemistry (1997), 67(2), 155-65
The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. These interactions are mediated ... [more ▼]
The interactions between tumor cells and laminin or other components of the extracellular matrix have been shown to play an important role in tumor invasion and metastasis. These interactions are mediated by different cell surface molecules, including the monomeric 67 kD laminin receptor. This molecule appears to be very peculiar since so, far only a full-length gene encoding a 37 kD precursor protein has been isolated and the mechanism by which the precursor reaches the mature form is not understood. Based on clinical data, which clearly demonstrate the importance of the receptor in tumor progression, studies were conducted to define the structure, expression, and function of this laminin receptor as a step toward developing therapeutic strategies that target this molecule. The data suggest that acylation of the precursor is the key mechanism in maturation of the 67 kD form. The function of the membrane receptor is to stabilize the binding of laminin to cell surface integrins, acting as an integrin-accessory molecule, although homology of the gene encoding the receptor precursor with other genes suggests additional functions. Downregulation of the receptor expression on tumor cells might open new therapeutic approaches to decrease tumor aggressiveness. [less ▲]Detailed reference viewed: 33 (0 ULg)
Galectin-1 Modulates Human Melanoma Cell Adhesion to Laminin
; ; et al
in Biochemical and Biophysical Research Communications (1995), 209(2), 760-7
Galectins constitute a gene family of beta-galactoside-specific lectins that show high homology in their carbohydrate-binding site. They have been postulated to be involved in many biological events, but ... [more ▼]
Galectins constitute a gene family of beta-galactoside-specific lectins that show high homology in their carbohydrate-binding site. They have been postulated to be involved in many biological events, but their specific functions are not yet well defined. Galectin-1 is a laminin binding protein that recognizes poly-N-acetyllactosamine chains on this major basement membrane glycoprotein. In this study, we analyzed the possibility that galectin-1 could modulate interactions between human melanoma cells and laminin. We demonstrated that A375 and A2058 cell lines express galectin-1 both intracellularly and on the cell surface. In an in vitro assay, recombinant galectin-1 increased melanoma cell attachment to laminin in a dose-dependent manner. This effect was abolished by lactose. Anti-galectin-1 inhibited adhesion of melanoma cells to laminin in a dose-dependent fashion. However, neither galectin-1 nor anti-galectin-1 antibody affected melanoma cell spreading on laminin in vitro. These data indicate that galectin-1 might participate in melanoma cell adhesion to laminin and therefore could be a modulator of invasion and metastasis. [less ▲]Detailed reference viewed: 18 (2 ULg)
Galectin-3, a Laminin Binding Protein, Fails to Modulate Adhesion of Human Melanoma Cells to Laminin
; ; et al
in Neoplasma (1995), 42(5), 215-9
Galectin-3 is a laminin binding protein which expression is altered in a variety of human carcinomas including colon, breast and endometrium. In these tumors, we consistently observed a down regulation of ... [more ▼]
Galectin-3 is a laminin binding protein which expression is altered in a variety of human carcinomas including colon, breast and endometrium. In these tumors, we consistently observed a down regulation of galectin-3 expression related to increased aggressiveness. Galectin-3 belongs to a family of galactose-binding lectins and binds laminin through its numerous poly-N-acetyllactosamine chains. To date, the exact role of galectin-3 in the complex interactions between cancer cells and laminin has not been clearly defined. Adhesion of melanoma cells to laminin is a critical event during tumor invasion and metastasis. In this study, we explore the possibility that galectin-3 could modulate attachment of two human melanoma cell lines to laminin. A2058 and A375 melanoma cell expressed galectin-3 on their surface as demonstrated by immunofluorescence, and attached to laminin in an in vitro assay. We demonstrate that neither recombinant galectin-3 nor an affinity purified antigalectin-3 antiserum altered adhesion of A2058 or A375 melanoma cells to laminin. Our data strongly suggest that galectin-3 is not a key element in adhesion of the melanoma cells to laminin. These results are not surprising in light of the observation that galectin-3 expression is down regulated in cancer and that increased adhesion to laminin is a constant feature of invasive cancer cells. [less ▲]Detailed reference viewed: 35 (1 ULg)
Inverse Expression of Two Laminin Binding Proteins, 67lr and Galectin-3, Correlates with the Invasive Phenotype of Trophoblastic Tissue
; ; et al
in Biochemical and Biophysical Research Communications (1994), 201(1), 388-93
