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M Ponec

Publications and source records attributed to M Ponec.

At least 145 records · Page 8Linked to original sources
PubMed1984

Defective low-density lipoprotein metabolism in cultured, normal, transformed, and malignant keratinocytes.

To obtain more information on differences in cellular behavior during the differentiation process, a number of types of epithelial cells with and without a defect in cornified envelope formation were compared as to the regulation of intracellular cholesterol synthesis by low-density lipoprotein (LDL) and the uptake and degradation of [125I]LDL. The following cells were cultured: normal skin fibroblasts (F), normal (K) and SV 40 transformed (SVK14) keratinocytes, and a number of squamous carcinoma cell (SCC) lines in which the defective terminal differentiation was found to occur in the following order of intensity: SCC-12F2 less than SCC-25 approximately equal to SCC-15 less than SCC-12B2 less than SCC-4. Compared with normal human fibroblasts, most of the cells under study showed a defective response to changes of the extracellular serum LDL concentration. The degree of inducibility of cholesterol synthesis after the cells were deprived of extracellular sources of cholesterol as well as the degree of the LDL-induced suppression of the intracellular cholesterol synthesis in cells preincubated in medium supplemented with lipoprotein-deficient serum decreased in the following order: F greater than SCC-4 greater than SCC-15 approximately equal to SCC-25 greater than SCC-12B2 congruent to SCC-12F2 greater than SVK14 approximately equal to K. A defect in LDL metabolism was found to be responsible for the partial or complete failure of LDL to regulate the cholesterol metabolism, because when sterol was delivered to all cell types in artificial nonlipoprotein form (i.e., as 25-hydroxycholesterol) a marked suppression of cholesterol synthesis was observed. For all SCC lines tested except SCC-12B2 good correlation was found between the degree of LDL-induced suppression of cholesterol synthesis and the decreasing ability of cells to differentiate into squames or cornified envelope-forming cells. The transformation of keratinocytes by SV 40 virus did not lead to any change in the response of the cells to changes in the extracellular LDL concentration since both the normal and the transformed keratinocytes showed the same response to LDL (i.e., no response).

Carcinoma, Squamous Cell↗
PubMed1984

Monoclonal antibodies with different specificities against cytokeratins. An immunohistochemical study of normal tissues and tumors.

Monoclonal antibodies against cytokeratins, isolated from human callus, were prepared. Here we describe the characterization of three of these monoclonal antibodies (Clones 77, 78, and 80) with special emphasis on the staining characteristics of a number of normal epithelial tissues and a series of tumors. On thin sections and in immunoblotting experiments our monoclonal antibodies showed different specificities. In immunoblots Clone 80 stained more bands in preparations of cytokeratin from callus, cultured keratinocytes, and a metastasis of a lung carcinoma than Clones 77 and 78. In this last cytokeratin preparation Clones 77 and 78 each stained a separate band not stained by Clone 80. In normal tissues Clone 80 stained all types of epithelia except myoepithelium and podocytes of glomeruli. Clone 78 stained mainly squamous epithelium. Clone 77 stained differentiated squamous epithelium, transitional epithelium, ductal epithelium, and parenchymatous epithelium. These results confirm the heterogeneity of cytokeratins. In neoplasms Clone 77 was positive with adenocarcinoma and squamous cell carcinoma. Clone 78 stained squamous cell carcinoma and well-differentiated adenocarcinoma. Clone 80 stained all epithelial tumors, including anaplastic carcinoma, and is therefore useful in tumor diagnosis.

Animals↗
PubMed1983

Biphasic entry of glucocorticoids into cultured human skin keratinocytes and fibroblasts.

The uptake of glucocorticoids by cultured human skin keratinocytes and fibroblasts was found to be a rapid, temperature-sensitive process. All glucocorticoids tested accumulated in the cells, and the ratio between the intracellular and extracellular concentrations (ci/co ratio) was higher than 1. For most of the glucocorticoids under study there was good correlation between the lipophilicity and the rate of uptake. Since the uptake of glucocorticoids seems to be unsaturable in the concentration range used and no competition was observed between these compounds for entry into the cells, it may be assumed that the uptake of glucocorticoids is essentially a simple diffusion process based on a distribution of glucocorticoids between a lipid-rich phase and water. The analysis of the uptake process revealed that the entry of glucocorticoids into cultured human skin fibroblasts and keratinocytes is a non-mediated passive diffusion process that involves two distinct steps: a rapid, non-specific, high-capacity association to the cell membrane followed by a slower internalization process associated with a stronger binding of glucocorticoids within the cell.

