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T Wieder

Publications and source records attributed to T Wieder.

At least 37 records · Page 2Linked to original sources
PubMed1997

Bcl-2 antagonizes apoptotic cell death induced by two new ceramide analogues.

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N-thioacetylsphingosine and FS-5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations > or = 14 microM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly-(ADP-ribose) polymerase (PARP). Additional experiments in bcl-2-transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl-2. Furthermore, neither N-thio-acetylsphingosine nor FS-5 induced PARP cleavage in bcl-2-transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.

Amides↗
PubMed1997

1alpha,25-dihydroxyvitamin D3 induces sphingomyelin hydrolysis in HaCaT cells via tumor necrosis factor alpha.

Treatment of the human keratinocyte cell line HaCaT with 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in the hydrolysis of sphingomyelin with peak elevations of ceramide levels after 2-3 h (Geilen, C. C., Bektas, M., Wieder, Th., and Orfanos, C. E. (1996) FEBS Lett. 378, 88-92). In the present paper, the mechanism underlying the effect of 1,25-(OH)2D3 on sphingomyelin hydrolysis was investigated. Using the cell culture supernatant of HaCaT cells treated with 1,25-(OH)2D3 for 2 h, it was possible to induce sphingomyelin hydrolysis as early as 30-60 min after addition to resting cells. Several lines of experimental evidence indicated that tumor necrosis factor alpha (TNFalpha) mediates sphingomyelin hydrolysis after 1,25-(OH)2D3 treatment: (i) 1,25-(OH)2D3 stimulated TNFalpha mRNA expression after 1 h, (ii) newly synthesized TNFalpha occurred 2 h after 1,25-(OH)2D3 treatment, (iii) indirect activation of sphingomyelin hydrolysis by the supernatant of 1, 25-(OH)2D3-treated HaCaT cells was abolished by preincubation of the supernatant with antibodies directed against TNFalpha, and (iv) preincubation of HaCaT cells with neutralizing antibodies directed against the 55-kDa receptor of TNFalpha blocked the ability of 1, 25-(OH)2D3 to induce sphingomyelin hydrolysis in HaCaT cells. These data demonstrate that 1,25-(OH)2D3 activated sphingomyelin hydrolysis by an autocrine mechanism via TNFalpha expression. Furthermore, this indirect mode of action may serve as an explanation for the delayed induction of sphingomyelin hydrolysis by vitamin D3.

Calcitriol↗
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↗
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

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↗
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↗
PubMed1995

The effect of two synthetic phospholipids on cell proliferation and phosphatidylcholine biosynthesis in Madin-Darby canine kidney cells.

The concentration-dependent effects of two different synthetic phospholipids on cell proliferation and phosphatidylcholine biosynthesis were compared in Madin-Darby canine kidney (MDCK) cells. The alkyllysophospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine and the alkylphosphocholine, hexadecylphosphocholine, inhibited cell proliferation with half-inhibitory concentrations (IC50) of 75 and 135 mumol/L, respectively. The agents also inhibited phosphatidylcholine biosynthesis in confluent and proliferating MDCK cells. The IC50 of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine was 40 mumol/L in confluent cells and 20 mumol/L in proliferating cells, whereas the IC50 of hexadecylphosphocholine was higher in both experimental systems (67 mumol/L in confluent cells and 40 mumol/L in proliferating cells). Further experiments revealed that the effect of both agents on phosphatidylcholine biosynthesis was reversible and that the inhibition was mediated by translocation of the rate-limiting enzyme of this pathway, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), from membranes to the cytosol, where it is inactive. The present findings suggest that the inhibition of phosphatidylcholine biosynthesis by both synthetic phospholipids might be related, in part, to their antiproliferative effects.

Animals↗
PubMed1994

Growth factors stimulate phosphorylation of CTP:phosphocholine cytidylyltransferase in HeLa cells.

