Biomedical subjects

J Kempenaar

Publications and source records attributed to J Kempenaar.

31 records · Page 2Linked to original sources

PubMed1989

Susceptibility of human male keratinocytes to MHC-restricted H-Y-specific lysis.

We studied the expression of the male-specific mH antigen H-Y on cultured human skin cells by investigating susceptibility to H-Y-specific cytolysis using conventional class I-restricted CTL clones in a modified cell-mediated cytotoxicity assay. In contrast to what was found in the rodent system, we observed H-Y-specific lysis of human male keratinocytes. Susceptibility for H-Y-specific lysis was efficiently enhanced by exposure of the keratinocytes to IFN-gamma. Our results demonstrate that human skin cells are equally sensitive for the activity of H-Y-specific CTLs as target cells of lymphoid origin. Finally, the cellular recognition of the H-Y mH antigen in the skin further supports its possible target function in the local graft versus host attack.

Cytotoxicity, Immunologic↗

PubMed1989

Toxicity screening of N-alkylazacycloheptan-2-one derivatives in cultured human skin cells: structure-toxicity relationships.

A number of N-alkylazacycloheptan-2-one derivatives, with the hydrocarbon chain lengths systematically varied from C2 to C16, were tested for their possible skin toxic effects. For this purpose, three in vitro cytotoxicity assays were used: (1) inhibition of proliferation of cultured human fibroblasts and keratinocytes; (2) inhibition of collagen contraction by human fibroblasts; and (3) cell morphology changes in confluent cultures of human fibroblasts and keratinocytes. With all assays used, the toxicity of N-alkylazacycloheptan-2-one derivatives increased from C2 to C8, remained constant at a hydrocarbon chain length between C8 and C14, and subsequently decreased with increasing alkyl chain length. A similar trend has been observed for flux enhancement of nitroglycerine in the presence of these N-alkylazacycloheptan-2-one derivatives, suggesting that with these compounds a parallelism exists between skin cell toxicity and penetration enhancing capacity. Since for practical use it is preferable to find a balance between skin toxicity and the penetration enhancement effect of a particular enhancer, it would be advisable to do QSAR studies of this kind with a number of congeners of a particular compound in order to optimize the choice. In this particular case, further modification of the N-alkylazacycloheptan-2-one structure might lead to an even better choice than the often propagated dodecyl derivative.

Cell Division↗

PubMed1989

Differentiation of cultured human keratinocytes: effect of culture conditions on lipid composition of normal vs. malignant cells.

Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover, cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid:neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on de-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes.

Cell Differentiation↗

PubMed1988

Lateral mobility of plasma membrane lipids in normal and transformed keratinocytes.

In this study we have examined possible differentiation-dependent modulations in plasma membrane lipid properties in normal keratinocytes, SV-40 transformed keratinocytes (SVK14) and a number of squamous carcinoma (SCC) cells. In normal keratinocytes the lateral diffusion coefficient of plasma membrane lipids (D) differs significantly for cells cultured permanently under low and normal Ca2+-conditions (5.16 x 10(-9) and 3.27 x 10(-9) cm2/s, respectively). When differentiation is induced by exposing low Ca2+-cultured cells to normal Ca2+ concentrations D increases to 7.07 x 10(-9) cm2/s during the initial hours of differentiation followed by a gradual sustained decrease to values also observed in cells cultured permanently under normal Ca2+-conditions. In SCC and SVK14 cells a similar initial transient increase in lateral lipid mobility is observed upon initiation of differentiation, but, in contrast to normal keratinocytes, no sustained decrease in D is seen upon prolonged culturing under normal Ca2+ conditions. The results indicate that the deficiency of the transformed cells to respond to Ca2+-induced differentiation might involve transformation-dependent alterations in membrane structure and function.

Calcium↗

PubMed1988

Proliferation and differentiation of human squamous carcinoma cell lines and normal keratinocytes: effects of epidermal growth factor, retinoids, and hydrocortisone.

Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca2+ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors.

Calcium↗

PubMed1988

Lipid composition of cultured human keratinocytes in relation to their differentiation.

The present study was undertaken to explore the possibility of the use of cultured human keratinocytes for the study of changes in lipid composition in relation to epidermal differentiation. In a submerged culture system, in which the stratification is incomplete, no significant differences have been found between the lipid composition of cells grown either at low calcium concentration (0.06 mM) (at which the keratinocyte differentiation is markedly retarded) or at normal calcium concentration (1.6 mM) (at which some differentiation takes place). Under these conditions the amount of phospholipids and sterols was high and that of ceramides was low. Furthermore, the acylglucosylceramides (AGC) and acylceramides (AC), the latter one known to be involved in water barrier function, were found to be absent. Contrary to this, both AGC and AC were found to be present in significant amounts in an air-exposed model using de-epidermized dermis (DED) as a substrate (in which, as judged from morphologic criteria, the extent of keratinocyte stratification is similar to that seen under the in vivo conditions). Fatty acid analysis revealed significantly lower content of 18:2 and higher content of 16:1 and 18:1 acids with all culture conditions used, as compared to the parent epidermis. This is probably a result of fatty acid levels and composition in fetal calf serum (which was used in the present study) that differ markedly from the in vivo situation. The 20:4 content was similar to that in the epidermis only in cells cultured under the submerged conditions, during which they have been found (Isseroff et al. 1987. J. Lipid Res. 28: 1342-1349) to be able to convert 18:2 to 20:4. In DED cultures, however, the 20:4 content was markedly lower. Under all culture conditions used, the triglyceride content was higher as compared to the non-cultured epidermis. The high content of triglycerides and the fatty acid composition of the various lipid fractions showed a resemblance with what is found in the epidermis in essential fatty acid-deficient animals. This resemblance was confirmed by electron micrographs which revealed the presence of some partially or completely empty lamellar bodies. The results of the present study suggest that the air-exposed culture model, in which the keratinocytes show a high extent of stratification, could be of great value in the study of epidermal lipid metabolism. However, further alterations in culture conditions are necessary to more closely approximate the lipid composition of noncultured epidermis.

Cell Differentiation↗

PubMed1987

Regulation of lipid synthesis in relation to keratinocyte differentiation capacity.

Cultured keratinocytes and squamous carcinoma cells provide a useful model system for studying the processes involved in the regulation of differentiation, as the differentiation capacity of the cells can be modulated experimentally by changing the extracellular calcium concentration. Furthermore, the squamous carcinoma cell lines exhibit a defect in their differentiation capacity which they express to different extents. In this paper, the effect of external lipoproteins has been studied on lipid synthesis in normal keratinocytes and three squamous carcinoma cell (SCC) lines which showed a decreasing capacity to differentiate in the order of normal keratinocytes greater than SCC-12F2 greater than SCC-15 greater than SCC-4. The ability of the cells to form cornified envelopes was taken as a measure of differentiation capacity. The rate of total lipid synthesis as well as the phospholipid-neutral lipid ratio decreased in the order SCC-4 greater than SCC-15 greater than SCC-12F2 greater than or equal to normal keratinocytes, clearly correlating with the differentiation capacity of the cells. Because of the high rate of phospholipid synthesis and the low rate of ceramide synthesis, it is concluded that, under these in vitro conditions used, the maturation of keratinocytes proceeds to a lesser extent than that seen under in vivo conditions. In proliferating cells, in which the low-density lipoprotein (LDL) receptor is operative to a high extent, the rate of lipogenesis, especially that of neutral lipids, responded dramatically to changes of extracellular lipoprotein concentration. In the presence of lipoproteins a marked decrease of cholesterol and triacylglycerol synthesis and an increase of cholesterol ester synthesis has been observed. On the other hand, in differentiating cells lipogenesis appeared to be independent of extracellular lipoproteins, due to the absence of the LDL uptake mechanism, the only exception being the synthesis of triacylglycerols, the rate of which could be modulated to a certain extent by extracellular lipoproteins. The results presented here demonstrate a close inverse relationship between the regulation of lipogenesis by extracellular lipoproteins and the ability of the cells to differentiate.

