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Biomedical subjects

C C Geilen

Publications and source records attributed to C C Geilen.

At least 55 records · Page 3Linked to original sources
PubMed1997

The phospholipid analogue hexadecylphosphocholine (HePC) inhibits proliferation of keloid fibroblasts in vitro and modulates their fibronectin and integrin synthesis.

Hexadecylphosphocholine (HePC), a topically effective compound, exerts a strong antiproliferative effect on neoplastic cells. In the present study we investigated (1) the antiproliferative effect of HePC on benign mesenchymal cells in vitro, using as examples normal and keloid fibroblasts, and (2) the influence of HePC on various functional properties of these cells, including phosphatidylcholine biosynthesis, their capacity to reorganize a three-dimensional collagen-I matrix, and their expression and synthesis of fibronectin and subunits of the beta 1 integrin family. Fibroblasts derived from keloids (kelfib) and from normal skin (nfib) were cultured in serum-containing medium and treated in the third passage with 50 mumol/l HePC. Proliferative activity was significantly (P < 0.05) more strongly inhibited in kelfib than in nfib under HePC treatment, whereas their phosphatidylcholine synthesis was inhibited to a similar extent. However, the ability of fibroblasts to contract a three-dimensional collagen-I lattice was significantly (P < 0.05) enhanced only in kelfib treated with HePC. These functional differences following treatment with HePC were paralleled by an upregulation of the alpha 2- and beta 1-integrin chains, and a downregulation of fibronectin synthesis and the alpha 5-subunit. Our results indicate a differential modulation of kelfib metabolism by HePC, suggesting a possible new therapeutic approach for keloid and hypertrophic scars in vivo.

Cell Division↗
PubMed1997

Ceramide signalling: regulatory role in cell proliferation, differentiation and apoptosis in human epidermis.

The stratum corneum of vertebrates is a major structural compartment that provides mechanical protection and prevents skin desiccation. The water barrier function of the stratum corneum was first reported in 1944, and this was shown later to be associated with multilayered lipid lamellae localized in the extracellular spaces. The major lipid components isolated from the cornified epidermal layers are ceramides, which belong to the class of sphingolipids, cholesterol and free fatty acids; their biosynthesis is in tight relationship with the cutaneous barrier function. In studies in which the barrier is artificially disturbed, lipid biosynthesis is found to be directly regulated by barrier permeability. As mentioned above, the ceramides involved in this process are located in the extracellular spaces of the upper epidermal layers, whereas sphingomyelin, the most common sphingolipid, is an integral part of the bilayer plasma membrane of the keratinocytes. During the last few years, however, increasing evidence has shown that sphingolipids may also take part in cell signalling, and the term 'sphingomyelin cycle' has been coined to describe this novel path-way of signal transduction. Intracellular messengers of the sphingomyelin cycle are ceramides as the products of an agonist-stimulated sphingomyelin hydrolysis. Increased levels of intracellular ceramides induce cell differentiation and/or apoptosis and reduce cell proliferation. In contrast to the extracellular barrier-forming ceramides which are complex partly O-acylated species containing long-chain fatty acids, intracellular signal-transducing ceramides are not O-acylated and have acyl chain lengths of 16 and 18 carbon atoms. We present here a review of our present knowledge on the sphingomyelin cycle as a possible signal transduction pathway in the human epidermis. We discuss the common origin of extracellular ceramides constituting the lipid barrier and of intracellular ceramides generated by agonist-stimulated sphingomyelin hydrolysis and serving as second messengers. A summary of alterations in sphingolipid metabolism and lipid composition of the epidermis in diseased skin is also given and the possible use of different sphingolipids for therapy is envisaged.

Apoptosis↗
PubMed1997

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids.

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10(-8) to 10(-5) M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 microM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 microM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture.

Animals↗
PubMed1997

L-ascorbic acid inhibits UVA-induced lipid peroxidation and secretion of IL-1alpha and IL-6 in cultured human keratinocytes in vitro.

