Ras antagonist inhibits growth and chemosensitizes human epithelial ovarian cancer cells.

The objective of this article was to determine whether human ovarian carcinoma cells (OVCAR-3) express significant amounts of Ras oncogene and active Ras-guanosine triphosphate (GTP) and, if so, whether the Ras inhibitor farnesyl thiosalicylic acid (FTS) inhibits their growth and chemosensitizes them to cisplatin. We assayed Ras and Ras-GTP in OVCAR-3 cells before and after FTS treatment. The effect of FTS on OVCAR-3 cell growth was assessed in terms of cell number. Because the OVCAR-3 cell line was derived from a patient who was refractory to cisplatin, we examined whether FTS enables cisplatin to induce death of these cells. Significant amounts of Ras and active Ras-GTP were expressed by OVCAR-3 cells and were reduced by 40% by FTS. FTS inhibited OVCAR-3 cell growth in a dose-dependent manner. When combined with cisplatin, FTS reduced the number of OVCAR-3 cells by 80%, demonstrating synergism between FTS and cisplatin. FTS, at a concentration range that allows downregulation of Ras and Ras-GTP in OVCAR-3 cells, also chemosensitizes these cells and inhibits their growth. These results suggest that ovarian carcinomas might respond well to Ras inhibition, both alone and when combined with cisplatin. The combined treatment would allow the use of smaller doses of chemotherapy, resulting in decreased cytotoxicity.

Treatment of MRL/lpr mice, a genetic autoimmune model, with the Ras inhibitor, farnesylthiosalicylate (FTS).

Activation and proliferation of lymphocytes requires the active signal transducer Ras. Activation of lymphocytes, associated with autoimmunity, may therefore be modified by S-farnesylthiosalicylic acid (FTS), a synthetic substance that detaches Ras from the inner cell membrane and induces its rapid degradation. The MRL/lpr mouse is a genetic model of a generalized autoimmune disease sharing many features and organ pathology with systemic lupus erythematosus (SLE) and the primary antiphospholipid syndrome (APS). The objective of the present study was to examine the effect of FTS on laboratory and clinical pathology in the MRL/lpr mouse. Female MRL/lpr (n = 50) and MRL/++ control (n = 35) mice were treated intraperitoneally with either FTS (5 mg/kg/day) or saline between 6 and 18 weeks of age. The mice were weighed, tested for proteinuria and lymphadenopathy, lymphocyte proliferation, antibodies, grip strength and behaviour in an open field. FTS treatment resulted in a 50% decrease in splenocyte proliferation to ConA, LPS and a disease specific antigen, beta(2)-glycoprotein-I, and in a significant decrease in serum antibody levels against cardiolipin and dsDNA. Proteinuria and grip strength were normalized and lymphadenopathy and postmortem lymph node and spleen weights were significantly reduced in FTS treated MRL/lpr mice. These findings indicate that modulation of Ras activation has a significant impact on the MRL/lpr model and may represent a new therapeutic approach for the treatment of systemic autoimmune diseases such as SLE and APS.

The Ras-pathway inhibitor, S-trans-trans-farnesylthiosalicylic acid, suppresses experimental allergic encephalomyelitis.

AIM: To evaluate the effects of the synthetic Ras-pathway inhibitor, S-trans-trans-farnesylthiosalicylic acid (FTS) on acute and chronic experimental autoimmune encephalomyelitis (EAE and CR-EAE). BACKGROUND: Treatment of EAE and MS is based on immunosuppression aiming at downregulation of the proliferating myelin-reactive lymphocytes. One of the pathways of lymphocyte activation involves the GTP-binding protein Ras. FTS destabilizes the attachment of Ras to the cell membrane, resulting in an inhibition of the Ras-mediated signal transduction pathways. MATERIALS AND METHODS: EAE was induced in SJL/J mice by immunization with spinal cord homogenate (MSCH) in adjuvant and two i.v. boosts of pertussis antigen and CR-EAE with passive transfer of proteolipid protein (PLP)-activated lymphocytes. Animals were treated daily starting either from the day of EAE-induction (or cell transfer) or at a later stage, with i.p. injections of FTS (5 mg/kg/day). The clinical severity of the disease was evaluated daily and scored using a 0-6 scale. RESULTS: In six separate experiments, 27 of the 38 (71.7%) vehicle-treated animals developed clinical signs of EAE compared to 17/38 (44.7%) of the FTS-treated mice (p=0.02, t-test). The maximal average score in the control group was 2.94+/-2.2, whereas in the FTS group it was significantly lower (1.63+/-2.2, p=0.01). Mortality was 26.3% and 10.5% in the two groups, respectively (p=0.03). When treatment was initiated at a later stage, just before the onset of the clinical signs, the protective effect was even more pronounced. A significant suppression of clinical signs was also observed in the CR-EAE model (p=0.02). Lymphocyte proliferation assays demonstrated a more than twofold decrease in the reactivity to myelin antigens (MBP and PLP) and downregulation of the activated lymphocytes (expressing the CD62L, and IA-k-MHC Class I markers and the Vb17 T-cell receptor) in the FTS-treated group; in vitro FTS suppressed the Ras activity of lymphocytes and inhibited the proliferative ability of the lymphocytes in a dose-dependent manner. CONCLUSIONS: FTS suppresses EAE by downregulation of myelin-reactive activated T-lymphocytes. Since FTS did not induce generalized immunosuppressive effects, it may offer significant advantages over the broad immunosuppressive modalities and may be a candidate treatment for autoimmune diseases, such as MS.

Membrane interactions of a constitutively active GFP-Ki-Ras 4B and their role in signaling. Evidence from lateral mobility studies.

Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonist S-trans, trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306-1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was followed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites.

C8-amino purine nucleosides. A well-defined steric determinant of glycosyl conformational preferences.

100 MHz proton magnetic resonance measurements were performed on dilute solutions of adenosine and guanosine and their 8MH2, 8-NHCH3 8-N(CH3)2 and 8-bromo derivatives. The chemical shift of the ribose C2'-H and especially the difference in chemical shifts between the C1'-H and C2'-H resonances clearly indicated whether the nucleoside exists in a syn glycosyl conformation (the C8-dimethylamino derivatives) or as a flexible syn-anti mixture (the monomethylamino and amino derivatives). The temperature dependent behavior of these indicators can be employed to define qualitative shifs in syn-anti equilibrium with temperature. An increased C1'-H-C2'-H chemical shift separation implies shift to more anti, a decreased separation a shift to more syn conformers.

Glycosyl conformational and inductive effects on the acid catalysed hydrolysis of purine nucleosides.

The log kobs vs. pH profiles were determined in the intermediate acidity region for the glycosyl hydrolysis of guanosine and its 8-amino, 8-monomethylamino, 8-dimethylamino and 8-bromo derivatives. The decreased rate of the 8-amino and enhanced rate of the 8-bromo compound compared to guanosine support an A type mechanism: base protonation followed by glycosyl bond cleavage. All three 8-amino guanosines exhibited log kobs - pH profiles clearly showing that both mono and di-base protonated nucleosides undergo hydrolysis. The 700 fold rate acceleration of 8-N(CH3)-guanosine compared to 8-NHCH3-guanosine and the 110 fold rate acceleration of 8-N(CH3)2-adenosine compared to 8-NHCH3-adenosine could be unequivocally attributed to the fixed syn glycosyl conformation of both 8-dimethylamino compounds and relief of steric compression upon hydrolysis in these molecules. The lack of anomerization of all substrates during the course of the reaction supports an A rather than a Schiff-base mechanism.