Calcium↗

PubMed1987

Differentiation of human keratinocytes: changes in lipid synthesis, plasma membrane lipid composition, and 125I-EGF binding upon administration of 25-hydroxycholesterol and mevinolin.

We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistently under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression.

Anticholesteremic Agents↗

PubMed1986

Glucocorticoids: binding affinity and lipophilicity.

The relative binding affinity of 35 steroids for the glucocorticoid receptor was determined in experiments in which the competition of various unlabeled steroids with either [6,7-3H]dexamethasone or [1,2-3H]hydrocortisone for the cytosolic glucocorticoid receptor of cultured human keratinocytes was measured. The data obtained were correlated with steroid lipophilicity, measured as the partition coefficient of the steroid between 1-octanol and pH 7.4 aqueous buffer. The introduction of various substituents on the steroid molecule induced changes in the binding affinity and was associated in some cases with concomitant changes in steroid lipophilicity. The substitution by a 17 alpha-OH or 21-OH group leads in all cases to a decrease in steroid lipophilicity and to an increase in affinity. In contrast, 17 alpha-OAc and especially 21-OAc substitution on hydrocortisone and betamethasone causes a decrease in the steroid affinity for the receptor and an increase in steroid lipophilicity. The elongation of the ester chain from acetate to valerate in both position C-17 and C-21 leads to the increase in both the binding affinity for the receptor and the lipophilicity of steroids. However, all 21-esters showed lower binding affinity than the parent alcohol. The binding affinity of the highly lipophilic 17 alpha, 21-diester was found to be lower than that of the 17 alpha-ester but higher than that of the 21-ester or of the parent alcohol. Only in the series of 17 alpha- and 21-esters is there a correlation between the binding affinity of steroids for the glucocorticoid receptor and their lipophilicity.

Cells, Cultured↗

PubMed1985

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes.

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.

Acetates↗

PubMed1985

SV 40-transformed (SVK14) and normal keratinocytes: similarity in the expression of low-density lipoprotein, epidermal growth factor, glucocorticoid receptors, and the regulation of lipid metabolism.

Transformation of normal keratinocytes by simian virus 40 (SV 40) leads to the establishment of epithelial cell lines that can be cultured in the absence of the feeder layer and do not become senescent in culture. The SVK14 cell line developed by Taylor-Papadimitriou et al can serve as a model for study of the modification of various cellular processes by certain pharmacologic and physiologic agents, because these cells resemble normal keratinocytes with respect to a variety of parameters related to proliferation and differentiation, as follows: The SVK14 cells show the same ability to form ionophore-induced cross-linked envelopes that is strongly suppressed when the calcium level in the culture medium is reduced. When cultured in a high-calcium medium, both cell types showed a high rate of de novo cholesterol synthesis that was independent of the extracellular lipoprotein concentration. Cells cultured in a low-calcium medium had a much lower rate of cholesterol synthesis, but this rate increased markedly in cells preincubated in lipoprotein-deficient (LPDS) medium and decreased again with the addition of increasing amounts of low-density lipoprotein (LDL). Both types of cell showed decreased ability to bind epidermal growth factor (EGF) and LDL during calcium-induced differentiation, the expression of LDL and/or EGF receptors being high in low-calcium and low in high-calcium cells. Addition of etretinate (0.05-5.0 microM) suppressed cholesterol synthesis and strongly stimulated triglyceride synthesis in both cell types without significantly affecting the rate of protein synthesis. The addition of small doses of glucocorticoids (10(-9) to 10(-6) M) led to stimulation and higher doses (up to 5 X 10(-5) M) to inhibition of cell proliferation.

Calcium↗

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↗

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↗