Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein

Laminin is a noncollagenour glycoprotein isolated from a transplantable mouse tumor producting basement membrane (BM). Purified antibodies to laminin do not cross-react with other known BM antigens including type IV collagen, fibronectin, bullous pemphigoid antigen, and a BM proteoglycan. Using immunofluorescence, laminin is localized in the BM zones of those human, chick, guinea pig, bovine, monkey, rat, and mouse tissues examined. Epithelial and endothelial cells in culture synthesize laminin while mesenchymal cells do not. By immunoelectron microscopy, laminin was localized to the lamina lucida of human epidermal BM and of mouse esophagus epithelial BM. The wide distribution of laminin among diverse tissues and species, and in early stages of embryonic development suggests that laminin is an ubiquitous component of basement membranes.     

Gene mapping of mouse laminin A and B2 subunits using mouse-Chinese hamster somatic cell hybrids

Laminin is a multichain extracellular matrix glycoprotein found primarily in basement membranes. The molecule is made up of three subunits, designated as A, B1, and B2. Using an 850-base cDNA probe for the mouse laminin A-chain and a 1000-base cDNA probe for the mouse laminin B2 chain, we have screened mouse—Chinese hamster somatic cell hybrids in order to assign the genes for each of these polypeptides to their respective mouse chromosome. We have determined that the mouse laminin B2-chain gene is located on chromosome 1 (confirming this assignment) and the laminin A-chain gene is located on chromosome 17.     

Loss of basement membrane components laminin and type IV collagen parallels the progression of oral epithelial neoplasia

Aims : To determine the immunohistochemical localization of basement membrane components laminin and type IV collagen in premalignant and malignant lesions of the oral epithelium.  

Methods and results

Formalin-fixed tissue sections of 12 epithelial hyperplasias with no dysplasia and 30 dysplasias, clinically diagnosed as leukoplakia and/or erythroplakia, as well as 50 invasive squamous cell carcinomas, were stained with mouse monoclonal antibodies to human laminin and type IV collagen. Statistical analysis showed that there was a linear trend for discontinuous distribution of laminin from epithelial hyperplasia to epithelial dysplasia and invasive squamous cell carcinoma ( P  < 0.001). Laminin staining showed a linear trend for discontinuity with increasing grade of dysplasia ( P  < 0.05) and was more frequently discontinuous in areas of deep tumour invasion than in central or superficial areas ( P  < 0.05). Brush-shaped thickening and reduplication of the basement membrane were also identified.  

Conclusions

Alterations in the distribution of laminin and type IV collagen in oral premalignant and malignant lesions indicate that the loss of continuity of the subepithelial basement membrane parallels the progression of the neoplastic transformation process in oral epithelium.     

Patterns of basement membrane laminin distribution in nonneoplastic and neoplastic thyroid tissue.

Laminin, a major basement membrane component, is typically absent or partially lost around the epithelial elements of most invasive carcinomas. To evaluate the distribution of laminin in both primary and metastatic thyroid tumors, we studied 14 benign thyroid lesions (eight adenomas, two Graves' disease, two Hashimoto's thyroiditis, one adenomatous hyperplasia, one nodular goiter), 20 carcinomas (seven papillary, six tall cell variant, four follicular, three Hürthle), and eight metastases (five tall cell variant, three follicular) utilizing a polyclonal antibody against highly purified, nidogen-free laminin. All benign lesions showed positive, linear immunostaining along basement membranes. Partial loss or absence of laminin was seen in the solid areas of all types of thyroid carcinomas examined; well-differentiated papillary and follicular tumors, as well as papillary and follicular areas of more poorly differentiated neoplasms, maintained linear laminin immunostaining in the papillary cores beneath the epithelial cells and around follicles. A similar correlation between laminin deposition and architectural organization was seen in metastatic lesions. Hürthle cell carcinomas had a unique fragmented, pericellular immunostaining pattern around individual tumor cells, suggesting uncontrolled laminin synthesis. Our findings suggest that preservation of laminin production in thyroid tumors reflects their degree of differentiation and that absence of laminin correlates with lack of structural organization rather than reflecting invasive and metastatic potential.     