Tumor invasion of host tissues and trophoblastic penetration of the endometrium share common biological features. Both processes involve the invasion of basement membranes, an event that is initiated by ... [more ▼]
Tumor invasion of host tissues and trophoblastic penetration of the endometrium share common biological features. Both processes involve the invasion of basement membranes, an event that is initiated by adhesion of cancer or trophoblast cells to basement membrane components and particularly to laminin. Adhesion to this latter glycoprotein is mediated through a variety of cell surface receptors. We have previously shown that the 67 kD Laminin Receptor (67LR) and a 31 kD Human Laminin Binding Protein, recently renamed galectin-3, are inversely modulated as the invasive phenotype of cancer cells progresses, with up regulation of the former, and down regulation of the latter, respectively. In this study, we examined the expression of these two proteins in 27 human trophoblastic specimens at different gestational ages using Northern and Western blot techniques. Expression of the 67LR increased from 7 weeks to a maximum at 12 weeks, when invasion is maximal, and then decreased. Expression of galectin-3 was inversely modulated by the gestational age, with a minimum expression at 12 weeks. Our data demonstrate that invasive trophoblast displays the same pattern of laminin binding proteins expression than invasive cancer cells, and further demonstrates that invasion of the extracellular matrix by trophoblast and cancer cells share common molecular mechanisms. [less ▲]Detailed reference viewed: 11 (2 ULg)
Cell localization and redistribution of the 67 kD laminin receptor and alpha 6 beta 1 integrin subunits in response to laminin stimulation: an immunogold electron microscopy study.
; ; et al
in Cell Adhesion and Communication (1994), 2(3), 201-9
The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa ... [more ▼]
The 67 kDa laminin receptor (67LR), one of several cell surface laminin-binding proteins, is involved in the interactions between cancer cells and laminin during tumor invasion and metastasis. A 37 kDa polypeptide (37LRP), previously identified as the 67LR precursor, is abundantly present in the cytoplasm and has been implicated in polysome formation. To better understand the cellular localization of the 67LR and its precursor, transmission electron microscopic studies of human melanoma A2058 cells were carried out using immunogold labeling and a variety of antibodies: (a) affinity purified antibodies directed against 37LRP cDNA-derived synthetic peptides; (b) anti-67LR monoclonal antibodies raised against intact human small cell lung carcinoma cells; and (c) monoclonal antibodies against the subunits of the integrin laminin receptor, alpha 6 beta 1. Double-labeling immunocyto-chemistry revealed that anti-67LR monoclonal antibodies as well as anti-37 LRP antibodies recognized antigens that were localized in the cytoplasm in electron dense structures. As expected, cell membrane labeling was also observed. Surprisingly, alpha 6 and beta 1 integrin subunits were detected in the same cytoplasmic structures positive for the 67LR and the 37LRP. After addition of soluble laminin to A2058 cells in suspension, the number of labeled cytoplasmic structures increased especially in the vicinity of the plasma membrane, and were exported onto the cell surface. Neither fibronectin nor BSA induced such an effect. The data demonstrate that the 67 LR and the 37 LRP antibodies detect colocalized antigens that are in cytoplasmic structures with alpha 6 beta 1 integrin.(ABSTRACT TRUNCATED AT 250 WORDS) [less ▲]Detailed reference viewed: 14 (0 ULg)
Differential Expression of the 67-Kd Laminin Receptor and 31-Kd Human Laminin-Binding Protein in Human Ovarian Carcinomas
; ; et al
in European Journal of Cancer (1994), 30A(8), 1096-9
The expression of the 67-kD laminin receptor (67LR) and the 31-kD human laminin-binding protein (HLBP31), two proteins involved in cancer cell laminin interaction, was evaluated on 30 ovarian cancer ... [more ▼]
The expression of the 67-kD laminin receptor (67LR) and the 31-kD human laminin-binding protein (HLBP31), two proteins involved in cancer cell laminin interaction, was evaluated on 30 ovarian cancer specimens. Expression of the 67LR was increased (up to 2.5-fold, in 87% of the patients), while HLBP31 expression was downregulated in cancer cells compared with the normal tissue, as detected by northern blotting and immunohistochemistry. The immunohistochemical study demonstrated that the 67LR was significantly overexpressed (P < 0.05) in the group of patients whose cytoreductive surgery was suboptimal, and those with poor clinical outcome. No correlation was observed between HLBP31 expression and clinicopathological features. Increased expression of the 67LR appears to correlate with the invasive phenotype of ovarian cancer cells and suggests a role of the latter in ovarian cancer invasion. [less ▲]Detailed reference viewed: 12 (0 ULg)
Interaction between the 67 kilodalton metastasis-associated laminin receptor and laminin.