Biological Transport, Active↗
PubMed1983

Cultured human skin fibroblasts and keratinocytes: differences in the regulation of cholesterol synthesis.

The regulation of cholesterol synthesis in cultured human skin fibroblasts and keratinocytes was compared, the incorporation of [14C]-acetate or [14C]-octanoate into [14C]-cholesterol being taken as a measure of de novo cholesterol synthesis. The two types of cultured cells differed in the following features of the regulation of cholesterol synthesis: (1) Keratinocytes synthesized 10-fold more cholesterol/mg cell protein. (2) Keratinocytes retained a greater amount of the de novo synthesized cholesterol intracellularly, and fibroblasts released it to a much higher degree into the culture medium. (3) When the extracellular environment was deprived of cholesterol, the intracellular synthesis remained virtually unchanged in keratinocytes but increased markedly in fibroblasts. (4) The low-density lipoproteins (LDL) that enter the cells by receptor-mediated endocytosis and are then degraded in lysosomes, liberate cholesterol, which in turn interferes with the intracellular cholesterol synthesis. The lipoproteins strongly suppress cholesterol synthesis in fibroblasts, but do not have this effect in keratinocytes. (5) When added to the culture medium, nonlipoprotein cholesterol produced no effect on cholesterol synthesis in keratinocytes, whereas fibroblasts showed a marked suppression of this synthesis. The addition of 25-hydroxycholesterol to the culture medium led to a strong suppression of cholesterol synthesis in both fibroblasts and keratinocytes. These findings suggest that in both cell types the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity can be suppressed by a sterol delivered to the cell in artificial nonlipoprotein form. (6) The amount of [125I]-LDL bound specifically to the cell membrane receptor and particularly the amount internalized and degraded by the cells is much lower in keratinocytes than in fibroblasts, as shown biochemically. In the ultrastructural studies no binding of LDL to keratinocytes was observed.

Cell Count↗
PubMed1982

Glucocorticoids and cultured human skin cells: specific intracellular binding and structure-activity relationships.

Cytosol fractions of both cultured human skin fibroblasts and epidermal keratinocytes contain macromolecules that bind glucocorticoids with high affinity. Using the cytosol of cultured keratinocytes good correlation has been found between the displacement of radiolabelled glucocorticoids (3H-hydrocortisone, 3H-dexamethasone and 3H-triamcinolone acetonide) by a variety of glucocorticoids and the clinical efficacy. The structure-binding relationship studies revealed the relative contribution of various substituents on the steroid molecule for the binding affinity of the steroids. The introduction of a double bond at C1-C2, the presence of 9 alpha-fluoro atom or 11 beta-hydroxy group and the esterification of the 17 alpha-hydroxy group caused an increase, whereas esterification of the hydroxy group on C21 leads to marked decrease of the affinity of the steroid.

Binding, Competitive↗
PubMed1981

Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte macrophages.

It is commonly assumed that the low density lipoprotein (LDL) degradation in extra-hepatic cells proceeds partly by way of the high-affinity LDL uptake process. In addition, it has been suggested that extra-hepatic LDL catabolism proceeds partly by way of preferential uptake and subsequent degradation of LDL by phagocytic scavenger cells. In order to study the effect of LDL and high density lipoprotein (HDL) on cholesterol metabolism in such scavenger cells, cultured human or pig monocytes were incubated with varying amounts of human or pig LDL, HDL and LDL/HDL mixtures, and the incorporation of [14C]acetate into cholesterol was measured. The addition of LDL suppressed endogenous cholesterol synthesis. Incubation of monocytes with LDL/HDL mixtures reduced the total amount of de novo synthesized cholesterol to the same extent as incubation with LDL alone. Our results suggest that in cultured monocyte macrophages HDL does not influence endogenous cholesterol synthesis, even in the presence of LDL.