Laminin production by murine melanoma cells: possible involvement in cell motility

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [³⁵S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [³⁵S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the M _r=950kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the M _r=400kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (M _r =200kD) and as a disulfide-linked B dimer (M _r=400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.     

Distribution of merosin, a laminin-related tissue-specific basement membrane protein, in human Schwann cell neoplasms

The expression of merosin and laminin was studied in human schwannomas, plexiform neurofibromas, and malignant schwannomas immunohistochemically using monoclonal antibodies. Merosin is a unique novel tissue-specific basement membrane protein found in basement membranes of trophoblast, striated muscle, and Schwann cells. Merosin is related to laminin, another basement membrane protein with which it shows a homologous C terminal domain. In schwannomas, merosin was only found in areas where tumor cells were in contact with stromal or vascular tissue. Laminin, however, was present in all tumor cell basement membranes. In plexiform neurofibromas large amounts of both merosin and laminin were seen in Schwann cell basement membranes. Very little of either protein was found in malignant schwannomas. Thus merosin is present almost exclusively in highly differentiated Schwann cell neoplasms, and its distribution is more restricted than that of laminin. The expression of merosin in plexiform neurofibromas and in the schwannoma cells juxtaposed to the mesenchymal cells suggests that this protein is induced epigenetically in well-differentiated cells in contact with connective tissue or vascular components.     

Separation and characterization of the subunits of the laminin of EHS sarcoma

A rapid and sensitive method was developed for the preparative separation of laminin subunits. Laminin was extracted and purified from mouse EHS sarcoma. On SDS-PAGE, the reduced and carboxymethylated molecule separated into two components corresponding to molecular weights of about 400 KDa (subunit A) and 200 KDa (subunit B). These two subunits were preparatively separated using heparin-agarose affinity chromatography. The larger subunit quantitatively adhered to the affinity column while the smaller one did not adhere. Amino acid analyses of the separated subunits showed distinct differences. Subunit B was further resolved into two distinct polypeptides of 200 KDa, B1 and B2, by means of reverse-phase HPLC. Although the amino acid compositions of B1 and B2 were very similar, the peptide maps generated by digestion of the B1 and B2 chains with Staphylococcus aureus V8 protease or by cyanogen bromide showed B1 and B2 to differ from each other. Thus, at least three different polypeptide subunits are present in this laminin and probably arise from separate gene origins. These studies provide a basis for the subsequent localization and analysis of the specialized structural and functional domains of laminin.     

Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.

We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1 integrin receptor recognizing laminin-5.     

Monoclonal antibodies against the GP-2 subunit of laminin

Two stable rat X mouse hybridoma lines have been isolated. These hybridoma lines produce IgG antibodies directed against the polypeptide portion of the GP-2 subunit of laminin. Antibodies produced by the hybridomas have been shown to be IgG 2b (lambda) and IgG 2a (kappa), respectively. In competition ELISA assays the monoclonal antibodies exhibited different binding affinities for laminin. Furthermore, the two antibodies were partially additive in their reactivity to laminin. Preliminary results also indicate that the antibodies recognized different antigenic determinants in laminin as determined by their reactivity to basement membranes in human and mouse tissues. The monoclonal antibody designated LAM-I stained a broad spectrum of human and mouse tissues; the other monoclonal antibody LAM-II reacted with mouse, but not human tissues. The results indicate that these monoclonal antibodies could be utilized to explore the organization of laminin in basement membranes of different tissues and species.     