The effect of insulin and epidermal growth factor on the phosphorylation of CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) was investigated in HeLa cells. For the first time, cytidylyltransferase phosphorylation was shown to be influenced by growth factors in cell culture experiments. The rephosphorylation of cytidylyltransferase after an oleate-mediated dephosphorylation and translocation to membranes was increased after 2 min in the presence of insulin or epidermal growth factor by 99% and 76%, respectively, compared with controls. However, the increased phosphorylation of cytidylyltransferase did not have an effect on its subcellular distribution. Furthermore, purified cytidylyltransferase preincubated with alkaline phosphatase is a substrate for p44mapk, a member of the mitogen-activated protein (MAP) kinase family downstream of the growth factor receptors, in vitro. In accordance with the in vivo data, in vitro phosphorylation of cytidylyltransferase by p44mapk occurred after 2 min.

Cell Membrane↗
PubMed1994

Identification of a putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase from rat liver.

A putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase (CT) was identified using two peptide-specific antibodies. One antibody (SA2) was raised against the N-terminus of CT (amino acid residues 1-17) and the other antibody (SA209) against an alpha-helical domain of the enzyme (amino acid residues 247-257). Both antibodies quantitatively immunoprecipitated CT from rat liver cytosol and showed specificity towards CT when octylglucoside extracts of rat liver cytosol were assessed by Western blot analysis. However, further experiments revealed that the antibodies had different characteristics. Whereas the antibody directed against the N-terminus of CT (SA2) did not influence CT/membrane interaction, the new antibody (SA209) against the alpha-helical domain of the enzyme interfered with this interaction. Our results provide experimental evidence that the alpha-helical domain (amino acid residues 228-287) of CT may serve as a membrane-interacting domain.

Animals↗
PubMed1994

Uptake, subcellular distribution and metabolism of the phospholipid analogue hexadecylphosphocholine in MDCK cells.

The uptake, subcellular distribution and metabolism of hexadecylphosphocholine was investigated using hexadecylphospho-[methyl-14C]choline as tracer. The phospholipid analogue was taken up in a time-, dose- and temperature-dependent manner and showed a high affinity for the plasma membrane in vivo and in vitro. After incorporation into the plasma membrane, hexadecylphosphocholine appeared in all subcellular membranes investigated but not in the cytosolic fraction. After 24 h, the detectable metabolites of incorporated hexadecylphosphocholine co-chromatographed with the phosphatidylcholine standard (34.3%), the phosphocholine standard (22.1%), the glycerophosphocholine standard (4.3%) and the betaine standard (3.6%).

Animals↗
PubMed1994

Determination of alkylphosphocholines by high-performance liquid chromatography with light-scattering mass detection.

A rapid and sensitive method for the determination of five different alkylphosphocholines including the antineoplastic phospholipid analogues hexadecylphosphocholine and octadecylphosphocholine is presented. The method is based on the separation of the lipids by high-performance liquid chromatography and quantitation by light-scattering mass detection. The lower limit of detection is approximately 50 pmol for each alkyphosphocholine tested. Quantitation is linear over the range 0.05-75 nmol. Hexane-isopropanol extracts of cultured cells can be applied to the column without further cleanup. The high resolution of separation and the sensitivity of detection render this method useful for pharmacokinetic investigations dealing with the uptake of alkylphosphocholines into different types of cells.

Animals↗
PubMed1994

N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase.

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.

Cell Membrane↗
PubMed1994

Phospholipid analogue hexadecylphosphocholine inhibits proliferation and phosphatidylcholine biosynthesis of human epidermal keratinocytes in vitro.

