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Lessons in signal transduction from a DNA tumor virus
ROLE OF ISOPRENOIDS IN TUMORIGENESIS Soma MR, Baetta R, Paoletti R, and Fumagalli R. Institute of Pharmacological Sciences, University of Milan, ITALY
LESSONS I N SIGNAL T R A N S D U C T I O N F R O M A DNA T U M O R VIRUS B. S. Sehaffhausen 1, M./Yoakim 1, K. Mullane 1, O. Gj0rupl, L. Cantley 2, T. M. Roberts 3, K. S. Campbell 3, W. Su 3 & D. Pallas3 1: Department of Biochemistry Tufts Univ. School of Medicine, Boston MA 02111; 2: Department of Medicine, Beth Israel Hospital Boston, MA ; 3: Department of Cellular and Molecular Biology, Dana-Father Cancer Institute, Boston, MA;
Growth factors are major regulators of cell proliferation, cell migration, differentiation, and extracellular matrix deposition and resorption. In regulating cell proliferation, most growth factors bind to specific cell surface receptors that are transmembrane tyrosyne kinases. The signal transduction of such receptors cause in cascade sequence activation of ras signaling to activate raf which in turn activates the MAP kinase pathway culminating in modulation of transcription factors necessary for initiating progression through G1 phase of the cell cycle. Ras and other signal transduction proteins activity is dependent on their localization in the plasma membrane through a postransational modification which covalently bind a lipid moiety (famesyl pyrophosphate or geranylgeranyl pyrophosphate) derived from mevalonate. Thus, pharmacological intervention at this bottle-neck target by means of direct and indirect inhibition of protein isoprenilation has garnered paramounting attention. Recently, we providedin vivo evidence that indirect inhibition of protein prenylation, by inhibition of HMGCoA reductase activity, resulted in a profound retardation of glioma growth rates induced by stereotaxical injection of C6 glioma cells in rat brain. Cytofluorimetric analysis of DNA from treated C 6 glioma cells in vitro showed an arrest/retardation in the G I and G2/M phases of the cell cycle. This effect was paralleled by Ras displacement from cell membrane. Moreover, inhibition of HMGCoA reductase activity in combination with classical chemotherapy (earmustine), showed a synergistic effect on glioma growth rates induced by stereotaxical injection of C6 glioma cells in rat brain, with no significant increase in toxicity as assessed by myelosuppression assay. Thus, the results provide in rive support that interfering with protein prenylation constitutes a novel approach with great potential for developing pharmacological agents with anticancer activity for brain tumor treatment.
Polyomavirus induces a wide variety of tumors in its natural host. The virus encodes a collection of activities that regulate cell . growth. In the case Of the nuclear large T antigen, such activities can be divided into Rb family-dependent and Rb family-independent activities that promote cell cycle progression. However, the major polyoma transforming protein is middle T antigen. Middle T transformation requires ras pathway function; dominant negative ras interferes with middle T transformation. Middle T action results from interactions with a collection of cell proteins that affect signal transduction. In a mamler dependent on interactions with tyrosine kinases of the src family, middle T associates with the adaptor SHC, phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C'~. All three signal tranducers can be found together in single complexes. Binding of Pt 3-K and SHC are critical for transformation, while PLC y binding is important under limiting serum conditions: For PI 3-K, a cycle of SH2 interactions with tyrosine phosphorylated sequences regulates inta-acellular trafficking. The association of middle T with SHC and PI 3-K means that there is more than one connection to ras pathways. The connections of these proteins to each other and to G protein signaling will be discussed. Finally, middle T also associates with protein phosphatase 2A. This provides an additional connection to ras signaling, since it contributes an additional mechanism for the activation of MAP kinase.
HYPOLIPIDEMIC DRUGS ON ATHEROSCLEROSIS AND ARTERIAL FUNCTION Paolo Rubba Inst.of Internal Medicine and Diseases of Metabolism Medical School,Federico Ii University, Naples, Italy
LIPIDS AND VASCULAR FUNCTION T.F. LOscher, Cardiovascular Research, Division of Cardiology, University Hospital/Inselspital, Bern, Switzerland
Lipids, and in particular low density lipoproteins (LDL)can profoundly effect vascular function. Qn endothelial cells LDL has particularly pronounced effects once it has been oxidized. Apparently it appears to bind to the scavenger receptor. After activation of this receptor, oxidized LDL is able to increase endothelin mRNA and production of endothelin peptide. Qn the other hand, oxidized LDL impairs the activity of the L-arginine nitric oxide (NO) pathway. Indeed, particularly after stimulation of serotonerglc receptors as well as c~2adrenergic receptors endothelin-dependent relaxations of porcine coronary arteries are severely reduced in the presence of oxidized LDL. Furthermore, it is likely that the intracellular mobilization of Larginine is blunted under these conditions. Finally, it has been proposed that hypertipidemia and atherosclerosis markedly increases the breakdown of nitric oxide by superoxide and other oxygen-derived free radicals produced within the endothelium under these conditions. Furthermore, LDL can activate platelets and therefore contribute to the pro-thrombotic state occurring after impairment of endothelial function in the presence of high local concentrations of LDL and oxidized LDL in particular. These effects of oxidized LDL are of particular importance since it appears that in the human atherosclerotic plaque, lipoproteins are present in their oxidized form. In vascular smooth muscle cells, LDL can increase proliferative responses of the cells thereby further contributing to the atherosclerotic process and plaque formation. " Lipid lowering therapy has been shown to reverse and normalize endothelial function in experimental animals as well as in the coronary circulation of patients with coronary artery disease. Hence, endothelial dysfunction occurring in the presence of hyperlipidemia appears at least in part to be reversible and approachable therefore for lipid lowering therapy. These changes in endothelial and vascular smooth muscle function in the presence of lipoproteins may importantly contribute to the development of atherosclerosis and its complications in the coronary and cerebral circulation.
