Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

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Cell surface heparan sulphate and adhesive property of sublines of rat ascites hepatoma AH7974

Journal of Cell Science ◽

10.1242/jcs.90.4.683 ◽

1988 ◽

Vol 90 (4) ◽

pp. 683-689 ◽

Cited By ~ 1

Author(s):

A. Kimura ◽

T. Kawaguchi ◽

T. Ono ◽

A. Sakuma ◽

Y. Yokoya ◽

...

Keyword(s):

Cell Surface ◽

Culture Medium ◽

Heparan Sulphate ◽

Soluble Form ◽

Foetal Calf Serum ◽

Serum Free Medium ◽

Free Medium ◽

Ascites Hepatoma ◽

Serum Free ◽

Rat Ascites Hepatoma

Two variants (74AD and 74FL) established from rat ascites hepatoma AH7974 were examined for the production of glycosaminoglycans in culture. There was no difference between the adhesive (74AD) and the floating (74FL) variants in quantity of glycosaminoglycans produced by their cultivation in minimum essential medium supplemented with 10% foetal calf serum. However, they were distinctly different in the distribution patterns of heparan sulphate. In 74FL, about 70% of total heparan sulphate was found in the culture medium in soluble form, whereas in 74AD, only 7% was found in the medium and the rest was in the cell-substratum complex. In a serum-free medium, 74AD cells grew without adhering to the substratum. After cultivation, more than 90% of total heparan sulphate was found in the cell-associated fractions and the rest in the substratum fractions. No heparan sulphate was detected in the culture medium. On the other hand, 74FL cells released heparan sulphate to the serum-free medium as much as to the serum-containing medium. The increase in amount of heparan sulphate in the culture medium of 74FL cells was supposed to be caused by failure of the cells to deposit heparan sulphate at the cell surface and not caused by increased production. Cell-substratum adhesion mechanisms involving cell surface heparan sulphate (heparan sulphate proteoglycan) and some serum intermediate(s) are discussed for 74AD cells.

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Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface

Biochemical Journal ◽

10.1042/bj2380721 ◽

1986 ◽

Vol 238 (3) ◽

pp. 721-728 ◽

Cited By ~ 27

Author(s):

D McV Ward ◽

J Kaplan

Keyword(s):

Growth Factor ◽

Cell Surface ◽

Transferrin Receptor ◽

Transferrin Receptors ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Surface Receptor ◽

Receptor Number ◽

Receptor Distribution

Incubation of serum-growth HeLa cells in serum-free medium causes a rapid (t1/2 3 min) 30-60% decrease in the binding of 125I-diferric transferrin to the cell surface. Addition of fetal bovine serum to cells in serum-free medium results in a rapid (t1/2 3 min) and concentration-dependent increase in binding activity. The loss or gain in ligand binding is a result of changes in surface receptor number rather than an alteration in ligand-receptor affinity. A variety of hormones (insulin, insulin-like growth factor, interleukin 1 and platelet-derived factor) were found to mimic the effect of serum on receptor number. The alteration in surface receptor number was found to be calcium-dependent. Changes in surface receptor number were independent of either receptor biosynthetic rate or the absolute cellular content of receptors. The effect of insulin or serum on Hela cell transferrin receptor distribution was unaffected by the presence of transferrin, demonstrating that receptor distribution in this cell type is ligand-independent. The ability of serum or insulin to modify surface transferrin receptor number was also observed in mouse L-cells, human skin fibroblasts, and J774 macrophage tumour cells. However, transferrin receptors on K562 and Epstein-Barr virus-transformed human lymphoblasts were unaltered by these agents. The quantities of receptors whose distribution is predominantly on the surface (i.e. epidermal growth factor or low density lipoprotein receptor) were unaltered by addition of the mitogenic agents. These results extend our previous studies [H.S. Wiley & J. Kaplan (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7456-7460] demonstrating that mitogenic agents can induce redistribution of receptor pools in selected cell types.

