scholarly journals Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

1981 ◽  
Vol 88 (2) ◽  
pp. 352-357 ◽  
Author(s):  
EG Hayman ◽  
E Engvall ◽  
E Ruoslahti
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Cell Attachment ◽  
Culture Media ◽  
Rat Kidney ◽  
Cell Types ◽  
Transformed Cells ◽  
Kidney Cells ◽  
Normal Cells ◽  
The Matrix

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.

scholarly journals Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells.

1982 ◽  
Vol 94 (1) ◽  
pp. 28-35 ◽  
Author(s):  
E G Hayman ◽  
A Oldberg ◽  
G R Martin ◽  
E Ruoslahti
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Rat Kidney ◽  
Heparan Sulfate Proteoglycan ◽  
Chondroitin Sulfate Proteoglycan ◽  
Transformed Cells ◽  
Kidney Cells ◽  
Sulfate Proteoglycan ◽  
Normal Rat

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

scholarly journals Loss of thrombospondin transcriptional activity in nickel-transformed cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 851-858 ◽  
Author(s):  
K Salnikow ◽  
S Cosentino ◽  
C Klein ◽  
M Costa
Keyword(s):  
Chinese Hamster ◽  
Restriction Enzymes ◽  
Cell Types ◽  
Suppressor Gene ◽  
Transformed Cells ◽  
Normal Cells ◽  
Nickel Compounds ◽  
Embryo Cells ◽  
Rb Gene

mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformed cells, suggesting that the promoter region of thrombospondin was less efficiently transcribed in Ni-transformed cells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumor suppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of an expression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondin promoter such that the expression was higher in normal cells than in transformed cells.

Polylactosaminoglycan modification of a small integral membrane glycoprotein, influenza B virus NB

1988 ◽  
Vol 8 (3) ◽  
pp. 1186-1196
Author(s):  
M A Williams ◽  
R A Lamb
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Gel Filtration ◽  
Cell Types ◽  
Influenza B Virus ◽  
Influenza B ◽  
Membrane Glycoprotein ◽  
Intact Cells ◽  
B Virus ◽  
Carbohydrate Chains

The structure of the carbohydrate components of NB, the small integral membrane glycoprotein of influenza B virus, was investigated. The carbohydrate chains of NB are processed from the high-mannose form (NB18) to a heterogeneous form of much higher molecular weight, designated NBp. Selection of this carbohydrate-containing form of NB with Datura stramonium lectin, its susceptibility to digestion by endo-beta-galactosidase, and determination of the size of NBp glycopeptides by gel filtration chromatography suggested that the increase in molecular weight is due to processing to polylactosaminoglycan. Investigation of the polypeptides produced by influenza B/Lee/40 virus infection of several cell types and another strain of influenza B virus suggested that the signal for modification to polylactosaminoglycan is contained in NB. Expression of mutants of NB lacking either one or both of the normal N-terminal sites of asparagine-linked glycosylation indicated that both carbohydrate chains are modified to contain polylactosaminoglycan. NBp and a small amount of unprocessed NB18 are expressed at the infected-cell surface, as determined by digestion of the surfaces of intact cells with various endoglycosidases. Unglycosylated NB, expressed either in influenza B virus-infected cells treated with tunicamycin or in cells expressing the NB mutant lacking both N-linked glycosylation sites, was expressed at the cell surface, indicating that NB does not require carbohydrate addition for transport.

STUDIES ON SMALL MOLECULAR WEIGHT ADHESION PROTEINS (SAPs) FROM CONNECTIVE TISSUES

1987 ◽  
Author(s):  
S Wasi ◽  
P Alles ◽  
D Gauthier ◽  
U Bhargava ◽  
J Farsi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyclonal Antibody ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Binding Capacity ◽  
Guanidine Hydrochloride ◽  
Cell Types ◽  
Type I ◽  
Cell Binding ◽  
Connective Tissues

