Biomedical subjects

M Ueffing

Publications and source records attributed to M Ueffing.

27 records · Page 2Linked to original sources

PubMed1996

Protein kinase C activation is involved in ultraviolet B irradiation-induced endothelial cell ICAM-1 up-regulation and lymphocyte-endothelium interaction in vitro.

Lymphocyte-endothelium interactions are pivotal steps in mediating inflammatory responses. The authors have analysed the influence of ultraviolet B (UVB) irradiation on intercellular adhesion molecule (ICAM)-1 expression on cells of the human microvascular endothelial cell line (HMEC)-1 and the intracellular signalling pathways involved. Flow cytometry revealed dose-dependent ICAM-1 up-regulation with maximum induced expression 24h after sublethal UVB irradiation of 10 mJ/cm2. While anti-tumour necrosis factor (TNF)-alpha antibodies or recombinant human interleukin (IL)-10 did not influence this response, anti-interferon (IFN)-gamma antibodies blocked the UVB-induced ICAM-1 up-regulation. Significant induction of intracellular/membrane-bound IFN-gamma was measured as early as 6 h post-UVB. Since previous work has shown a differential role of protein kinase C (PKC) in cytokine induced ICAM-1 expression, the effect of a selective bisindolylmaleimide-derived PKC-inhibitor (GF109203X) was studied. Ultraviolet B-induced ICAM-1 up-regulation was effectively blocked by the PKC-inhibitor, whereas a PKA-inhibitor was ineffective. Moreover, immunofluorescence analysis showed a radiation-induced membrane translocation of PKC-alpha, indicative of enzyme activation, in HMEC-1 cells already 30 min post-UVB. The functional relevance of the UVB-induced ICAM-1 expression and involvement of PKC in this process was demonstrated in an adhesion assay with peripheral blood mononuclear cells. In conclusion, UVB-induced ICAM-1 expression on human endothelial cells involves PKC-dependent pathways and can be prevented by a PKC-inhibitor. The use of PKC-inhibitors as additive modulators in immune reactions may bear clinical potential. The mechanisms of IFN-gamma induction in endothelial cells by UVB deserve further investigation.

Cell Adhesion↗

PubMed1995

Critical involvement of transmembrane tumor necrosis factor-alpha in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin.

In this report, we show that ionizing radiation (IR) at a clinically relevant dose (4 Gy) causes apoptosis in macrovascular and microvascular human endothelial cells. Treatment of irradiated cells with a low dose of bacterial endotoxin (LPS), similar to the levels observed in serum during endotoxemia, enhanced the rate of apoptosis, although LPS alone was unable to induce programmed cell death. The cytokine and endotoxin antagonist interleukin-10 (IL-10) reduced the rate of LPS + IR-induced apoptosis to levels obtained with irradiation alone. Using neutralizing antibodies against tumor necrosis factor-alpha (TNF), we could show crucial involvement of TNF in the LPS-mediated enhancement of IR-induced apoptosis, but not in the IR-induced apoptosis per se. However, further analysis strongly suggested the transmembrane form of TNF (mTNF), but not soluble TNF, to be accountable for the LPS-mediated cytotoxic effects. Studies with anatagonistic receptor specific antibodies clearly showed that TNF receptor type I (TR60) is essential and sufficient to elicit this effect. These findings are of potential clinical importance because they may disclose a relevant mechanism that leads to endothelial damage after radiotherapy or total body irradiation used for conditioning in bone marrow transplantation and that may thus contribute to transplant related complications, especially in association with endotoxemia or related inflammatory states.

Animals↗

PubMed1995

Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. Possible molecular mechanisms.

We have previously reported that two closely related protein kinase C (PKC) isoforms, PKC alpha and PKC beta I, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W. O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKC beta I lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKC alpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKC alpha and PKC beta I. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKC beta I differed from those overexpressing PKC alpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKC beta I may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.

Animals↗

PubMed1995

Rapid microplate mitogenic assay using a meltable scintillation wax.

