GGTI-298 induces G0-G1 block and apoptosis whereas FTI-277 causes G2-M enrichment in A549 cells.

The mechanism by which the geranylgeranyltransferase I inhibitor GGTI-298 and the farnesyltransferase inhibitor FTI-277 inhibit human tumor growth is not known. Herein, we demonstrate that in the human lung adenocarcinoma A549 cells, GGTI-298 induced a G1-G0 block whereas FTI-277 induced an enrichment in the G2-M phase of the cell cycle. Although FTI-277, GGTI-298, and compactin inhibited A549 cell growth, only GGTI-298 and compactin induced apoptosis as demonstrated by four criteria: 4',6-diamidine-2-phenylindoledihydrochloride staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, DNA fragmentation assay, and flow cytometry. Furthermore, the involvement of geranylgeranylated proteins in apoptotic pathways was confirmed by demonstrating that geranylgeraniol was able to block the ability of compactin to induce apoptosis. These results suggest that protein geranylgeranylation is critical for the control of programmed cell death and that, in A549 cells, farnesylated and geranylgeranylated proteins are involved in G2-M and G0-G1, respectively.

Farnesol and geranylgeraniol induce actin cytoskeleton disorganization and apoptosis in A549 lung adenocarcinoma cells.

The effects of exogenous isoprenoids were investigated on A549 human lung adenocarcinoma cells. Among the tested isoprenoids, only farnesol and geranylgeraniol induce actin cytoskeleton disorganization, growth inhibition, and apoptosis. In contrast, desmosterol leads only to growth inhibition. We show that all tested isoprenoids are potent inhibitors of HMG CoA reductase activity, the sterols being the most powerful while they induce neither F-actin disorganization nor apoptosis. Thus the molecular mechanisms induced by farnesol and geranylgeraniol appear independent of reductase regulation. Our results point out the specific role of farnesol and geranylgeraniol on actin cytoskeleton organization and apoptosis in adenocarcinoma cells.

Competitive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells.

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

Differential regulation of fibroblast growth factor (FGF) receptor-1 mRNA and protein by two molecular forms of basic FGF. Modulation of FGFR-1 mRNA stability.

To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the beta-actin promoter were used to infect a pancreatic cell line (AR4 2J) which does not produce FGF-2. Saturation and competition binding studies with 125I-FGF-2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. Kd values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, -2, and -3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3-fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF-2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2.

Inhibition of human tumor cell growth in vitro and in vivo by a specific inhibitor of human farnesyltransferase: BIM-46068.

Oncogenic mutations of the ras gene leading to constitutive activation of downstream effectors have been detected in a large spectrum of human cancers (pancreas, thyroid, colon and NSCLC). Membrane anchorage of Ras required for functional activity in signal transduction is facilitated by post-translational modifications resulting in covalent attachment of a farnesyl group to the cysteine in the C-terminal CAAX motif. This attachment is mediated by farnesyltransferase (FTase). Here, we report a novel series of potent FTase inhibitors, where the tetrapeptide CAAX motif has been modified by incorporation of a thiazolidine carboxylic acid moiety followed by reduction of the 1st and 2nd peptide bonds to a secondary and tertiary amine, respectively. The C-terminal carboxylate was converted to esters for improved cellular penetration. These compounds showed specific inhibition of purified human FTase enzyme, inhibition of proliferation in vitro in a large spectrum of human tumor cell lines and inhibition of growth of human tumor xenografts in athymic nude mice. In addition, in regard to a panel of cell lines, using the Compare analysis to determine the Pearson coefficient correlation, the anti-proliferative spectrum of BIM-46068 has been shown to be distinct from the profile of typical chemotherapeutic agents.