Integrin Dependence of Calu-1 Cell Motility on Endothelial Extracellular Matrix Proteins

We recently showed that the motility of the malignant Calu-1 human epidermoid lung carcinoma cells correlates to their expression levels of α2, α3, α6, and β1 integrin subunits. To determine a causative relationship underlying this correlation, here we measured Calu-1 cell adhesion to and migration on laminin, collagen IV, human umbilical vein endothelial cell monolayers, and endothelial cell extracellular matrix in the presence of function-blocking antibodies against the suspect integrin subunits. Blocking individual α subunits did not affect adhesion to or motility on laminin, but when used in pair-wise combinations, monoclonal antibody treatments significantly decreased tumor cell motility on, without diminishing adhesion to, laminin and the other substrates. Blocking all three α subunits at once or the β1 subunit alone abolished migration on laminin; however, the latter treatment also abolished adhesion, whereas the former treatment did not. By contrast, blocking the β1 subunit significantly reduced motility on collagen IV, endothelial cell monolayers, and endothelial cell extracellular matrix, but always without affecting adhesion. These results suggest a separation of roles and mechanisms of different integrins in adhesion and motility.     

Integrin VLA-6 (αβ1) mediates adhesion of mouse bone marrow-derived mast cells to laminin

The development of mast cells from bone marrow precursors and their function as the mucosal- or connective-tissue-type mast cell are critically dependent on micro environmental factors. Extracellular matrix proteins, such as collagen, fibronectin, and laminin, may represent insoluble components of the microenvironment. Recent studies have described multiple isoforms of laminin isolated from different tissues. In the present study, adhesion of mouse bone marrow-derived mast cells (BMMC) and long-term mast cell lines to Engelbreth-Holm-Swarm (EHS) tumor laminin, rat laminin, human merosin, and human placental laminin was compared. The greatest level of adhesion was found with human laminin as the substrate. By use of a newly prepared mouse VLA-α6 integrin-specific mAb (MA6) together with the previously described mAb GoH3, VLA-6 (α6β1) integrin was found to be expressed and utilized by BMMC and long-term mast cell lines. VLA-6 has been described as a major laminin receptor with roles in diverse cell functions including cell growth and differentiation. BMMC have been shown to express a 32/67-kDa laminin receptor. Therefore, in addition to the 32/67-kDa laminin receptor described in early studies, BMMC also express VLA-6 integrin, which may have roles in the regulation of their development.     

Laminin和survivin的表达与胆囊癌临床病理特征的关系

目的:研究laminin和survivin蛋白在原发性胆囊癌组织中的表达情况,及其与癌组织类型、病理分级和转移状况的关系。 方法:应用免疫组织化学方法检测49例原发性胆囊癌、21例胆囊腺瘤和13例慢性胆囊炎组织中laminin和survivin蛋白的表达。 结果:胆囊黏膜内癌或原位癌细胞基底膜laminin线性染色完整；NevinⅡ期胆囊癌组织表现为基底膜不完整,变薄,断裂,或缺损；临床NevinⅢ、Ⅳ、Ⅴ期胆囊癌，则无基底膜形成，在肿瘤组织周围，laminin表达类型呈碎片状或断线状和连续线状，部分癌组织内laminin染色消失和癌细胞浆内有laminin弱染色。胆囊癌组织中survivin阳性表达率明显高于胆囊腺瘤和慢性胆囊炎组织，但survivin的阳性表达与胆囊癌细胞分化程度、病理分级和转移无关(P>0.05)。且胆囊癌组织中laminin的连续线断裂或缺失,与胆囊癌组织中survivin的表达情况无相关性。 结论:胆囊癌基质中laminin的表达类型与胆囊癌的侵袭和转移有关，而survivin在胆囊癌中表达增加，但两者之间似乎无关联。      

Ultrastructural Localization of Laminin and Type IV Collagen in Normal Human Breast

The extracellular matrix components laminin and type IV collagen have both been localized in the basement membrane of the normal human breast ductule. Breaks in the continuity of these components occur in breast carcinomas and have been implicated in tumor metastasis. Using a postembedding ultrastructural immunogold technique, laminin and type IV collagen were distributed within the basal lamina surrounding the normal human breast ductule. Both components were present diffusely along the basal lamina and were not localized to particular regions, and neither were present between epithelial and myoepithelial cells. Laminin binding of these cells thus probably occurs only at the basal aspect where they are in contact with the basal lamina and is not involved in the cell-cell adhesion between epithelial and myoepithelial cells. This study provides a basis for further ultrastructural investigations of extracellular matrix components in normal and neoplastic breast tissue.     

