Cell growth inhibition by farnesyltransferase inhibitors is mediated by gain of geranylgeranylated RhoB.
Recent results have shown that the ability of farnesyltransferase inhibitors (FTIs) to inhibit malignant cell transformation and Ras prenylation can be separated. We proposed previously that farnesylated Rho proteins are important targets for alternation by FTIs, based on studies of RhoB (the FTI-Rho hypothesis). Cells treated with FTIs exhibit a loss of farnesylated RhoB but a gain of geranylgeranylated RhoB (RhoB-GG), which is associated with loss of growth-promoting activity. In this study, we tested whether the gain of RhoB-GG elicited by FTI treatment was sufficient to mediate FTI-induced cell growth inhibition. In support of this hypothesis, when expressed in Ras-transformed cells RhoB-GG induced phenotypic reversion, cell growth inhibition, and activation of the cell cycle kinase inhibitor p21WAF1. RhoB-GG did not affect the phenotype or growth of normal cells. These effects were similar to FTI treatment insofar as they were all induced in transformed cells but not in normal cells. RhoB-GG did not promote anoikis of Ras-transformed cells, implying that this response to FTIs involves loss-of-function effects. Our findings corroborate the FTI-Rho hypothesis and demonstrate that gain-of-function effects on Rho are part of the drug mechanism. Gain of RhoB-GG may explain how FTIs inhibit the growth of human tumor cells that lack Ras mutations. (+info)
Human geranylgeranyl diphosphate synthase. cDNA cloning and expression.
Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA. (+info)
The geranylgeranyltransferase I inhibitor GGTI-298 induces hypophosphorylation of retinoblastoma and partner switching of cyclin-dependent kinase inhibitors. A potential mechanism for GGTI-298 antitumor activity.
The geranylgeranyltransferase I inhibitor GGTI-298 has recently been shown to arrest human tumor cells in the G1 phase of the cell cycle, induce apoptosis, and inhibit tumor growth in nude mice. In the present manuscript, we provide a possible mechanism by which GGTI-298 mediates its tumor growth arrest. Treatment of the human lung carcinoma cell line Calu-1 with GGTI-298 results in inhibition of the phosphorylation of retinoblastoma protein, a critical step for G1/S transition. The kinase activities of two G1/S cyclin-dependent kinases, CDK2 and CDK4, are inhibited in Calu-1 cells treated with GGTI-298. Furthermore, GGTI-298 has little effect on the expression levels of CDK2, CDK4, CDK6, cyclins D1 and E, but decreases the levels of cyclin A. GGTI-298 increases the levels of the cyclin-dependent kinase inhibitors p21 and p15 and had little effect on those of p27 and p16. Most interesting is the ability of GGTI-298 to induce partner switching for several CDK inhibitors. GGTI-298 promotes binding of p21 and p27 to CDK2 while decreasing their binding to CDK6. Reversal of partner switching and G1 block was observed after removal of GGTI-298. Furthermore, GGTI-298 treatment results in an increased binding of p15 to CDK4, which is paralleled with decreased binding to p27. The results demonstrate that the GGTI-298-mediated G1 block in Calu-1 cells involves increased expression and partner switching of CDK inhibitors resulting in inhibition of CDK2 and CDK4, and retinoblastoma protein phosphorylation. (+info)
Reduced lung tumorigenesis in human methylguanine DNA--methyltransferase transgenic mice achieved by expression of transgene within the target cell.
Human methylguanine-DNA methyltransferase (MGMT) transgenic mice expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) in lung were crossbred to A/J mice that are susceptible to pulmonary adenoma to study the impact of O6-methylguanine (O6mG)-DNA adduct repair on NNK-induced lung tumorigenesis. Expression of the chimeric human MGMT transgene in lung was identified by northern and western blot analysis, immunohistochemistry assay and enzymatic assay. AGT activity was 17.6 +/- 3.2 versus 1.2 +/- 0.4 fmol/microg DNA in lung of MGMT transgenic mice compared with non-transgenic mice. Immunohistochemical staining with anti-human AGT antibody showed that human AGT was expressed throughout the lung. However, some epithelial cells of bronchi and alveoli did not stain for human AGT, suggesting that the human MGMT transgene expression was heterogeneous. After 100 mg/kg NNK i.p. injection in MGMT transgenic mice, lung AGT activity remained much higher and levels of lung O6mG-DNA adducts in MGMT transgenic mice were lower than those of non-transgenic mice. In the tumorigenesis study, mice received 100 mg/kg NNK at 6 weeks of age and were killed 44 weeks later. Ten of 17 MGMT transgenic mice compared with 16 of 17 non-transgenic mice had lung tumors, P < 0.05. MGMT transgenic mice had lower multiplicity and smaller sized lung tumors than non-transgenic mice. Moreover, a reduction in the frequency of K-ras mutations in lung tumors was found in MGMT transgenic mice (6.7 versus 50% in non-transgenic mice). These results indicate that high levels of AGT expressed in mouse lung reduce lung tissue susceptibility to NNK-induced tumorigenesis due to increased repair capacity for O6mG, subsequently, decreased mutational activation of K-ras oncogene. Heterogeneity in the level of AGT expressed in different lung cell populations or other forms of carcinogenic DNA damage caused by NNK may explain the residual incidence of lung tumors in MGMT transgenic mice. (+info)
RAS and leukemia: from basic mechanisms to gene-directed therapy.