; ; et al
in Kidney International (1993), 43(1), 30-7
Normal and neoplastic cells interact with laminin via a variety of cell surface proteins. The specific binding sites on laminin for each particular cell surface laminin-binding protein have not yet been ... [more ▼]
Normal and neoplastic cells interact with laminin via a variety of cell surface proteins. The specific binding sites on laminin for each particular cell surface laminin-binding protein have not yet been identified. In this study, the interaction between laminin and the high affinity metastasis-associated 67 kD laminin receptor (67 LR) was investigated by electron microscopy using the rotary shadowing technique. Laminin receptor that was purified from human colon carcinoma metastases appeared as a globular structure with a diameter of 5.2 +/- 0.8 nm. The 67 LR specifically bound to laminin on its long arm close to the intersection of the long and the short arms. There was no specific interaction of bovine serum albumin with laminin. Biochemical confirmation of the rotary shadowing experiments included slot blot solid phase assays in which [I125]-labeled 67 LR bound in a dose dependent manner to laminin as well as to the chymotrypsin resistant (C1) fragment of laminin that contains a short piece of the long arm. [I125]-labeled 67 LR did not bind to the pepsin resistant (P1) fragment of laminin that did not contain that segment on the long arm. This study therefore identifies the binding site on laminin for the 67 kD metastasis-associated laminin receptor as a region on the long arm of laminin close to the intersection of the four arms. [less ▲]Detailed reference viewed: 8 (0 ULg)
Enhancement of metastatic potential of murine and human melanoma cells by laminin receptor peptide G: attachment of cancer cells to subendothelial matrix as a pathway for hematogenous metastasis.
; ; et al
in Journal of the National Cancer Institute (1993), 85(3), 235-40
BACKGROUND: Stable anchorage of circulating cancer cells to the vasculature is a critical step in the formation of hematogenous metastases. Although the basement membrane glycoprotein laminin clearly ... [more ▼]
BACKGROUND: Stable anchorage of circulating cancer cells to the vasculature is a critical step in the formation of hematogenous metastases. Although the basement membrane glycoprotein laminin clearly plays a crucial role in this event, the exact interactive pathways among cancer cells, laminin, and the vessel wall have not been elucidated. In a previous study, we identified synthetic peptide G, which contains the laminin-binding domain of the 67-kd laminin receptor and which inhibits tumor cell adhesion to endothelial cells. PURPOSE: To assess the role of the interaction between laminin and the 67-kd laminin receptor in hematogenous metastasis formation, we studied the effect of peptide G on melanoma cell behavior in vivo and in vitro. METHODS: The effect of peptide G and control peptides was studied in vivo on lung retention and colonizing potential of murine (B16BL6) and human (A2058) melanoma cells injected intravenously in C57BL/6 and nude mice, respectively. In addition, their effect on cell adhesion and chemotaxis to laminin and on binding of iodine 125-labeled laminin to cells was studied in vitro. RESULTS: In vivo, pretreatment of cells with peptide G resulted in a two- to 10-fold significant increase in the number of experimental lung metastases. A significant relative increase in lung retention of peptide G-treated tumor cells was observed 48 hours after injection, although after 4 hours a partial reduction was observed. In vitro, peptide G significantly increased laminin binding and cancer cell adhesion to laminin and subendothelial matrix, whereas chemotaxis to laminin was significantly inhibited. CONCLUSIONS: Peptide G differentially affected the biological response of cancer cells to laminin. In vitro, it increased laminin binding and cell adhesion to laminin and subendothelial matrix, whereas it inhibited cell chemotaxis to laminin. In vivo, the overall effect of peptide G was an augmentation of lung metastasis. IMPLICATIONS: Our findings suggest that direct adhesion of tumor cells to the subendothelial matrix is a main pathway for hematogenous metastases and that tumor cell-matrix interaction may be more relevant than tumor cell-endothelial cell attachment in this process. [less ▲]Detailed reference viewed: 9 (0 ULg)
Genes involved in tumor invasion and metastasis are differentially modulated by estradiol and progestin in human breast-cancer cells.