Alternate splicing at the 3'-end of the human pancreatic tumor-associated mucin MUC4 cDNA

MUC4 is a membrane-bound mucin and is considered as the human homologue of the rat sialomucin complex (SMC). The deduced structural organization of the wild type-MUC4 cDNA (WT-MUC4) sequence revealed two subunits: a large amino mucin type subunit (MUC4alpha) and a transmembrane subunit (MUC4beta). MUC4beta is a membrane-bound growth factor like subunit and contains three EGF-like domains. The MUC4 gene is expressed in several normal tissues like trachea, lung, and testis. It is not expressed in a normal human pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors and tumor cell lines. Recently, we have demonstrated the presence of alternative splice events in the 3'-end of the MUC4 cDNA that generated new putative variants (sv1-sv10) in normal human testis and in a pancreatic tumor cell line (HPAF). In search of MUC4 variant(s) that are specific to pancreatic adenocarcinoma, we investigated the splicing phenomena in the MUC4 cDNA sequence by using a large panel of pancreatic tumor cell lines. We have identified ten alternative splice events located downstream to the central large tandem repeat domain. These splice events generated 12 variant species (sv4, sv9, sv10-18, and sv21) of MUC4 cDNAs. The deduced amino acid sequence of these variant MUC4 cDNAs revealed two distinct types: a family of secreted and a membrane-associated variant form. Among the members of MUC4 secreted variant family, three (sv4, sv12, and sv13) of ten showed a short 144 residue COOH-terminus compared to 1154 residues in WT-MUC4. The variants with this short COOH-terminus (144 residues) was found in 37% (4/11) of the tumor lines. The putative membrane-bound variant sv10 was detected in 37% (4/11) pancreatic tumor cell lines but not in any normal human tissues. In conclusion, we have identified novel splice variant(s) of MUC4 in pancreatic adenocarcinoma.     

Immunohistochemical detection of laminin in 98 human breast carcinomas: a light and electron microscopic study

The distribution of laminin was studied in 98 breast carcinomas with antilaminin and the avidin-biotin-peroxidase complex method. Laminin was observed within vascular and epithelial basement membranes. Laminin displayed a continuous linear pattern in intraductal carcinomas, and it was heterogeneously distributed, with a discontinuous linear pattern, in invasive carcinomas. No intracellular laminin staining was detected. Electron microscopic study showed laminin immunostaining in the lamina densa of basement membranes in nonneoplastic breast tissue. In tumors, laminin immunostaining frequently revealed multilayered basement membranes and abnormal multilayered basement membranes in blood vessels in the tumor stroma. These data suggest that laminin immunostaining, as a new approach to the heterogeneous basement membrane changes occurring in carcinomas, should permit better understanding of cell diffusion processes and of stroma-tumor cell interactions. The consistent extracellular distribution of laminin in contact with the stroma indicates that the latter plays an important role in the assembly of basement membrane components.     

Ultrastructural localization of laminin and type IV collagen in normal human breast

The extracellular matrix components laminin and type IV collagen have both been localized in the basement membrane of the normal human breast ductule. Breaks in the continuity of these components occur in breast carcinomas and have been implicated in tumor metastasis. Using a postembedding ultrastructural immunogold technique, laminin and type IV collagen were distributed within the basal lamina surrounding the normal human breast ductule. Both components were present diffusely along the basal lamina and were not localized to particular regions, and neither were present between epithelial and myoepithelial cells. Laminin binding of these cells thus probably occurs only at the basal aspect where they are in contact with the basal lamina and is not involved in the cell-cell adhesion between epithelial and myoepithelial cells. This study provides a basis for further ultrastructural investigations of extracellular matrix components in normal and neoplastic breast tissue.     