We investigated the antioxidative effect of L-ascorbic acid on lipid peroxidation and on secretion and mRNA expression of IL-1alpha and IL-6 after UVA irradiation (20 J/cm2) in cultured human keratinocytes. Lipid peroxidation was measured by (i) high performance liquid chromatography with UV detection of malondialdehyde (MDA) at 256 nm and (ii) spectrometric measurement of thiobarbituric acid-reactive substances (TBARS). To evaluate UV-induced cytotoxicity, we assessed cell membrane damage by measuring lactate dehydrogenase (LDH) release. UVA-induced lipid peroxidation in cultured human keratinocytes was inhibited by ascorbic acid in a concentration-dependent manner: MDA protein equivalent was reduced by 47% (10(-6)), compared to keratinocytes not exposed to L-ascorbic acid (p < 0.05), and the TBARS showed a concentration-dependent decrease of 49% (10(-6) M) in L-ascorbic acid-supplemented cultures compared to controls (p < 0.05). LDH release was decreased by 45% in L-ascorbic acid-supplemented keratinocyte cultures, indicating protection against cell death (p < 0.05). L-Ascorbic acid was able to downregulate IL-1alpha mRNA expression in both UVA-irradiated and nonirradiated cells; however, IL-6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L-ascorbic acid. These findings indicate a major cell-protective effect of L-ascorbic acid on UVA-induced lipid peroxidation and the secretion of pro-inflammatory cytokines by UVA-irradiated human keratinocytes.

Ascorbic Acid↗
PubMed1997

Current use and future potential role of retinoids in dermatology.

Since their introduction 15 years ago, retinoids have been increasingly used for topical and systemic treatment of psoriasis and other hyperkeratotic and parakeratotic skin disorders, keratotic genodermatoses, severe acne and acne-related dermatoses, and also for therapy and/or chemoprevention of skin cancer and other neoplasia. Oxidative metabolites of vitamin A (retinol) are natural retinoids present at low levels in the peripheral blood. Synthetic retinoids are classified into 3 generations including nonaromatic, monoaromatic and polyaromatic compounds. They are detectable in plasma 30-60 minutes after systemic administration, and reach maximum concentrations 2 to 4 hours later. Elimination half-life is 10 to 20 hours for isotretinoin, 80 to 175 days for etretinate and 2 to 4 days for, trans-acitretin; the latter, however, partially converts into etretinate. Retinoid concentrations in skin are rather low in contrast to subcutaneous fat tissue. Intracellularly, retinoids interact with cytosolic proteins and specific nuclear receptors. Two classes of nuclear receptors have been suggested to mediate retinoid activity at the molecular level, RARs and RXRs. The expression of retinoid receptors is tissue specific; skin mainly espresses RAR gamma and RXR alpha. Retinoids affect epidermal cell growth and differentiation as well as sebaceous gland activity and exhibit immunomodulatory and anti-inflammatory properties. Current retinoid research targets the development of receptor-selective retinoids for tailoring and/or improving their therapeutic profile. Currently, tretinoin is used systemically for acute promyelocytic leukaemia, etretinate and acitretin for psoriasis and related disorders, as well as other disorders of keratinisation and isotretinoin for seborrhoea, severe acne, rosacea and acneiform dermatoses. Systemic retinoids are also applied for chemoprevention of epithelial skin cancer and cutaneous T cell lymphoma. The major adverse effect of retinoids is teratogenicity; all other adverse effects are dose-dependent and controllable. Contraception is, therefore, essential during retinoid treatment in women of child-bearing age. Clinical monitoring requires physical examination for adverse effects every 3 to 4 weeks and proper laboratory investigations, also including analysis of retinoid bioavailability in selected cases. Topical retinoids are rapidly developing at present and seem promising for the future; their clinical application includes acne, aging, photodamage, precanceroses, skin cancer and disorders of skin pigmentation. The development of receptor-specific retinoids for topical treatment of psoriasis and/or acne may lead to interesting new compounds based on our current concepts of retinoid function.