There is definite evidence that lipid lowering treatment reduces the incidence of cardiovascular events, particularly in men with already existent symptomatic cardiovascular disease. The favorable influence of lipid lowering drugs in reducing the progression of arterial disease, as evaluated by quantitative angiography, is also well established. However, the discrepancy between significant reduction of cardiovascular events and relatively small changes in the severity of arterial stenoses, still requires additional investigation. Some explanations for this discrepancy have been advocated on the basis of the results of controlled clinical trials. They include: a) Lipid-lowering treatment inhibits the formation of new lesions which are likely to be responsible for the majority of new cardiovascular events. b) Lipid-lowering drugs stabilize already existent atherosclerotic plaques, making them' less prone to overimposed thrombosis. c) Arterial remodeling , during lipid lowering treatment, leads to underestimation by angiographic methods of extensive changes occurring an the arterial wall. (d) Small changes in lumen diameter, following lipid-lowering treatment are markers of a pronounced ~mprovement of the vasodilatory capacity of the arteries, which is detectable only by functional tests. While little additional information is expected from angiographic follow-up trials, new answers are expected to come from researches using arterial wall rather than lumen parameters as end-points. There is also great need of larger studies oh functional end-points, exploring possible effects of different lipid-lowering drugs on the vasodilatory capacity of the arteries. In order to fill this knowledge gap new functional end-points should be identified, possibly taking advantage of non-invasive investigation of the arterial response to vasodilatory stimuli. Both ultrasound and plethysmographic methods are currently on the way of being adequately validated and standardized.
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LESSONS I N SIGNAL T R A N S D U C T I O N F R O M A DNA T U M O R VIRUS B. S. Sehaffhausen 1, M./Yoakim 1, K. Mullane 1, O. Gj0rupl, L. Cantley 2, T. M. Roberts 3, K. S. Campbell 3, W. Su 3 & D. Pallas3 1: Department of Biochemistry Tufts Univ. School of Medicine, Boston MA 02111; 2: Department of Medicine, Beth Israel Hospital Boston, MA ; 3: Department of Cellular and Molecular Biology, Dana-Father Cancer Institute, Boston, MA;
Growth factors are major regulators of cell proliferation, cell migration, differentiation, and extracellular matrix deposition and resorption. In regulating cell proliferation, most growth factors bind to specific cell surface receptors that are transmembrane tyrosyne kinases. The signal transduction of such receptors cause in cascade sequence activation of ras signaling to activate raf which in turn activates the MAP kinase pathway culminating in modulation of transcription factors necessary for initiating progression through G1 phase of the cell cycle. Ras and other signal transduction proteins activity is dependent on their localization in the plasma membrane through a postransational modification which covalently bind a lipid moiety (famesyl pyrophosphate or geranylgeranyl pyrophosphate) derived from mevalonate. Thus, pharmacological intervention at this bottle-neck target by means of direct and indirect inhibition of protein isoprenilation has garnered paramounting attention. Recently, we providedin vivo evidence that indirect inhibition of protein prenylation, by inhibition of HMGCoA reductase activity, resulted in a profound retardation of glioma growth rates induced by stereotaxical injection of C6 glioma cells in rat brain. Cytofluorimetric analysis of DNA from treated C 6 glioma cells in vitro showed an arrest/retardation in the G I and G2/M phases of the cell cycle. This effect was paralleled by Ras displacement from cell membrane. Moreover, inhibition of HMGCoA reductase activity in combination with classical chemotherapy (earmustine), showed a synergistic effect on glioma growth rates induced by stereotaxical injection of C6 glioma cells in rat brain, with no significant increase in toxicity as assessed by myelosuppression assay. Thus, the results provide in rive support that interfering with protein prenylation constitutes a novel approach with great potential for developing pharmacological agents with anticancer activity for brain tumor treatment.