; ; et al
in International Journal of Cancer = Journal International du Cancer (1992), 52(4), 653-7
Invasion of basement membranes by cancer cells is a critical step in metastasis, which requires the coordinated expression of specific genes such as laminin receptors and metalloproteinases. Estradiol and ... [more ▼]
Invasion of basement membranes by cancer cells is a critical step in metastasis, which requires the coordinated expression of specific genes such as laminin receptors and metalloproteinases. Estradiol and progesterone modulate the clinical progression of steroid-sensitive breast cancers; however, little is known about the molecular regulation of the invasive phenotype by these hormones. We therefore examined the effects of 10 nM estradiol and/or 10 nM progestin R5020 on the expression of 2 non-integrin laminin binding proteins, the 67-kDa laminin receptor (67LR) and HLBP31 as well as the 72-kDa type-IV collagenase (MMP-2) and its inhibitor, TIMP-2, in steroid-receptor-positive (T47D and MCF-7) and -negative (MDA-MB 231) human breast-cancer cells. The relative steady-state level of 67LR mRNA was increased 2- to 3-fold by estradiol in both MCF-7 (p < 0.001) and T47D (p < 0.001) cells, also by R5020, alone or in combination with estradiol, in T47D cells (p < 0.001) and to a much less extent in MCF-7 cells. HLBP31 mRNA and protein levels were increased 2- to 3-fold (p < 0.001) by R5020 alone or in combination with estradiol, but not by estradiol alone. None of the steroid treatments affected the expression or activity of MMP-2. Interestingly, however, TIMP-2 mRNA levels and protein expression in MCF-7 and T47D cells were 50% down-regulated (p < 0.001) by treatment with R5020 or R5020 plus estradiol, but not by treatment with estradiol alone. None of these genes were modulated in steroid-independent MDA-MB231 cells. The data suggest that estradiol and progesterone might act as coordinators regulating specific genes in the steroid-sensitive breast-cancer cell, leading to the acquisition of the metastatic phenotype. [less ▲]Detailed reference viewed: 19 (0 ULg)
Inverse modulation of steady-state messenger RNA levels of two non-integrin laminin-binding proteins in human colon carcinoma.
Castronovo, Vincenzo ; ; et al
in Journal of the National Cancer Institute (1992), 84(15), 1161-9
BACKGROUND: Interactions between cells and the basement membrane glycoprotein laminin are altered during colon cancer progression. Colon carcinoma and normal mucosa cells express a variety of laminin ... [more ▼]
BACKGROUND: Interactions between cells and the basement membrane glycoprotein laminin are altered during colon cancer progression. Colon carcinoma and normal mucosa cells express a variety of laminin-binding proteins, including the 67-kd laminin receptor (67 LR) and a 31-kd human laminin-binding protein (HLBP31) homologous to the 31-kd human IgE-binding protein/galactoside-binding lectin. PURPOSE: To investigate whether various laminin-binding proteins are differentially expressed in human colon carcinoma, we studied messenger RNA (mRNA) levels of the 67 LR and HLBP31 in matched tumor and adjacent normal mucosa samples from a series of 21 patients. METHODS: Total cellular RNA from tumor and normal mucosa was isolated and analyzed by Northern and slot blot hybridization. In addition, HLBP31 protein levels were assessed by the immunoblot technique. Quantitative laminin affinity chromatography was also used to measure the synthesis of HLBP31 protein in five human cancer cell lines. RESULTS: The steady-state mRNA level of HLBP31 was downregulated (i.e., decreased) in 18 of 21 human colon carcinomas compared with the level in their corresponding normal colonic mucosa. On average, the level of HLBP31 mRNA was decreased 50% +/- 30% (+/- SD) in the colon cancers. The mean ratio of colon cancer HLBP31 mRNA to adjacent normal mucosa HLBP31 mRNA was twofold lower in primary tumors of patients with metastases (0.3 +/- 0.2 SD) than in primary tumors of patients free of metastatic lesions (0.6 +/- 0.2 SD). The differences between the two groups of patients were statistically significant (P less than .05, Wilcoxon-Mann-Whitney test). We have previously shown that the ratio of colon cancer 67 LR mRNA to corresponding normal mucosa 67 LR mRNA was increased in the same patient population. When the two ratios (ratio of cancer to normal HLBP31 mRNA and ratio of cancer to normal 67 LR mRNA) were compared, HLBP31 mRNA/67 LR mRNA was significantly lower (P less than .05) in primary tumors with metastases (mean +/- SD, 0.3 +/- 0.2) than in primary cancers without metastases (mean +/- SD, 0.7 +/- 0.5). The steady-state level of HLBP31 mRNA was directly correlated with the amount of HLBP31 protein in both colon tissue samples and human cancer cell lines. CONCLUSION: HLBP31 mRNA expression in colon cancer tissues is modulated inversely to that of 67 LR mRNA expression. The down-regulation of HLBP31 appears to be associated with the metastatic capabilities of colon cancer cells. IMPLICATIONS: Prospective studies on a large cohort should determine if the systematic detection of HLBP31 and 67 LR protein and/or mRNA can be a valuable adjunct in the prognostic evaluation of primary colon cancers. [less ▲]Detailed reference viewed: 10 (0 ULg)
Extracellular matrix receptors and mouse skin carcinogenesis: altered expression linked to appearance of early markers of tumor progression.
; ; et al
in Cancer Research (1992), 52(10), 2966-76
Interaction of cells with the basement membrane is important for cell proliferation and differentiation. Disruption of the basement membrane is an early event during progression of benign tumors to cancer ... [more ▼]
Interaction of cells with the basement membrane is important for cell proliferation and differentiation. Disruption of the basement membrane is an early event during progression of benign tumors to cancer. Using the techniques of immunohistochemistry and immunofluorescence, we show that cell-matrix interactions via the cell surface integrin receptors alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 4, the Mr 67,000 laminin receptor (67LR) laminin-binding protein, and the secreted matrix protein laminin are strictly regulated during differentiation of mouse epidermis. While alpha 6 beta 4 and alpha 5 beta 1 are polarized to the basal surface of basal cells in contact with the basement membrane, alpha 3 beta 1 and the non-integrin 67LR are primarily detected in the cell periphery of suprabasal cells, where cell to cell contacts are found. Sequential changes in expression of matrix receptors occur following multistage carcinogenesis of mouse skin. In an analysis of benign and malignant skin tumors induced by chemical carcinogens or oncogene transduction, we found that alpha 3 beta 1 and alpha 5 beta 1 as well as the non-integrin 67LR are sequentially down-regulated in the progression from benign to malignant, while alpha 6 beta 4 is the predominant receptor expressed in the carcinomas. Tumor expression of alpha 6 beta 4 is not polarized and is dissociated from its colocalized normal partner bullous pemphigoid antigen, which remains restricted to the basement membrane. The changes in matrix receptors are linked to appearance of keratin 13 in suprabasal regions, but always in alpha 6 beta 4 negative cells. The predominance of alpha 6 beta 4 in the proliferating cells during progression is associated with decreased expression of keratin 13 in carcinomas. These results suggest that matrix interactions with its receptors are important determinants of ordered differentiation in normal skin and show characteristic alterations during carcinogenesis that parallel changes in differentiation of the tumors. [less ▲]Detailed reference viewed: 8 (0 ULg)
Identification of a 14-kDa laminin binding protein (HLBP14) in human melanoma cells that is identical to the 14-kDa galactoside binding lectin.