Forecasting↗
PubMed1996

Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases.

Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.

CDC2 Protein Kinase↗
PubMed1996

Determination of cytidine 5'-monophospho-N-acetylneuraminic acid pool size in cell culture scale using high-performance anion-exchange chromatography with pulsed amperometric detection.

A simultaneous determination of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) and its metabolic products, cytidine, CMP and Neu5Ac by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Carbo-Pac PA1 column is described. Preparation of the samples involved a single purification step of the crude cell extract on DEAE-Sepharose. The method is adequate to quantify the amount of CMP-Neu5Ac produced by one culture dish; equivalent to 6.10(6) cells. In addition, a method for desalting and recovery of the separated material was developed to determine the cellular concentration of CMP-Neu5Ac in Madin Darby canine kidney (MDCK) cells. The addition of 5 mM N-acetylmannosamine to the culture medium gave rise to a 6.4-fold elevation of this value.

Animals↗
PubMed1996

The antitumor phospholipid analog, hexadecylphosphocholine, activates cellular phospholipase D.

Hexadecylphosphocholine (HePC), a glycerol-free phospholipid analog, belongs to a new class of drugs that demonstrate selective anticancer activity. The mechanisms underlying the anticancer activity are unclear. To investigate possible signal transduction relationships we examined the influence of HePC on cellular phospholipid metabolism. When HePC was added to cultured human breast fibroblasts (CCD-986-SK cells) that had been radiolabeled with fatty acid, phosphatidylethanol (PEt, the transphosphatidylation product of phospholipase D (PLD)) formation was stimulated as early as 5 min after addition. In cells labeled with [3H]choline, HePC treatment caused release of choline-containing metabolites to the culture medium, concurrent with PEt formation. HePC also elicited formation of diacylglycerol (DG) which, after 30 min increased 3.5-fold over control. As little is known regarding HePC and PLD, attention was directed towards studies on PC metabolism by PLD. PEt formation was shown to be optimal at 20-50 microM HePC, and structure-activity studies showed HePC to be more potent than either lyso-phosphatidylcholine or 1-hexadecyl-2-O-methyl-rac-glycero-3-phosphocholine for PLD activation. PLD activity induced by HePC was totally inhibited by cellular pretreatment with phorbol dibutyrate, and 59% diminished by pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor. Our results demonstrate for the first time that HePC activates PLD, and suggest that PKC participates in this response. The relationship of PLD to the anticancer properties of HePC may be clinically relevant to drug actions.

Antineoplastic Agents↗
PubMed1996

c-Ha-ras oncogene expression increases choline uptake, CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine biosynthesis in the immortalized human keratinocyte cell line HaCaT.

The effect of c-Ha-ras transfection on phosphatidylcholine biosynthesis of the keratinocyte cell line HaCaT was investigated. It was shown that ras-transfection caused a 3-fold increase of choline incorporation into phosphatidylcholine. By investigating the mechanisms underlying this phenomenon, two targets were obtained. First, the choline uptake was elevated by 2-fold in ras-transfected HaCaT cells as compared with untransfected HaCaT cells, and second, the activity of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, was increased by 43%. Stimulation of HaCaT cells and ras-transfected HaCaT cells with oleate revealed that the increased activity of cytidylyltransferase might be due to a higher level of enzyme. In these experiments, a 75% increase of the specific activity of fully stimulated, membrane-bound cytidylyltransferase was found in ras-transfected HaCaT cells. Choline kinase which has been previously described as a target of ras-transfection in fibroblasts was unaffected.

Cell Division↗
PubMed1996

The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes.