Polyomavirus induces a wide variety of tumors in its natural host. The virus encodes a collection of activities that regulate cell . growth. In the case Of the nuclear large T antigen, such activities can be divided into Rb family-dependent and Rb family-independent activities that promote cell cycle progression. However, the major polyoma transforming protein is middle T antigen. Middle T transformation requires ras pathway function; dominant negative ras interferes with middle T transformation. Middle T action results from interactions with a collection of cell proteins that affect signal transduction. In a mamler dependent on interactions with tyrosine kinases of the src family, middle T associates with the adaptor SHC, phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C'~. All three signal tranducers can be found together in single complexes. Binding of Pt 3-K and SHC are critical for transformation, while PLC y binding is important under limiting serum conditions: For PI 3-K, a cycle of SH2 interactions with tyrosine phosphorylated sequences regulates inta-acellular trafficking. The association of middle T with SHC and PI 3-K means that there is more than one connection to ras pathways. The connections of these proteins to each other and to G protein signaling will be discussed. Finally, middle T also associates with protein phosphatase 2A. This provides an additional connection to ras signaling, since it contributes an additional mechanism for the activation of MAP kinase.
HYPOLIPIDEMIC DRUGS ON ATHEROSCLEROSIS AND ARTERIAL FUNCTION Paolo Rubba Inst.of Internal Medicine and Diseases of Metabolism Medical School,Federico Ii University, Naples, Italy
LIPIDS AND VASCULAR FUNCTION T.F. LOscher, Cardiovascular Research, Division of Cardiology, University Hospital/Inselspital, Bern, Switzerland
Lipids, and in particular low density lipoproteins (LDL)can profoundly effect vascular function. Qn endothelial cells LDL has particularly pronounced effects once it has been oxidized. Apparently it appears to bind to the scavenger receptor. After activation of this receptor, oxidized LDL is able to increase endothelin mRNA and production of endothelin peptide. Qn the other hand, oxidized LDL impairs the activity of the L-arginine nitric oxide (NO) pathway. Indeed, particularly after stimulation of serotonerglc receptors as well as c~2adrenergic receptors endothelin-dependent relaxations of porcine coronary arteries are severely reduced in the presence of oxidized LDL. Furthermore, it is likely that the intracellular mobilization of Larginine is blunted under these conditions. Finally, it has been proposed that hypertipidemia and atherosclerosis markedly increases the breakdown of nitric oxide by superoxide and other oxygen-derived free radicals produced within the endothelium under these conditions. Furthermore, LDL can activate platelets and therefore contribute to the pro-thrombotic state occurring after impairment of endothelial function in the presence of high local concentrations of LDL and oxidized LDL in particular. These effects of oxidized LDL are of particular importance since it appears that in the human atherosclerotic plaque, lipoproteins are present in their oxidized form. In vascular smooth muscle cells, LDL can increase proliferative responses of the cells thereby further contributing to the atherosclerotic process and plaque formation. " Lipid lowering therapy has been shown to reverse and normalize endothelial function in experimental animals as well as in the coronary circulation of patients with coronary artery disease. Hence, endothelial dysfunction occurring in the presence of hyperlipidemia appears at least in part to be reversible and approachable therefore for lipid lowering therapy. These changes in endothelial and vascular smooth muscle function in the presence of lipoproteins may importantly contribute to the development of atherosclerosis and its complications in the coronary and cerebral circulation.
There is definite evidence that lipid lowering treatment reduces the incidence of cardiovascular events, particularly in men with already existent symptomatic cardiovascular disease. The favorable influence of lipid lowering drugs in reducing the progression of arterial disease, as evaluated by quantitative angiography, is also well established. However, the discrepancy between significant reduction of cardiovascular events and relatively small changes in the severity of arterial stenoses, still requires additional investigation. Some explanations for this discrepancy have been advocated on the basis of the results of controlled clinical trials. They include: a) Lipid-lowering treatment inhibits the formation of new lesions which are likely to be responsible for the majority of new cardiovascular events. b) Lipid-lowering drugs stabilize already existent atherosclerotic plaques, making them' less prone to overimposed thrombosis. c) Arterial remodeling , during lipid lowering treatment, leads to underestimation by angiographic methods of extensive changes occurring an the arterial wall. (d) Small changes in lumen diameter, following lipid-lowering treatment are markers of a pronounced ~mprovement of the vasodilatory capacity of the arteries, which is detectable only by functional tests. While little additional information is expected from angiographic follow-up trials, new answers are expected to come from researches using arterial wall rather than lumen parameters as end-points. There is also great need of larger studies oh functional end-points, exploring possible effects of different lipid-lowering drugs on the vasodilatory capacity of the arteries. In order to fill this knowledge gap new functional end-points should be identified, possibly taking advantage of non-invasive investigation of the arterial response to vasodilatory stimuli. Both ultrasound and plethysmographic methods are currently on the way of being adequately validated and standardized.
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