Castronovo, Vincenzo ; ; et al
in Archives of Biochemistry & Biophysics (1992), 297(1), 132-8
The carbohydrate moieties present on laminin play a crucial role in the multiple biological activities of this basement membrane glycoprotein. We report the identification of a human laminin binding ... [more ▼]
The carbohydrate moieties present on laminin play a crucial role in the multiple biological activities of this basement membrane glycoprotein. We report the identification of a human laminin binding protein with an apparent molecular mass of 14 kDa on sodium dodecyl sulfate-polyacrylamide gels that was found, after purification and amino acid microsequencing, to be identical to the previously described 14-kDa galactoside binding soluble L-14 lectin. We have designated this human laminin binding protein as HLBP14. HLBP14 was purified from human melanoma cells in culture by laminin affinity chromatography and gel electroelution. We demonstrate that HLBP14 binds specifically to the poly-N-acetyllactosamine residues of murine laminin and does not bind to other glycoproteins that do not contain such structures, such as fibronectin. HLBP14 was eluted from a murine laminin column by lactose, N-acetyllactosamine, and galactose but not by other control saccharides, including glucose, fucose, mannose, and melibiose. It did not bind to laminin treated with endo-beta-galactosidase. Lactose also eluted HLBP14 off a human laminin affinity column, implying that human laminin also contains poly-N-acetyllactosamine residues. On immunoblots, polyclonal antibodies raised against HLBP14 recognized HLBP14 as well as 31- and 67-kDa molecules that are also laminin binding proteins, indicating that these proteins share common epitopes. L-14, a dimeric lactose binding lectin, is expressed in a wide variety of tissues. Although the expression of this molecule has been linked to a variety of biological events, the elucidation of its specific functions has been elusive. The observation that HLBP14, a human cancer cell laminin binding protein, is identical to L-14 strongly suggests that the functions attributed to this lectin could be mediated, at least in part, through its ability to interact with the poly-N-acetyllactosamine residues of laminin. HLBP14 could potentially play a role during tumor invasion and metastasis by modulating the interactions between cancer cells and laminin. [less ▲]Detailed reference viewed: 20 (0 ULg)
Detection of laminin receptor mRNA in human cancer cell lines and colorectal tissues by in situ hybridization.
; ; Castronovo, Vincenzo et al
in American Journal of Pathology (1992), 141(5), 1073-83
The 67-kd high-affinity laminin receptor (67 LR) is a gene product whose expression appears to be associated with the invasive and metastatic phenotype of a variety of human cancer cells. Northern blot ... [more ▼]
The 67-kd high-affinity laminin receptor (67 LR) is a gene product whose expression appears to be associated with the invasive and metastatic phenotype of a variety of human cancer cells. Northern blot hybridization has been routinely used to quantify the level of 67 LR mRNA from total cellular RNA extracts of homogenized tissue specimens or in vitro grown cell populations. This technique is useful to assess the average expression of the 67 LR mRNA of a particular sample but does not provide information about expression in specific cell types nor about heterogeneity of expression from cell to cell. In this study, we analyzed the expression of 67 LR mRNA in four human cancer cell lines with varying degrees of expression of 67 LR protein (renal cancer A-704, breast carcinoma MCF-7/4 and MCF-7/7, and pancreatic cancer Panc-1) using in situ hybridization performed with 67 LR riboprobes. Total cellular RNA was simultaneously extracted from the cell lines and hybridized on Northern blots with a 67 LR cDNA probe to assess the validity of the mRNA detection by in situ hybridization. Sixty-seven LR mRNA expression was higher in Panc-1 and MCF-7/4 cells than in MCF-7/7 and renal carcinoma A-704. There was a direct correlation (R2 = 0.88) between the in situ hybridization analysis and the mRNA levels detected by Northern blot analysis. The in situ hybridization method showed a heterogeneous expression of the 67 LR mRNA in the four cell lines with different subpopulations of cells showing a range from negative to high levels of the message. Sixteen freshly frozen human colorectal tissues (seven adenocarcinomas, five matched normal mucosae, and four adenomas) were also analyzed by in situ hybridization. The 67 LR mRNA was localized in normal and neoplastic epithelial cells. Adenocarcinoma cells showed a 1.6- to 5-fold higher expression (P < 0.02 according to the Wilcoxon-Mann-Whitney test) than did epithelial colonic cells from normal mucosae or adenomas. The signal tended to be stronger in poorly differentiated carcinomas and carcinomas with metastases than in moderately differentiated and nonmetastatic tumors. We conclude that the high expression of 67 LR mRNA in colorectal tumors is due to an increased production by tumor cells. Furthermore, in situ hybridization is an effective method to detect the expression of LR mRNA in cultured cell lines as well as in frozen tissue sections. [less ▲]Detailed reference viewed: 9 (0 ULg)
Biosynthesis of the 67 kDa high affinity laminin receptor.