The alkylphospholipid hexadecylphosphocholine (HePC), member of a new class of antineoplastic drugs, has been previously shown to exert cytotoxic effects on neoplastic cell lines in vitro, and a selective antineoplastic activity has been reported after topical application of HePC in vivo, in particular on skin metastases of human mammary carcinomas. Preliminary observations suggest that HePC might also be beneficial in the treatment of non-neoplastic skin diseases characterized by epidermal hyperplasia such as psoriasis. Therefore, we investigated whether HePC might inhibit the proliferation of normal human keratinocytes, and whether its effects might be dependent upon the proliferative status of the treated cells. Moreover, its effects on phosphatidylcholine biosynthesis were studied in keratinocytes. HePC dose-dependently decreased cell numbers, thymidine incorporation, and protein synthesis when applied during the growth phase of keratinocytes grown in serum-free medium, with a minimal inhibitory dose of 10(-7) mol/l for thymidine incorporation, 3 x 10(-7) mol/l for cell numbers, and 10(-6) mol/l for 35S-methionine incorporation. No major differences were observed when keratinocytes were grown under high-Ca++ conditions. In contrast, slowly proliferating confluent keratinocyte cultures showed growth inhibition only after 10(-4) mol/l HePC. Phosphatidylcholine biosynthesis was dose-dependently inhibited by HePC with a half inhibitory concentration of 3 x 10(-6) mol/l, and with translocation of the rate-limiting enzyme. CTP:phosphocholine cytidylyltransferase, to the cytosol, where the enzyme is inactive. These data show a pronounced antiproliferative effect of HePC also on proliferating non-malignant keratinocytes, and are compatible with its possible action on hyperproliferative skin disorders.

Cell Division↗
PubMed1994

Phospholipid analogues: side chain- and polar head group-dependent effects on phosphatidylcholine biosynthesis.

In recent studies we showed that the phospholipid analogue hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis by affecting the translocation of the rate-limiting enzyme of phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), to membranes, where it is active (Geilen et al. 1992. J. Biol. Chem. 267: 6719-6724). The present study was performed to investigate the structure-dependency of this effect. It is shown that the inhibitory properties of phospholipid analogues are dependent on their alkyl side chain length (dodecylphosphocholine < tetradecylphosphocholine < hexadecylphosphocholine < heptadecylphosphocholine < octadecylphosphocholine > eicosadecylphosphocholine). Furthermore, it is demonstrated that this inhibition of phosphatidylcholine biosynthesis by phospholipid analogues is also dependent on the polar head group (hexadecylphosphocholine >> hexadecylphosphoethanolamine = hexadecylphosphoserine). These effects result from an inhibition of the CTP:phosphocholine cytidylyltransferase and are not due to an inhibition of choline uptake or differences in the cellular uptake of the phospholipid analogues investigated.

Animals↗
PubMed1993

Antagonism of phorbol-ester-stimulated phosphatidylcholine biosynthesis by the phospholipid analogue hexadecylphosphocholine.

The antagonization of phorbol 12-myristate 13-acetate (PMA)-stimulated phosphatidylcholine (PtdCho) biosynthesis by the phospholipid analogue hexadecylphosphocholine (HePC) in MDCK cells was investigated and compared with the corresponding influence in HeLa cells. In both cell lines, PMA-stimulated PtdCho biosynthesis was antagonized by 50 microM HePC. However, subsequent experiments provided evidence that PMA enhances PtdCho biosynthesis by at least two mechanisms: (i) by stimulation of choline uptake and (ii) by translocation of CTP:choline phosphate cytidylyltransferase to membranes. In MDCK cells, 5 nM PMA caused a 4-fold increase in [methyl-3H]choline incorporation into PtdCho, which was paralleled by an approx. 2-fold stimulation of choline uptake. These data indicate that choline uptake might play an important role in the regulation of PtdCho biosynthesis in this cell line, especially since we could not detect any significant increase in membrane-bound cytidyltransferase activity in PMA-treated MDCK cells. In contrast, enhanced PtdCho biosynthesis in HeLa cells is achieved by a 2-fold increase in particulate cytidylyltransferase activity after PMA stimulation. Translocation of cytidylyltransferase from the cytosol to membranes is therefore important in HeLa cells. Nevertheless, in both cell lines, the main target of HePC seems to be the translocation process. In MDCK cells, addition of 50 microM HePC decreases membrane-bound cytidylyltransferase activity by about 45%, compared with control cells and PMA-treated cells. In HeLa cells, PMA-induced translocation of cytidylyltransferase to membranes is totally abolished by HePC.

Animals↗