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Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082418 ◽

1978 ◽

Vol 36 (2) ◽

pp. 310-311

Author(s):

W. Liebrich

Keyword(s):

Hela Cells ◽

Calf Serum ◽

Glass Tube ◽

Serum Free Medium ◽

Nacl Solution ◽

Free Medium ◽

Minimum Essential Medium ◽

Serum Free ◽

All Solutions ◽

Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

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Hemoglobin Enhances the Binding of Bacterial Endotoxin to Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650565 ◽

1996 ◽

Vol 76 (02) ◽

pp. 258-262 ◽

Cited By ~ 12

Author(s):

Robert I Roth

Keyword(s):

Endothelial Cells ◽

Binding Protein ◽

Bacterial Endotoxin ◽

Dependent Manner ◽

Serum Free Medium ◽

Free Medium ◽

Human Endothelial Cells ◽

Serum Factors ◽

Serum Free ◽

Lps Binding

SummaryHuman endothelial cells, when incubated with bacterial endotoxin (lipopolysaccharide, LPS), modify their surface in association with prominent production of procoagulant tissue factor (TF) activity. This deleterious biological effect of LPS has been shown previously to be enhanced approximately 10-fold by the presence of hemoglobin (Hb), a recently recognized LPS binding protein that causes disaggregation of LPS and increases the biological activity of LPS in a number of in vitro assays. The present study was performed to test the hypothesis that Hb enhances the LPS-induced procoagulant activity of human umbilical vein endothelial cells (HUVEC) by increasing LPS binding to the cells. The binding of 3H-LPS to HUVEC was determined in the absence or presence of Hb or two other known LPS-binding proteins, human serum albumin (HSA) and IgG. LPS binding was substantially increased in the presence of Hb, in a Hb concentration-dependent manner, but was not increased by HSA or IgG. Hb enhancement of LPS binding was observed in serum-free medium, indicating that there was no additional requirement for any of the serum factors known to participate in the interaction of LPS with cells (e.g., lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD14 (sCD14)). Hb enhancement of LPS binding also was observed in the more physiologic condition of 100% plasma. LPS-induced TF activity was stimulated by Hb, but not by HSA or IgG. In serum-free medium, TF activity was not stimulated under any of the conditions tested. Ultrafiltration of LPS was dramatically increased after incubation with Hb but not with HSA or IgG, suggesting that LPS disaggregation by Hb was responsible for the enhanced binding of LPS to HUVEC and the subsequent stimulation of TF activity.

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A study of serum free medium culture on the cloning efficiency of human bone marrow myeloid and erythroid progenitors

Pathology ◽

10.3109/00313029009063553 ◽

1990 ◽

Vol 22 (3) ◽

pp. 145-148 ◽

Cited By ~ 1

Author(s):

Z-H. Wu ◽

L.A. Dowton ◽

G. Georgiou ◽

D.D.F. Ma

Keyword(s):

Bone Marrow ◽

Human Bone ◽

Human Bone Marrow ◽

Cloning Efficiency ◽

Serum Free Medium ◽

Free Medium ◽

Erythroid Progenitors ◽

Serum Free ◽

Medium Culture

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THE INFLUENCE OF SERUM ON THE UPTAKE, CONVERSION AND ACTION OF DIHYDROTESTOSTERONE IN RAT PROSTATE GLANDS IN ORGAN CULTURE

Journal of Endocrinology ◽

10.1677/joe.0.0640289 ◽

1975 ◽

Vol 64 (2) ◽

pp. 289-297 ◽

Cited By ~ 17

Author(s):

ILSE LASNITZKI ◽

HILARY R. FRANKLIN

Keyword(s):

Organ Culture ◽

Horse Serum ◽

Target Tissue ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Human Pregnancy ◽

Human Male ◽

Columnar Cells

SUMMARY The influence of serum on the uptake, conversion and action of dihydrotestosterone in relation to the sex steroid binding protein, TeBG, has been investigated in rat ventral prostates in organ culture. The organs were incubated with [1,2-3H]dihydrotestosterone in: (1) serum-free medium, (2) horse serum, foetal and newborn bovine serum or (3) human male and human pregnancy serum. With all sera the uptake of dihydrotestosterone fell with rising serum concentration, at first steeply and then more gradually. At the same concentration, the uptake was significantly lower in explants incubated with human pregnancy serum than in those kept with human male serum. The conversion of dihydrotestosterone to androstanediol followed the same pattern and less androstanediol was formed in the presence of pregnancy serum. Since pregnancy serum contains higher amounts of TeBG than male serum, the lowered uptake suggests that only the free hormone was available to the target organ. Addition of unlabelled dihydrotestosterone resulted in a higher uptake than that measured in explants incubated with the labelled steroid only. The effect of the human sera on uptake and conversion was correlated with the androgenic activity of dihydrotestosterone applied at physiological concentrations and expressed as the percentage of secretory columnar cells present. The degree of maintenance closely corresponded to the uptake of the hormone. In serum-free medium, the number of columnar cells approached the values found in vivo, with male serum their number, though reduced, was still substantial, with pregnancy serum it was extremely low. It is concluded that the amounts of TeBG present in serum regulate the supply of the hormone to the target tissue and thus control its biological action.

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