ggti-298 and geranylgeraniol

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.

Topics: Apoptosis; Benzamides; Caspase 3; Caspases; Cell Death; Cells, Cultured; Diterpenes; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracellular Signaling Peptides and Proteins; Lovastatin; Methionine; Polyisoprenyl Phosphates; Protein Prenylation; Protein Serine-Threonine Kinases; rho-Associated Kinases; rhoA GTP-Binding Protein; Umbilical Veins

Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC(50) approximately 1-2 microM) alpha(4)beta(1)-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the beta(1) integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates beta(1) integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, alpha(6)beta(1)-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion.

Topics: Benzamides; Cell Adhesion; Diterpenes; Fibronectins; Humans; Integrins; Jurkat Cells; Leukocytes; Lovastatin; Methionine; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Tetradecanoylphorbol Acetate; U937 Cells

In order to assess the relative contributions of farnesylated and/or geranylgeranylated proteins on cell cycle progression from G1 to S phase we designed potent and selective farnesyltransferase (FTI-277) and geranylgeranyltransferase-I (GGTI-298) inhibitors. Flow cytometry studies showed that treatment of NIH3T3 cells with GGTI-298 or lovastatin, which inhibits both protein farnesylation and geranylgeranylation, arrested cells in G0/G1 whereas cells treated with FTI-277 progressed normally through the cell cycle. [3H]thymidine incorporation studies showed that mevalonate and geranylgeraniol, but not farnesol, released the lovastatin G1 block. Furthermore, mevalonate release of the lovastatin G1 block was inhibited by GGTI-298 but not by FTI-277. These results demonstrate that geranylgeranylated proteins are required for cells to proceed from G1 to S phase, and that farnesylated proteins do not play an essential role in the G1 to S phase transition

Topics: Actins; Alkyl and Aryl Transferases; Animals; Benzamides; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Diterpenes; DNA; Enzyme Inhibitors; Farnesol; Fibroblasts; G1 Phase; Genes, ras; GTP-Binding Proteins; Lovastatin; Methionine; Mevalonic Acid; Mice; Protein Prenylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; rap GTP-Binding Proteins; ras Proteins; Resting Phase, Cell Cycle; S Phase; Transferases