| 1. |
- Magnusson, Karl-Eric, et al.
(author)
-
Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA Class 1, HLA Class 2, and neoplastic epithelial antigens) in the apical cell membrane
- 1990
-
In: Journal of Cellular Physiology. - : John Wiley & Sons. - 0021-9541 .- 1097-4652. ; 143:2, s. 381-390
-
Journal article (peer-reviewed)abstract
- We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic calcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subuni)-labelled ganglioside GM1 (diffusion coefficient, D [x 108] = 0.8–0.9 cm2s−1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 109] = 2 cm2s−1; R = 60–70%). However, antibody-labelled β2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was × 1.4 and R × 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were × 0.60 and × 0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobil-ity of different membrane components.
|
|
| 2. |
- Nånberg, Eewa, 1957-, et al.
(author)
-
Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation
- 1990
-
In: Journal of Cellular Physiology. - : Wiley-Blackwell Publishing Inc.. - 0021-9541 .- 1097-4652. ; 143:2, s. 232-242
-
Journal article (peer-reviewed)abstract
- To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.
|
|
| 3. |
- Parrow, Vendela, et al.
(author)
-
Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells
- 1992
-
In: Journal of Cellular Physiology. - : Wiley-Blackwell Publishing Inc.. - 0021-9541 .- 1097-4652. ; 152:3, s. 536-544
-
Journal article (peer-reviewed)abstract
- SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.
|
|
| 4. |
- Westergren-Thorsson, G, et al.
(author)
-
Proliferation of cultured fibroblasts is inhibited by L-iduronate-containing glycosaminoglycans
- 1991
-
In: Journal of Cellular Physiology. - : Wiley. - 0021-9541. ; 147:3, s. 30-523
-
Journal article (peer-reviewed)abstract
- We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth-promoting effects in densely seeded cultures.
|
|