The possible role of sphingomyelin cycle for the regulation of cell proliferation was investigated in human keratinocytes. The time-dependent breakdown of sphingomyelin was observed in the immortalized human keratinocyte cell line HaCaT as well as in primary human keratinocytes thereby providing evidence that the sphingomyelin cycle might be of importance in the epidermis. Peak levels of 20-30% sphingomyelin hydrolysis were measured 3 h after treatment of the cells with 1 alpha,25-dihydroxyvitamin D3 or with the vitamin D3 analogue, calcipotriol. The decrease of sphingomyelin upon addition of vitamin D3 or calcipotriol was accompanied by an approximately 70% increase of ceramide in the cells. The effects of vitamin D3 and calcipotriol on sphingomyelin breakdown were paralleled by their antiproliferative potency. Furthermore, the cell-permeable ceramide, N-acetylsphingosine, and natural ceramide inhibited cell proliferation of human keratinocytes. The results presented suggest that induction of the sphingomyelin cycle represents one mechanism mediating the therapeutic effect of calcipotriol in treatment of psoriasis.

Calcitriol↗
PubMed1996

[Clinical fluconazole and itraconazole resistance of oro-gastrointestinal candidiasis in a patient with AIDS].

We report on a 32-year-old male patient with advanced acquired immunodeficiency syndrome (AIDS), who had severe candidiasis of the gastrointestinal tract. Treatment with fluconazole, 200 mg/day, was introduced. After oral intake of fluconazole over 5 months itraconazole 200 mg/day was given for 1 month. However, fungal infection still persisted. The antifungal activity of fluconazole, itraconazole and ketoconazole against Candida albicans was evaluated by means of the microdilution test by determining the 90% inhibitory concentration of each drugs. A high minimal inhibitory concentration (MIC) was detected for fluconazole (50 micrograms/ml) revealing fluconazole resistance. The susceptibility to itraconazole was borderline (MIC 0.125 micrograms/ml) and that to ketoconazole was markedly lowered (MIC 0.25 micrograms/ml). Plasma levels of itraconazole were also found to be lowered. In HIV patients the gastrointestinal absorption of azole derivatives is often reduced. Therefore, the clinical resistance of Candida albicans to itraconazole can be explained by reduced susceptibility after azole therapy and also by the decreased absorption of the drug in HIV patients.

AIDS-Related Opportunistic Infections↗
PubMed1996

[Detection of Helicobacter pylori in dermatoses. Clinical incidental finding or pathogenetic association?].

Recently, several reports have been describing a possible relation between Helicobacter pylori infection of the gastric mucosa and dermatological diseases. Associations have been reported for urticaria, roascea, Sjögren's syndrome and Schönlein-Henoch purpura. In this paper we review the current knowledge on Helicobacter pylori infection and its relevance for skin diseases, especially its clinical and pathophysiological aspects.

Gastritis↗
PubMed1996

The mitogen-activated protein kinases system (MAP kinase cascade): its role in skin signal transduction. A review.

Mitogen-activated protein (MAP) kinases are proline-directed kinases which are downstream components of a pathway involving p21ras and the serine/threonine kinase Raf-1. They represent an important link between the signal transduction processes at the level of the plasma membrane and the final nuclear events. Not only various growth factors and cytokines, but also other signals such as UV-light or extracellular matrix components are able to activate MAP kinases. We believe that the MAP kinase cascade may play a significant role in regulating cell proliferation and differentiation in human epidermis. In this review we summarize the rapidly increasing knowledge in this field of signal transduction and discuss some very recent results on MAP kinases and their role in skin biology.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Cell-matrix interactions of normal and transformed human keratinocytes in vitro are modulated by the synthetic phospholipid analogue hexadecylphosphocholine.