Studies on human laminin and laminin-collagen complexes

Intact human laminin and laminin type IV collagen complexes were extracted from term placental membranes and their structures were examined by electroimmunoblot and by rotary shadowing electron microscopy. Rotary shadowing electron microscopy revealed a structure of human laminin which is essentially similar to the cruciform structure of the mouse tumor (EHS) laminin, but with some notable differences. The observed lengths of the short arms in the human laminin are different. One short arm has an average length of 34 nm and another short arm a length of 42 nm. In the mouse laminin all three short arms are of equal length. The average length of the long arm is 97 nm, which is longer than that of mouse tumor laminin (77 nm). The distal portion of the long arm has two clearly separated globular domains instead of the single one observed with tumor laminin. Human laminin was found in various states of aggregation including dimers, trimers and higher aggregates. Laminin molecules appeared to attach to a point in type IV collagen located 87 nm from C-terminus, or at 174 nm from C-terminus and one with less frequency, at 251 nm from C-terminus. A small number of molecules appeared to bind at the N-terminus of the collagen. These laminin-collagen interactions occurred via the distal globular domains of both the short and long arms of laminin. The data suggest that human laminin extracted from placenta is structurally different from that isolated from the mouse EHS tumor.     

Immunohistochemical localization of basement membrane type VII collagen and laminin in neoplasms of the head and neck

The distribution pattern of the basement membrane components type VII collagen and laminin, was studied immunohistochemically in normal human head and neck tissues and in a series of benign and malignant tumours from the same site. Using monoclonal antibodies, a basement membrane containing type VII collagen and laminin could be demonstrated beneath the epithelial cell layer in 16 normal head and neck tissues from different localizations. Unlike type VII collagen, laminin was also abundantly present around blood vessels and muscle fibres. With respect to 42 squamous cell carcinomas studied, type VII collagen and laminin were present in basement membranes surrounding small and large tumour fields, independent of the tumour grade. Type VII collagen was demonstrated in the cytoplasm of tumour cells in 36% of the cases, while the antibody to laminin displayed a basement membrane staining pattern mainly. Both antibodies showed a staining gradient in more than half of the cases, with strong staining in the centre of the tumour and weakening of the staining towards the tumour periphery. In a series of 22 salivary gland tumours consisting of 19 pleomorphic adenomas and three adenoid cystic carcinomas, the distribution pattern of type VII collagen and laminin was very heterogeneous. Laminin was present in 17 and type VII collagen in 10 of 19 cases of pleomorphic adenoma, mostly scattered throughout the tumour fields. In the tumours positive for type VII collagen areas with little or no positivity were also found. A correlation between type VII collagen positivity and the presence of basal cell keratin 14 positivity was noticed in the majority of cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biological relevance of laminin 5gamma2 expression at the invading edge of colonic carcinomas

Previous studies have shown the presence of dilated neoplastic glands with cellular gaps called glandular pores (GPs) and laminin 5gamma2 expression at the invading edge of colonic carcinomas. We now extended our studies to explore a possible association between GP formation and laminin 5gamma2 expression at the invading edge of colonic carcinomas. Immunostain was performed on sections of five consecutive neoplastic glands with and without GPs from 86 colonic adenocarcinomas to assess the expression of laminin 5gamma2. Neoplastic glands with GPs were observed in 85% (73/86) of the tumors. Laminin 5gamma2 was expressed in 92% (335/365) of the neoplastic glands with GPs but only in 17% (63/365) of the neoplastic glands without GPs (p<0.05). Laminin 5gamma2 was overexpressed in the cells at the free ends of the pores in 88% of the neoplastic glands with GPs, but only in 14% of those without pores (p<0.05). Hence, at the growing edge of colonic carcinomas, laminin 5gamma2 was frequently expressed in neoplastic glands having GPs. Remarkably, the tumor cells at the free ends of the GPs overexpressed laminin 5gamma2, indicating increased production of this adhesion-migration macromolecule. The results suggest a close interaction between this adhesion-migration macromolecule, PG formation and the local progression of colonic carcinomas.