We have identified a family of low molecular weight proteins with cell attachment properties in a variety of soft and mineralised connective tissues (Wong et al., Biochem. J. 232, 119, 1985). For further characterisation of these proteins we extracted porcine bones with 4 M guanidine hydrochloride and purified the proteins on a series of gel filtration columns The purifed SAPs comprise three bands with Mr -14 000 -17 000. All three proteins bound to heparin-sepahrose in both the presence and absence of 4M urea, and when eluted with 2 M NaCl they retained their cell binding capacity. These proteins promoted the adhesion and spreading of a variety of cell types, including normal fibroblasts, osteoblasts, and epithelial cells, and tumour (osteosarcoma) cells. On Western blotting SAPs did not cross-react with antibodies against fibronectin, laminin or type I collagen; however, they were recognised by a monoclonal antibody to human vitronectin, a polyclonal antibody to bovine vitronectin and polyclonal antibody to human somatomedin B. Dose response experiments indicated that maximum attachment of human gingival fibroblasts occurred in the presence or absence of fetal bovine serum on wells precoated with 2.5 μg/cm2 of SAPs. Attachment of cells to these proteins was partially inhibited by the synthetic pentapeptide Gly-Arg-Gly-Asp-Ser. Utilising the nitrocellulose cell binding assay of Hayman et al (J. Cell. Biol. 95, 20, 1982), the cell attachment to these proteins could be completely inhibited by heparin (100 units/mL) whereas up to 1000 units/mL of heparin had no inhibitory effect on cell attachment to fibronectin and vitronectin. The occurrence of these proteins in a variety of connective tissues and their recognition by different cell types may reflect their general biological role in adhesive mechanisms in both hard and soft connective tissues. Currently, we are investigating the relationship between SAPs and vitronectin, since it is possible that SAPs represent a tissue-processed form of vitronectin or may be novel attachment proteins with regions of homology with vitronectin

scholarly journals Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors.

1988 ◽  
Vol 107 (6) ◽  
pp. 2351-2361 ◽  
Author(s):  
J Lawler ◽  
R Weinstein ◽  
R O Hynes
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Attachment ◽  
Solid Phase ◽  
Rat Kidney ◽  
Muscle Cells ◽  
Cell Types ◽  
Adhesive Properties ◽  
Adhesive Proteins

Thrombospondin is a 420,000-D glycoprotein that has recently been shown to have several properties in common with the members of a class of adhesive proteins. To characterize further the adhesive properties of thrombospondin, we have studied its ability to support cell attachment. Thrombospondin adsorbed to plastic dishes supports the attachment of human endothelial and smooth muscle cells and the monocyte-like cell line (U937) as well as normal rat kidney cells. The majority of attached cells do not spread on the solid-phase thrombospondin. The attachment of all four cell types to thrombospondin is abolished if the assay is performed in the presence of EGTA, although the cells still attach to fibronectin. If thrombospondin is adsorbed to the dishes in the presence of EGTA and then washed with buffer containing calcium before addition of the cells, attachment is still markedly inhibited, indicating that calcium affects the conformation and function of thrombospondin. Attachment of all four cell types is also markedly inhibited by the synthetic peptides gly-arg-gly-asp-ser-pro (GRG-DSP) and gly-arg-gly-asp-ala-cys (GRGDAC) but not by the control peptide gly-arg-gly-glu-ser-pro (GRG-ESP). Affinity chromatography of n-octylglucoside extracts of surface-labeled endothelial cells or smooth muscle cells on thrombospondin-Sepharose and GRG-DSP-Affigel columns was used to identify an integrin complex related to glycoprotein IIb-IIIa as an RGD-dependent receptor for thrombospondin. In addition, a monoclonal antibody (LM609) that blocks attachment of endothelial cells to vitronectin, fibrinogen, and von Willebrand factor also inhibits attachment of endothelial cells to thrombospondin. These data indicate that the attachment of cells to thrombospondin is mediated by RGD and calcium-dependent mechanisms and is consistent with the hypothesis that the GRGDAC sequence in thrombospondin is a site for interaction with an integrin receptor of the beta 3 subclass.

scholarly journals The nature of conditioning nutrients for human malignant melanoma cultures

1983 ◽  
Vol 62 (1) ◽  
pp. 249-266
Author(s):  
K.A. Ellem ◽  
G.F. Kay
Keyword(s):  
Molecular Weight ◽  
Cell Density ◽  
Cell Attachment ◽  
Cell Types ◽  
Fresh Medium ◽  
Low Molecular Weight ◽  
High Concentration ◽  
Cell Content ◽  
Human Malignant Melanoma ◽  
Serum Factors