Castronovo, Vincenzo ; ; et al
in Biochemical and Biophysical Research Communications (1991), 177(1), 177-83
High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone ... [more ▼]
High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone and obtained by in vitro translation of hybrid-selected laminin receptor mRNA, has been immunologically identified in cancer cell extracts as the putative precursor of the 67 LR. In this study, we used affinity purified antibodies developed against cDNA-deduced 37 LRP synthetic peptides in pulse chase experiments and demonstrated a precursor-product relationship between the 37 LRP and the 67 LR. Immunoblot, pulse chase and immunofluorescence experiments showed that transient transfection of the full length 37 LRP cDNA clone induced a dramatic increase in the synthesis of the 37 LRP but not of the mature 67 LR. We propose that the 67 LR results from the association of two gene products: the 37 LRP and a polypeptide yet to be identified. [less ▲]Detailed reference viewed: 9 (2 ULg)
Laminin receptor complementary DNA-deduced synthetic peptide inhibits cancer cell attachment to endothelium.
Castronovo, Vincenzo ; ;
in Cancer Research (1991), 51(20), 5672-8
Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 ... [more ▼]
Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 high-affinity receptor (67 LR) could play a major role in this process. Scatchard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000 high-affinity receptors that mediate, at least in part, the attachment of highly invasive melanoma cells. This endothelial cell-melanoma cell interaction was significantly inhibited by soluble laminin and by anti-laminin antibodies. Peptide G, an eicosapeptide derived from the complementary DNA sequence of the 67 LR precursor (IPCNNKGAHSVGLMWWMLAR) that specifically binds to laminin and presumably contains the active ligand-binding site of the receptor, specifically prevented attachment of the melanoma cells to both the bovine aortic endothelial cell monolayer and human umbilical vein endothelium. Thus, peptide G may selectively interfere with the metastatic cascade by inhibiting tumor cell attachment to endothelium via the laminin-67 LR pathway and is a potential new antimetastatic agent. [less ▲]Detailed reference viewed: 10 (0 ULg)
Functional domains of the 67-kDa laminin receptor precursor.
Castronovo, Vincenzo ; ;
in Journal of Biological Chemistry (1991), 266(30), 20440-6
We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of ... [more ▼]
We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin. [less ▲]Detailed reference viewed: 15 (0 ULg)
Molecular inhibition of cancer cell invasion and metastasis.
Castronovo, Vincenzo ; ; et al
in Princess Takamatsu Symposia (1991), 22
A group of coordinated cellular processes, not just one gene product, is responsible for invasion and metastasis, the most life-threatening aspect of cancer. It is now recognized that negative factors may ... [more ▼]
A group of coordinated cellular processes, not just one gene product, is responsible for invasion and metastasis, the most life-threatening aspect of cancer. It is now recognized that negative factors may be just as important as positive elements. Genetic changes causing an imbalance of growth regulation lead to uncontrolled proliferation necessary for both primary tumor and metastasis expansion. However, unrestrained growth does not, by itself, cause invasion and metastasis. This phenotype may require additional genetic changes. Thus, tumorigenicity and metastatic potential have both overlapping and separate features. Invasion and metastasis can be facilitated by proteins which stimulate tumor cell attachment to host cellular or extracellular matrix determinants, tumor cell proteolysis of host barriers, such as the basement membrane, tumor cell locomotion, and tumor cell colony formation in the target organ for metastasis. Facilitory proteins may act at many levels both intracellularly and extracellularly, but are counterbalanced by factors which can block their production, regulation or action. A common theme has emerged: in addition to loss of growth control, an imbalanced regulation of adhesion, proteolysis, and motility appears to be required for invasion and metastasis. Re-equilibrating the expression of the genes involved in these tumor invasion related events could potentially constitute the basis for new anti-cancer therapeutic strategies. [less ▲]Detailed reference viewed: 9 (0 ULg)