Showing papers by "Kari Alitalo published in 1982"

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells.

Abstract: Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.

Pericellular Matrix in Malignant Transformation

Abstract: Publisher Summary This chapter describes the properties of the major defined matrix components, and considers their role for the cell phenotype The principal function of the matrix is to give mechanical support and to anchor cells in tissue type-specific structures, but it may also have other duties, such as that of a selective permeability barrier The composition of the matrix surrounding cells, the pericellular matrix, is closely dependent on the cell type and the degree of its differentiation It has been becoming increasingly evident that altered or defective cell surface-matrix interactions may be salient features of the malignant phenotype Failure to maintain an intact basal lamina may be involved in the neoplastic disorganization of tissue architecture and development of invasive tumors The chapter emphasizes on the pericellular matrix components that seem to be involved in cell adhesion, and less on the extracellular matrix material Furthermore, recent results on epithelial cells, proteolysis of basal laminae, and interstitial matrix, and to the matrix components that are characteristic of some tumor cells are also discussed in the chapter

Deposition of Basement Membrane Proteins in Attachment and Neurite Formation of Cultured Murine C‐1300 Neuroblastoma Cells

Abstract: The deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C-1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell-associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high-molecular weight proteins in the two cultures were markedly different. We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell–to–substratum interactions in C-1300 cell cultures.

Deposition of an intermediate form of procollagen type III (pN-collagen) into fibrils in the matrix of amniotic epithelial cells.

Abstract: We have followed the deposition and maturation of the pericellular matrix of amniotic epithelial cell cultures for up to eight weeks using metabolic labeling and immunoelectron microscopy. This matrix contains mainly collagen type III and fibronectin. Cleavage of the carboxypropeptide occurred after secretion of the procollagen molecules into the medium but was not accompanied by a significant release of the aminopropeptide. The early matrix, as isolated from the cultures by a deoxycholate procedure, contained collagenous proteins predominantly composed of pN alpha 1(III) chains, which still possessed the aminopropeptide, and only little material in the form of alpha 1(III) chains. The relative amount of alpha 1(III) chains increased during subsequent days of culture. Electron microscopy showed two types of structures in the matrix: thin fibrils, ranging from 10 to 30 nm in diameter, with no apparent cross-striation, and 50-500 nm thick bundles composed of filamentous and amorphous material. In the fibrils, immunoferritin electron microscopy showed a regular staining for the aminopropeptide of procollagen type III with a periodicity of 71 nm. These collagenous fibrils did not stain for fibronectin which was found in the bundles. Since most of the aminopropeptide in the matrix appeared covalently linked as pN-collagen, we conclude that the deposition of this intermediate form of procollagen is a general mechanism in collagen type III fibrillogenesis.

Pericellular matrix and cell surface glycoproteins of virus-transformed mouse epithelial cells.

Abstract: Mouse embryo Mus musculus castaneous epithelial cells, transformed with Moloney murine sarcoma virus (MSV) or with ecotropic murine leukemia virus (MuLV), were analyzed for production of pericellular matrix glycoproteins. The nontransformed, MSV-transformed, and MuLV-transformed cells produced fibronectin, laminin, type I collagen, and small amounts of type III collagen when studied by immunofluorescence using specific antibodies. The virus-transformed epithelial cells produced enhanced amounts of fibronectin into their growth media. Nontransformed M. musculus castaneous epithelial cells mainly produced type I collagen, as shown by metabolic labeling and polypeptide analysis. A significant increase in the glycoprotein production was seen by the MuLV-transformed cells, whereas small changes in the collagen production were apparent after MSV transformation. MuLV-transformed cells produced increased amounts of type I collagen and also some collagenous polypeptides that comigrated with procollagen type IV chains. The ratio of the procollagen type I chains deposited in the matrix was altered in transformed cells. Radioactive surface labeling of the cells revealed changes of the high-molecular-weight glycoproteins in both the MSV- and the MuLV-transformed cells. Unlike virus-transformed fibroblastic cells, these transformed epithelial cells deposited and retained connective tissue glycoproteins in their pericellular matrices. The results indicate that viral transformation modulates the pericellular matrix and surface glycoproteins of cultured mouse epithelial cells. The ability of virus-transformed epithelial cells to deposit pericellular matrices is a major difference between them and virus-transformed fibroblastic cells.

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Abstract: Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.

PericellularMatrixandCell SurfaceGlycoproteins of Virus transformedMouseEpithelialCells

Abstract: Mouse embryo Mus musculus castaneous epithelial cells, transformed with Moloney murine sarcoma virus (MSV) or with ecotropic murine leukemia virus (MuLV), were analyzed for production of pericellular matrix glycoproteins. The nontrans formed, MSV-transformed, and MuLV-transformedcells pro duced fibronectin, laminin, type I collagen, and small amounts of type III collagen when studied by immunofluorescenceusing specific antibodies. Thevirus-transformed epithelial cellspro duced enhanced amounts of fibronectin into their growth me dia. NontransformedM. muscu!us castaneaus epithelial cells mainly produced type I collagen, as shown by metabolic label ing and polypeptide analysis. A significant increase in the glycoprotein production was seen by the MuLV-transformed cells,whereas smallchanges inthecollagen production were apparent after MSV transformation. MuLV-transformed cells produced increased amountsof type I collagen and also some collagenous polypeptides that comigrated with procollagen type IV chains. The ratio of the procollagen type I chains deposited in the matrix was altered in transformedcells. Radio active surface labeling of the cells revealed changes of the high-molecular-weightglycoproteins in both the MSV- and the MuLV-transformedcells. Unlike virus-transformed fibroblastic cells, these transformedepithelial cells deposited and retained connective tissue glycoproteins in their pericellular matrices. The results indicate that viral transformation modulates the pericellular matrix and surface glycoproteins of cultured mouse epithelial cells. The ability of virus-transformedepithelial cells to deposit pericellular matrices is a major difference between them and virus-transformedfibroblastic cells.