Jouni Lohi

Laminin-5 in the progression of carcinomas.

The basement membrane (BM) separates epithelial elements from the surrounding stroma. BM is dynamic in regulation of epithelial cells differentiation as well as their organization into 3‐dimensional tissues. In these functions, among the molecules of the BM, laminins are especially dynamic. Laminins are distributed in a spatially and temporally regulated manner in various epithelial tissues. Various changes in the laminin distribution accompany the malignant transformation of epithelia. The role of the BM and laminins in the progression of carcinomas is not well understood. The BM has been suggested to act as a mechanical barrier against carcinoma cell invasion. BM laminins may play an active role in regulating the migration and proliferation of the carcinoma cells. Laminin isoform laminin‐5 expression is typical for some invasive carcinomas and it may act as a ligand for invading carcinoma cells. Neoexpression of laminin‐5 has also been associated to proliferative activity of the carcinoma cells. Integrins α3β1 and α6β4 are probable cell surface receptors acting with laminin‐5 in the regulation of carcoma cell invasion and proliferation.

Basement membrane laminin‐5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for α3β1 integrin

Interplay between laminin‐5 (Ln‐5) and its integrin (Int) receptors α2β1, α3β1 and α6β4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immuno‐reactivity for chains of Ln‐5 (α3‐β3‐γ2) and Ln‐10 (α5‐β1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln‐5 appeared to accumulate along the invading edges of carcinomas, while Ln‐10 was mostly absent. Immunoreactivity for Ln α1 chain, a component of Lns‐1 and ‐3, was not seen in adenomas or carcinomas. Immunoreactivity for α2, α6, β1 and β4 Ints was found in all tumors and that for α3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln‐5. Immunoblotting of carcinoma tissues showed that the γ2 chain of Ln‐5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln‐5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function‐blocking MAbs to α3 and β1 Int subunits, but not those to Int α2 or α6 subunits, significantly inhibited the adhesion of cells to Ln‐5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln‐5, produced by carcinoma cells, is a major BM component and the cells use the α3β1 Int complex for adhesion to Ln‐5.

Expression of type IV collagen α1(IV)–α6(IV) polypeptides in normal and developing human kidney and in renal cell carcinomas and oncocytomas

Type IV collagen trimer is a major component of basement membranes (BMs). It is composed of polypeptides named α1(IV)–α6(IV) chains. Chains α1,2(IV) are widely expressed in BMs while α3(IV)–α6(IV) are more restricted in human tissues. We have now studied by immunohistochemical means the distribution of collagen IV chains in fetal and adult human kidney, in oncocytomas, in renal cell carcinomas (RCCs) and their metastases and in experimental xenografts of human tumors. α1,2(IV) chains were found in all BMs of fetal and adult kidney as well as of renal tumors, while α3(IV)–α6(IV) chains were found in BMs of distal segments of developing and mature tubules. α3(IV)–α5(IV) chains were seen also in BMs of developing fetal glomeruli after the capillary loop stage. Most of the RCCs and their metastases showed occasional expression of α3(IV)–α6(IV) with papillary variants showing only expression of α5(IV) chain. There was a distinct expression of α3(IV)–α5(IV) chains in BMs of 3 oncocytomas. In 2 of them a variable expression of the α6(IV) chain was seen. In 3 of 4 xenografts, immunoreactivity for human‐specific monoclonal antibody (MAb) for α1,2(IV) was seen in the BM‐like structures. No α3–α6(IV) was seen in any of the xenografts, while polyclonal antiserum for type IV collagen presented immunoreactivity in BMs of all xenografts. Our results show that oncocytomas and most of the RCCs express scarce variants of type IV collagen containing α3(IV)–α6(IV) chains. In experimental xenograft tumors, both implanted RCC cells and host stromal cells have a capacity to produce type IV collagen. Int. J. Cancer 72:43–49, 1997.

Laminins, tenascin and type VII collagen in colorectal mucosa

SummaryThe distribution of different laminin polypeptides, type VII collagen and tenascin has been studied in adult and foetal colorectal mucosa by using the indirect immunofluorescence technique. Immunoreactivity for laminin α1 chain was located to basement membranes of epithelia, muscularis mucosae, and blood vessels, respectively in different segments of adult colon and rectum. Laminin β1 and γ1 chains were additionally expressed in lamina propria. Laminin α2 chain was also found in lamina propria around the pericyptal fibrollasts. Immunoreactivity for laminin β2 chain was restricted to basement membranes in the muscularis mucosae and arteries. Laminin α3 and β3 chains, suggestive for laminin-5, were confined especially to surface epithelial basement membranes. Immunoreactivity for type VII collagen was confined to basement membrane of surface epithelium in a punctate manner, while that for tenascin was seen slightly more broadly in the basement membrane zone and also in the muscular layer. The distribution of laminin chains in 16-week-foetal colon mostly resembled that of corresponding adult tissue, although immunoreactivities for laminin α2 and β2 chains were lacking. Type VII collagen and the high molecular weight isoform of tenascin also absent from the foetal colon. The results show that the basement membrane of the surface epithelium of colon and rectum express the components of epithelial adhesion complex, laminin-5 (α3-β3-γ2) and type VII collagen, resembling in this respect small intestine and stomach while laminin-2 (α2-β1-γ1) appears to be associated with pericryptal fibroblasts, and laminin-1 (α1-β1-γ1) widely in most basement membranes.

Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers

Integrin dimer α6β4 is a transmembrane component of an epithelial cell adhesion complex that consists of hemidesmosomes (HDs), basement membrane (BM)‐associated laminin‐5 (Ln‐5), and anchoring filaments/type VII collagen, all of which are absent from normal thyroid follicular epithelium. In the present study, the expression of epithelial cell adhesion complex antigens in thyroid tumours was investigated using immunohistochemistry. In addition to integrin subunits α6 and β4, immunoreactivity was found for all chains of Ln‐5, α3, β3 and γ2, type VII collagen and hemidesmosomal antigen, HD1, in most thyroid carcinomas associated with tumour anaplasia and papillary growth pattern and located at the border of parenchymal cells and connective tissue or blood vessel walls. In addition, a more restricted expression of bullous pemphigoid antigens 180 and 230 (BP180 and BP230), constituents of HDs, was found in some papillary and anaplastic carcinomas and atypical adenomas. Adhesion complex antigens were located to regions of cells which were immunoreactive for cytokeratin (ck)‐5 and proliferating cell nuclear antigen Ki‐67. The results suggest that in thyroid carcinomas, the emergence of adhesion complex antigens is associated with squamous differentiation and high proliferative activity.

Expression of laminin in renal‐cell carcinomas, renal‐cell carcinoma cell lines and xenografts in nude mice

We studied the expression of laminin (Ln) chains (α1–α3, β1–β3, γ1) in human renal‐cell carcinomas (RCC), papillary renal neoplasms (PRN) and oncocytomas, in RCC cell lines and their xenografts. In RCCs the basement membranes (BM) showed immunoreactivity for chains of Ln‐1 (α1‐β1‐γ1). Only in well‐differentiated RCCs could vessel BMs be distinguished from those of carcinoma cell islets. RCCs and oncocytomas also exhibited an abundant immunoreactivity for Ln β2 chain in both vessel and tumor cell BMs, while Ln α2 chain was not seen in any renal tumors. In distinction from RCCs, PRNs presented a strong BM immunoreactivity for Ln α3 and β3 chains and for Ln‐5, as well as lack of Ln β2 chain. A more variable reactivity for Ln‐5 was seen in oncocytomas. As PRNs and oncocytomas have been suggested to originate from collecting ducts, it is notable that in normal human kidney, we could detect immunoreactivity for Ln‐5 and its chains only in BM of the tubules of the loop of Henle. In immunoprecipitation experiments, an abundant production of Ln‐1, but not of Ln‐5, was seen in cultured RCC cells, while in xenografts of the same cells BM‐confined immunoreactivity for both Ln‐1 and Ln‐5 was seen. Ln β2 chain was produced by 2 of the 4 RCC cell lines in culture but was found only in 1 of the xenografted tumors.

Laminin chains in the basement membranes of human thymus

SummaryIn recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassalls corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β4 integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16--19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only α3, αv and β1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, β3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin α1, β1, γ1 and β2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin β2 chain. Furthermore, immunoreactivity for the laminin α2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen α1 and α2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are α3β1, αvβ1 and/or αvβ3 mediating adhesion to laminin-1 (α1-β1- γ1) and type IV collagen trimer α12 (IV)

Renal cell carcinomas and pancreatic adenocarcinomas produce nidogen in vitro and in vivo

The production of nidogen by four renal cell carcinoma (RCC) and three pancreatic adenocarcinoma (PAc) cell lines has been studied in cell culture and in xenografted tumours in nude mice. In RCC cells, immunoreactivity for nidogen was seen only after exposure to monensin to induce cytoplasmic accumulation of secretory proteins. In PAc cells, immunoreaction was also detectable in control cells. Immunoblotting of control and monensin‐exposed cells and immunoprecipitation of culture media of radioactively labelled cells demonstrated the production of nidogen polypeptide of Mr ca. 150000 by six of the seven cell lines. Basement membranes (BMs) and stroma of the xenografted tumours derived from these six cell lines demonstrated immunoreactivity for both human and mouse nidogen, as revealed with species‐specific antibodies. The ability of the cells to produce nidogen in vitro and deposit in vivo was positively correlated with high histological grade of the xenografted tumours, although the small number of cell lines studied calls for further studies to confirm this. The distribution of nidogen in human RCC and PAc specimens was also studied by immunohistochemistry. There was strong immunoreactivity for nidogen in tumour stroma, BM of carcinoma cell nests, and endothelial basal lamina, but no conclusions could be drawn regarding histological grade and immunostaining patterns, because stromal production could not be ruled out. The results show that nidogen is produced by human carcinoma cells both in vitro and in vivo. Copyright

Hemidesmosomal Molecular Changes in Dermatitis Herpetiformis; Decreased Expression of BP230 and Plectin/HD1 in Uninvolved Skin

Recent BP230-knockout experiments with subsequent blistering and recently identified plectin/HD1 mutations in epidermolysis bullosa simplex patients suggest that defective expression of BP230 and plectin/HD1 may predispose to blister formation in human skin. We have studied the expression of the epithelial adhesion complex as well as the basement membrane and anchoring fibril antigens in uninvolved dermatitis herpetiformis skin to find out if alterations can be detected in these structures predisposing to the blister formation typical of the disease. Ten uninvolved dermatitis herpetiformis skin specimens, which all showed clear granular deposits of IgA under the basement membrane in direct immunofluorescence and five normal skin specimens, were studied by indirect immunofluorescence technique. Six uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for BP230 and four uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for plectin/HD1. All five skin controls showed strong immunoreactions for BP230 and plectin/HD1. Other hemidesmosomal proteins including BP180 and integrin α6β4, as well as basement membrane proteins laminin-5, laminin-1, nidogen and type IV collagen, and the anchoring fibril protein type VII collagen showed a normal strong expression. Our results suggest that alterations in BP230 and plectin/HD1 may contribute or predispose to blister formation in dermatitis herpetiformis skin.