From a human melanoma line (MM96), showing some dependence of its rate of growth and cell attachment on serum concentration, sublines were selected for even greater dependence on serum factors. These sublines were used to identify the production of substances by other melanoma cells in culture that would supplement or replace the requirement for serum. Most of the sublines showed higher colony-forming efficiency in medium conditioned by one of several cell types in the presence of a low concentration of serum (2.5%) compared with fresh medium containing a high concentration of serum (10%). The conditioning factor(s) were found to be moderately heat-stable, nonlipophilic, and to be of low molecular weight (less than or greater than 400). Screening of a variety of non-essential low molecular weight nutrients, which have been reported to potentiate the growth of a variety of cell types in low-density culture, was positive for the MM96 sublines only for pyruvate. In particular, L-alanine, L-serine, putrescine and alpha MSH (melanocyte-stimulating hormone) were ineffective. Despite the problems of comparing conditioned media with fresh medium, a reasonable correlation between the stimulatory effect and the cell content of added 2-oxocarboxylates was apparent. As would be anticipated, MM96 cultures showed a population density-dependent enhancement of growth up to a cell density of 2 to 4 × 10(4) cells cm-2. Further increase in the initial cell density of these cultures led to a decline in growth rate. An important additional observation was that simple dilution of the ingredients of RPMI1640 with phosphate-buffered saline or Hanks' balanced salt solution led to a reversal of growth inhibition accompanying a serum shift-down.

scholarly journals Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia

1991 ◽  
Vol 99 (2) ◽  
pp. 213-225 ◽  
Author(s):  
S.C. Mueller ◽  
W.T. Chen
Keyword(s):  
Cell Surface ◽  
Early Stage ◽  
Matrix Material ◽  
Transformed Cells ◽  
Thin Sections ◽  
Rous Sarcoma ◽  
The Matrix ◽  
Intracellular Compartments ◽  
Beta 1 Integrin ◽  
Cell Invasiveness

We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, beta 1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, beta 1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing beta 1 and several alpha chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, beta 1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with beta 1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of beta 1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.

Secretion of Erythropoietin from Microencapsulated Rat Kidney Cells: Preliminary Results

1993 ◽  
Vol 16 (7) ◽  
pp. 557-560 ◽  
Author(s):  
J. Koo ◽  
T.M.S. Chang
Keyword(s):  
Cell Culture ◽  
Trypan Blue ◽  
Culture Media ◽  
Rat Kidney ◽  
Free Cell ◽  
Kidney Cells ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Free Cells ◽  
The Media

Rat kidney epithelial cells were microencapsulated within alginate-poly(L)lysinealginate membrane. The microencapsulated cells were incubated using a culture media containing cobalt and another without cobalt. The viability was measured by trypan blue exclusion test. Secretion of erythropoietin (EPO) was measured by radioimmunoassay (RIA). Viability of free cells was 53%. The viability of microencapsulated cells increased to 72% after 12 days of incubation and remained at this level. Samples of the culture media were collected every 2 days for RIA. Samples within the microcapsules were collected by breaking the microcapsules open. RIA of these samples showed the following for the media containing cobalt. Between day 16 and day 32 the concentrations of EPO were 5.3 mU/ml inside and 18.3 mU/ml outside the microcapsule. The medium from the same number of free cells contained 21.2 mU/ml of EPO. Culture media without cobalt collected during the same period contained 1.8 mU/ml inside and 9.9 mU/ml outside the microcapsules. The free cell culture with this media during the same period contained 8.3 mU/ml.

Surface Modification and Electrophoresis of Normal and Transformed BHK21 Cells

1974 ◽  
Vol 14 (1) ◽  
pp. 203-214
Author(s):  
A. L. LATNER ◽  
G. A. TURNER
Keyword(s):  
Electrophoretic Mobility ◽  
Cell Types ◽  
Transformed Cells ◽  
Normal Cells ◽  
Neuraminidase Treatment ◽  
Total Histone ◽  
Electrophoretic Mobilities ◽  
Formaldehyde Treatment ◽  
The Mean ◽  
Number Of Cells

The mean electrophoretic mobilities at pH 7.5 of virus-transformed (Py6) and normal BHK21 cells were very similar, whether they were harvested mechanically or by the use of trypsin. After formaldehyde treatment, there was significantly increased mobility in both cell types; the transformed cells showed significantly the greater change. After neuraminidase treatment, the mean electrophoretic mobility was decreased to the same extent in both types of cell. Treatment with neuraminidase and formaldehyde had no effect on the mean electrophoretic mobility of the normal cells but slowed the transformed variety. The mobility in histone solution had no relationship to histone concentration but was statistically correlated with the amount of histone per cell, calculated from total histone present divided by the total number of cells; a linear relationship being obtained with the normal cells but an initial plateau was demonstrated with the transformed cells. The normal cell line showed a similar plateau after neuraminidase treatment. The possible significance of these results is discussed.

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