Acetates↗
PubMed1981

Corticoids and cultured human epidermal keratinocytes: specific intracellular binding and clinical efficacy.

Cytosol of cultured human epidermal keratinocytes contains macromolecules, that bind corticoids with high affinity. Binding constants were in the same range for cultured keratinocytes originating from different individuals and did not change during serial cell cultivation. At 0 degree C and using 3H-triamcinolone acetonide as ligand, we obtained the following values; apparent Bmax = 160- 250 fmoles/mg protein and Kdiss = 3.1-5.0 nM; with 3H-dexamethasone Bmax = 200-350 fmoles/mg protein, Kdiss = 6.0-11.1 nM, and with 3H-hydrocortisone Bmax = 140-220 fmoles/mg protein, Kdiss = 17-25 nM. There was an indication that the binding capacity of the receptor system is higher the younger the age of the skin donor. A number of steroids and corticoids commonly used in dermatological practice were tested with respect to displacement of 3H-triamcinolone acetonide, 3H-dexamethasone, and 3H-hydrocortisone from binding sites in cytosol. Good correlation between clinical efficacy and specific binding was observed for all 3 ligands. Other steroids such as 17-beta estradiol and nandrolone did not show any affinity for the corticoid binding system. Progesterone displace 3H-corticoids from their binding sites, but the IC50 was of one order of magnitude larger.

Adrenal Cortex Hormones↗
PubMed1980

Corticoids and human skin fibroblasts: intracellular specific binding in relation to growth inhibition.

The binding of 3H-triamcinolone acetonide to soluble macromolecules of cultured human skin fibroblasts was studied in an attempt to explain the mechanism underlying the inhibitory effects of glucocorticoids on cell growth. The results were as follows: Cultured human skin fibroblasts contain in cytosol a high affinity binding system for glucocorticoids. Various glucocorticoid derivatives competed for specific binding of 3H-triamcinolone acetonide. In some but not all instances this competition was related to the clinical efficacy of the derivatives under study and to their potency for the inhibition of cell growth. A specific glucocorticoid binding system was detectable in steroid-sensitive, low-density cell cultures (apparent Bmax = 200 fmoles/mg protein). The number of steroid binding sites was lower in high-density cell cultures (apparent Bmax = 125 fmoles/mg protein). The sensitivity to growth inhibition by glucocorticoids was markedly decreased in the high-density cell cultures. There were no differences in the affinity constants between these cell cultures (Kdiss. = 3.3 X 10-9 M). When cells were grown in medium containing glucocorticoid, renewal of the incubation medium led to disappearance of the growth-inhibitory effects, whereas specific binding was not affected. Nandrolone, an inhibitor of cell growth, abolished the growth-inhibitory effects of glucocorticoids but did not displace 3H-triamcinolone acetonide from its binding sites. The results suggest that in addition to a mechanism mediated by a glucocorticoid binding system with receptor like properties also other factors as well appear of relevance for the control of cell growth. These factors may be beyond the actual binding process of steroid and involve the action at the level of genomic expression of the cell.

Animals↗
PubMed1979

Effects of glucocorticosteroids on cultured human skin fibroblasts. III. Transient inhibition of cell proliferation in the early growth stages and reduced susceptibility in later growth stages.

An immediate depression of the rate of cell proliferation occurred upon addition of glucocorticosteroids to cultures of human skin fibroblasts in the early growth stages. A reduced sensitivity or even insensitivity of the fibroblasts to growth inhibition inhibition was found upon the addition of the steroids at later stages of cell growth, when the cell density has increased. The inhibition in the early growth stages is transient and is most pronounced if the cultured medium is not renewed. This transient inhibition is not due to the development of steroid-resistant cell lines, and resembles the effect called tachyphylaxis, as is also observed in vasoconstriction tests.

Betamethasone Valerate↗
PubMed1979

Penetration of various corticosteroids through epidermis in vitro.