PURPOSE AND DESIGN: The purpose of this review is to provide an overview of the literature linking Ras signaling pathways and leukemia and to discuss the biologic and potential therapeutic implications of these observations. A search of MEDLINE from 1966 to October 1998 was performed. RESULTS: A wealth of data has been published on the role of Ras pathways in cancer. To be biologically active, Ras must move from the cytoplasm to the plasma membrane. Importantly, a posttranslational modification--addition of a farnesyl group to the Ras C-terminal cysteine--is a requisite for membrane localization of Ras. Farnesylation of Ras is catalyzed by an enzyme that is designated farnesyltransferase. Recently, several compounds have been developed that can inhibit farnesylation. Preclinical studies indicate that these molecules can suppress transformation and tumor growth in vitro and in animal models, with little toxicity to normal cells. CONCLUSION: An increasing body of data suggests that disruption of Ras signaling pathways, either directly through mutations or indirectly through other genetic aberrations, is important in the pathogenesis of a wide variety of cancers. Molecules such as farnesyl transferase inhibitors that interfere with the function of Ras may be exploitable in leukemia (as well as in solid tumors) as novel antitumor agents. (+info)
Molecular forceps from combinatorial libraries prevent the farnesylation of Ras by binding to its carboxyl terminus.
INTRODUCTION: Ras is one of the major oncogenes. In order to function properly it has to undergo post-translational processing at its carboxyl terminus. It has been shown that inhibitors of farnesyl transferase, the first enzyme in the processing chain, can suppress the transforming activity of oncogenic Ras. RESULTS: We have identified molecular forceps, branched peptidic molecules, from combinatorial libraries that bind to the carboxyl terminus of Ras and interfere with its farnesylation without inhibiting the farnesyl transferase. The active molecules were selected by a screening against the carboxy-terminal octapeptide of Ras. CONCLUSIONS: The implications of our findings are twofold. First, we demonstrate that it is possible to prevent enzymatic transformations by blocking the enzyme's access to its substrate using a synthetic small molecule to mask the substrate. Second, we show that it is feasible to derive molecules from combinatorial libraries that bind a specific epitope on a protein by selecting these molecules with the isolated peptide epitope. (+info)
Effect of the hypocholesterolemic agent YM-16638 on cholesterol biosynthesis activity and apolipoprotein B secretion in HepG2 and monkey liver.
YM-16638 ([[5-[[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl]thio]-1,3,4-++ +thiadiazol-2-yl] thio] acetic acid) showed a strong hypocholesterolemic effect in humans and monkeys. To clarify the mechanism of this hypocholesterolemic effect, the action of YM-16638 on cholesterol biosynthesis in the cultured human hepatoma cell line HepG2 and cynomolgus monkey liver was examined. Cholesterol biosynthesis activity derived from [14C]acetic acid, [3H/14C]mevalonic acid or [14C]isopentenyl pyrophosphate substrates was significantly decreased, but not that from [3H]farnesyl pyrophosphate or [3H]squalene substrates in HepG2 cells treated with YM-16638. Simultaneously, treatment of these cells with YM-16638 changed neither the rate of apolipoprotein B synthesis from [35S]methionine nor its secretion. In addition, the activities of hepatic cholesterol biosynthesis enzymes HMG-CoA reductase, mevalonate kinase (MK), isopentenyl pyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPPS), squalene synthase and squalene epoxidase were measured in monkeys fed a diet supplemented with YM-16638. Among these enzymes, MK, IPPI and FPPS activities in the YM-16638-treated group significantly decreased by 38%, 56% and 30%, respectively, when compared to those from control animals receiving no drug treatment. These results indicate that YM-16638 has the characteristics of a cholesterol biosynthesis inhibitor. (+info)
Leukocyte O6-alkylguanine-DNA alkyltransferase from human donors is uniformly sensitive to O6-benzylguanine.
O6-Alkylguanine-DNA alkyltransferase (AGT) is the key DNA repair protein responsible for resistance to chloroethylating and methylating agents that attack at the O6 position of guanine. O6-Benzylguanine (BG), a potent inhibitor of AGT, has recently entered clinical trials. A number of point mutations and at least one human polymorphism within AGT are associated with AGT resistance to inactivation by BG. In this study, we evaluated AGT inhibition by BG in an in vitro assay of peripheral blood mononuclear cell AGT from 56 normal donors, 42 Caucasians, and 14 Japanese. AGT activity ranged from 2.7 to 21.9 fmol/microg DNA and was similar in Japanese and Caucasian donors. Depletion of AGT by BG was uniform in all donors with mean ED50s of 037 microM BG in Caucasians and 0.36 microM BG in Japanese. To determine whether the gly160arg AGT polymorphism described in the Japanese population, and recently shown to be BG resistant, could be detected by this assay, we mixed purified gly160arg AGT protein with blood mononuclear cell extract and measured in vitro BG inactivation. The ED50 for the mixture of the gly160arg AGT and mononuclear cell extract was 9 microM BG. On the basis of results in 56 donors, we conclude that BG-resistant AGT, defined as an ED50 in mononuclear cells of >1 microM BG, is present in 0 of 56 donors, (95% confidence interval, 0-6%), suggesting that polymorphisms producing AGT-resistant BG are unusual in humans. (+info)