We have investigated the effect of the synthetic phospholipid analogue, hexadecylphosphocholine (HePC), a member of a new class of membrane-affecting antiproliferative drugs, on adhesion to extracellular matrix components, reorganization of three-dimensional collagen lattices, and proliferative activity of human keratinocyte populations in vitro. Six transformed keratinocyte lines were compared with their normal counterparts from interfollicular epidermis. Adhesion to collagen types I and IV, laminin, and fibronectin was weakest in normal keratinocytes (binding of 3-10% of the cells), followed by HaCaT and HaCaT-II/3 (25-30% binding), and the squamous carcinoma lines and SV-40 transformed keratinocytes (35-50% binding). Treatment with non-toxic doses of 10-20 mumol/l HePC led to a clear inhibition of the adhesive interactions by 50-75% in all populations. The impaired adhesion capacity was paralleled by a clear decrease of the ability of all keratinocyte populations to contract three-dimensional collagen lattices, an experimental model system for the reorganization of extracellular matrices. Histological examination revealed that these effects were due to a reduced cell number in the collagen lattices, a finding underscored by significant inhibition of proliferative activity of all keratinocyte populations by HePC. Furthermore, HePC led to marked changes of cellular morphology in the contracted gels. FACS analysis revealed that the impaired interaction with components of the extracellular matrix was not due to a specific downregulation of beta 1-integrins, the major cell-surface receptors for the respective matrix proteins. In conclusion, our results provide new insights into the potential action of HePC in a variety of skin disorders. Decreased proliferative activity and changes of cellular morphology, combined with impaired interactions with extracellular matrix proteins and a consecutive loss of matrix organization capacity, may cause suppression of different hyperproliferative skin diseases.

Antineoplastic Agents↗
PubMed1995

Influence of extracellular matrices on ganglioside pattern of two hepatoma cell lines with different adhesive properties.

A cell culture model was developed to investigate the involvement of gangliosides in cell-matrix adhesion. Two cell lines with different adhesive properties derived from solid Morris hepatoma 7777 were established. Cultured in horse serum-containing medium, the adhesive cell line (MH 7777A) adheres and spreads on uncoated culture dishes, whereas the revertant cell line (MH 7777A > N) does not adhere and grows in suspension. The adhesiveness of both cell lines is dependent on the coating protein used (none, bovine serum albumin, fibronectin or collagen I) and the horse serum concentration in the culture medium. Both cell lines, although of the same origin, differed in their ganglioside composition. The most abundant ganglioside of both MH 7777A and MH 7777A > N cell lines was fucosyl-GM1, 0.78 and 0.72 microgram per mg cellular protein, respectively. The GM3 and GD1a content of MH 7777A > N cells was significantly higher than that of MH 7777A cells. Furthermore, a matrix-dependency of the ganglioside pattern of both cell lines was demonstrated.

Animals↗
PubMed1995

Two new sphingomyelin analogues inhibit phosphatidylcholine biosynthesis by decreasing membrane-bound CTP: phosphocholine cytidylyltransferase levels in HaCaT cells.

The effects of two newly synthesized sphingomyelin analogues on phosphatidylcholine biosynthesis were investigated in the immortalized human keratinocyte cell line HaCaT. N-Acetyl-erythro-sphingosine-1-phosphocholine (AcSM) and N-octanoyl-erythro-sphingosine-1-phosphocholine (OcSM) inhibited the incorporation of choline into phosphatidylcholine with half-inhibitory concentrations (IC50) of 6 micrograms/ml and 10 micrograms/ml respectively. Further experiments revealed that AcSM and OcSM interfered with the translocation of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), in HaCaT cells and inhibited cytidylyltransferase activity in vitro. Despite the fact that OcSM was a potent inhibitor of cytidylyltransferase in vitro, its effects on phosphatidylcholine biosynthesis and translocation of cytidylyltransferase in HaCaT cells were less pronounced as compared with AcSM. Finally, we showed that the comparatively strong effects of AcSM in cell culture experiments were due to the uptake of large amounts of this sphingomyelin analogue into the cells. The results presented demonstrate that the activity of cytidylyltransferase may be negatively regulated by a high ratio of choline head group-containing sphingolipids.

Alkaline Phosphatase↗