The penetration through the epidermis in vitro of various topically used corticosteroids was compared. The amount penetrating through the epidermis from an ethanolic solution showed good correlation with the polarity of the corticosteroids under study. Hydrocortisone, the most polar corticosteroid, penetrates the epidermis the most rapidly; clobetasone butyrate, the least polar, the slowest. Other corticosteroids, i.e., hydrocortisone-17-butyrate, triamcinolone acetonide, and clobetasol-17-propionate, form an intermediate group whose penetration rates and polarities decrease in the indicated sequence. The corticosteroid generally accepted as having greater clinical efficacy in creams or ointments did not permeate better from an ethanolic solution in vitro.

Administration, Topical↗
PubMed1979

Effects of glucocorticosteroids on cultured human skin fibroblasts. V. Influence of anabolic steroids on the inhibitory effects of clobetasol-17-propionate on cell proliferation and collagen synthesis.

As previously found, the glucocorticosteroid clobetasol-17-propionate inhibits cell proliferation during the early growth stage of normal baby foreskin fibroblasts and collagen synthesis in confluent cultures of these cells. The degree of inhibition of cell proliferation decreases with increasing cell density and, moreover, is transient. The anabolic steroids nandrolone and nandrolone-phenyl-propionate have similar effects on these cells. Likewise the magnitude of the inhibition is dose-dependent. When present together the two types of drug do not act in an additive manner. Even at low concentrations the anabolic steroids abolish the inhibitory effect of the glucocorticosteroid on cell proliferation. Furthermore, in this case only the inhibitory effect of the glucocorticosteroid on collagen synthesis is found and there is no further increase in this effect due to the presence of the anabolic steroids. Our results imply that the use of low concentrations of anabolic steroids combined with glucocorticosteroids in topical application to the skin may abolish some of the undesirable side effects of the glucocorticosteroids.

Betamethasone↗
PubMed1977

Effects of glucocorticosteroids on primary human skin fibroblasts. I. Inhibition of the proliferation of cultured primary human skin and mouse L929 fibroblasts.

Various glucocorticosteroids were added to logarithmically growing cultures of primary human skin fibroblasts and of mouse L929 fibroblasts. These steroids inhibited proliferation of the human fibroblasts at concentrations which fall in a range expected to occur during the topical treatment of skin disorders. In terms of the concentrations required for the inhibition hydrocortisone was least and clobetasol-17-propionate most effective. All other steroids studied (hydrocortisone-17-butyrate, triamcinolone acetonide, betamethasone-17-valerate and hydrocortisone-21-acetate) showed medium effectiveness. Fluorination as such may not enhance the inhibitory effect. The inhibition was independent of the source (baby foreskin or adult arm skin) and passage number (7th to 13th or 15th and 16th passage, respectively) of the cells. The possible relationship between the inhibition of cell proliferation by such steroids and their therapeutic effect in psoriasis and their atrophic side effects is discussed. Mouse L929 fibroblasts were affected at 10(3)--10(4)-fold lower steroid concentrations and the range of the effective concentrations was 10(4)--10(5) times as wide as that for the primary human skin fibroblasts. It was concluded that these mouse fibroblasts are a poor model system for the study of in vivo effects of glucocorticosteroids in man.

Administration, Topical↗
PubMed1977

Effects of glucocorticosteroids on primary human skin fibroblasts. II. Effects on total protein and collagen biosynthesis by confluent cell cultures.

Confluent cultures of normal primary human skin fibroblasts were incubated with various glucocorticosteroids which are in current use clinically for the treatment of various skin disorders. For all steroids concentrations were found at which collagen hydroxyproline formation was inhibited, while total protein synthesis was little affected. The concentration effective for inhibition was highest for hydrocortisone and lowest for clobetasol-17-propionate. All other steroids (hydrocortisone-17-butyrate, triamcinolone acetonide and betamethasone-17-valerate) showed medium effectiveness. Fluorination as such was not a factor in the degree of inhibition. The inhibition observed was shown to be independent of concomitant specific effects on cell proliferation or cell turnover. The possible implications of these findings on the therapeutic effects in psoriasis and the frequently occurring atrophic side-effects of these steroids are discussed.

Administration, Topical↗