To combine the advantages of solid scintillation and the use of 96-well plates, an in-plate cell proliferation assay was designed using scintillation wax. Messy, conventional liquid wastes are not produced, thereby making solid scintillation safer and more convenient. Additionally, the use of optimized, 96-well ViewPlates for inplate counting procedures allows the monitoring of cell morphology during the course of the stimulation experiment. The scintillation wax, FlexiScint, was used for counting [3H]thymidine incorporation in an adherent cell line and compared to liquid scintillation counting. The data presented here demonstrate that this new assay produces results comparable to those obtained by conventional methods.

Animals↗

PubMed1994

Mechanism of inhibition of Raf-1 by protein kinase A.

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

3T3 Cells↗

PubMed1991

Effects of a fecapentaene on protein kinase C.

Protein kinase C (PKC) is a Ca2(+)- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). PKC can be activated in vitro by phosphatidylserine (PS) plus Ca2+. We report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca2+. In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by Ca2+ and PS. It can also either enhance or inhibit activation of PKC by the tumor promoter teleocidin, depending on the assay conditions. These results are of interest since fecapentaene is known to be a potent mutagen that is produced by Bacteroides species present in the lumen of the human colon. The present studies raise the possibility that this compound might also play a role in colon cancer by altering the activity of PKC.

Animals↗

PubMed1991

Reversible inhibition of a thyroid-specific trans-acting factor by Ras.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

Animals↗

PubMed1990

Inhibition of rodent protein kinase C by the anticarcinoma agent dequalinium.

Dequalinium has previously been shown to be an anticarcinoma agent (M. J. Weiss et al., Proc. Natl. Acad. Sci. USA, 84: 5444-5448, 1987). The present study demonstrates that it can inhibit protein kinase C-beta 1 isolated from an overproducing cell line with a 50% inhibitory concentration of 8-15 microM. Further examination of the inhibition by using structural analogues of dequalinium reveals that the length of the methylene bridge between the two quinaldinium moieties, the presence of the ring substituents, and the bipartite character of the compound each contributes to the inhibitory potency. Related studies show that the analogues display the same rank order of inhibitory potency when tested with the trypsin-generated catalytic fragment of the enzyme, indicating that dequalinium inhibits kinase activity through an interaction with the catalytic subunit. Further studies argue that the ability of a given analogue to inhibit phosphotransferase activity correlates with its ability to compete with [3H]phorbol-12,13-dibutyrate binding on the intact enzyme (50% inhibitory concentration of 2-5 microM). This suggests that the inhibitor is either binding directly to the regulatory subunit as well, or that due to its interaction with the catalytic subunit, dequalinium produces an indirect effect on sites defined by phorbol ester binding. Kinetic analysis revealed that inhibition is noncompetitive with respect to ATP or phosphatidylserine. Studies conducted with types I, II, and III rat brain isozymes, resolved by hydroxylapatite chromatography, demonstrate that dequalinium inhibits each of them with similar potency (50% inhibitory concentration of 11 microM) and imply that the site of contact on the enzyme is a highly conserved region. Morphology studies with dequalinium in intact cells demonstrate that the inhibitor can protect control cells against phorbol ester-induced morphology changes but cannot protect protein kinase C-overproducing cells, suggesting that an elevation in protein kinase C levels alone is sufficient to overturn the protection conferred by dequalinium. On the basis of these results, we propose that protein kinase C could be a critical in vivo target of dequalinium.

Animals↗

PubMed1990

Growth factor-induced DNA synthesis in cells that overproduce protein kinase C.

In previous studies (Housey et al.: Cell 52:343-354, 1988), our laboratory demonstrated that a cell line R6-PKC3 that stably overproduces high levels of the beta 1 isoform of PKC displayed several abnormalities in growth control, and these phenotypic changes were also markedly enhanced when the cells were exposed to TPA. The present studies indicate that these cells also display marked changes in their response to certain growth factors. A striking finding was that several agents when tested alone in serum-free medium, including EGF, PDGF, TPA, teleocidin, and OAG, stimulated DNA synthesis in quiescent R6-PKC3 cells but had a negligible effect in quiescent R6-C1 cells, a vector control cell line with normal levels of PKC. R6-PKC3 cells also show an exaggerated response to very low concentrations of serum, when compared to R6-C1 control cells. These studies provide direct genetic evidence that alterations in cellular levels of PKC can markedly influence the responses of cells to specific growth factors.

Animals↗