For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.

The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Ratl/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Ratl/ras and normal Ratl cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Ratl/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.

Small-molecule inhibitors of the housekeeping enzyme farnesyltransferase (FT) suppress the malignant growth of Ras-transformed cells. Previous work suggested that the activity of these compounds reflected effects on actin stress fiber regulation rather than Ras inhibition. Rho proteins regulate stress fiber formation, and one member of this family, RhoB, is farnesylated in vivo. Therefore, we tested the hypothesis that interference with RhoB was the principal basis by which the peptidomimetic FT inhibitor L-739,749 suppressed Ras transformation. The half-life of RhoB was found to be ϳ2 h, supporting the possibility that it could be functionally depleted Isoprenylated proteins, which constitute ϳ0.5% of the proteins in the cell, fall into two classes that are characterized by the presence of either farnesyl (C 15 ) or geranylgeranyl (C 20 ) isoprenoids (for reviews, see references 18 and 32). Isoprenylation is required for the biological function and efficient membrane association of these proteins, possibly because of a role of the isoprenyl groups in protein-protein interactions (33). The signal for isoprenyl modification is located in the C terminus of the polypeptide and dictates not only the specificity of the isoprenoid but also further modifications which occur.The housekeeping enzyme farnesyltransferase (FT) is one of at least three isoprenyl-protein transferase activities that have been identified (for reviews, see references 5, 10, 35, and 48). FT recognizes on its substrates a C-terminal CAAX motif (where C is cysteine, A is typically an aliphatic amino acid, and X is any amino acid) and catalyzes transfer of the C 15 isoprenoid from farnesyl PP i to the CAAX cysteine. The farnesylated product is a substrate for additional modification by enzymes which proteolyze the terminal three amino acids and carboxymethylate the new C terminus. Some farnesylated proteins (e.g., H-Ras) are further modified by the addition of palmitate to an additional cysteine(s) that is proximal to the CAAX motif. In these cases, palmitylation appears to increase the avidity of membrane association (20) but is dispensable for biological activity (26).Since FT is responsible for farnesylating oncogenic Ras (8), small-molecule inhibitors of FT were sought as possible cancer therapeutics (16,17). Recently, FT inhibitors have been demonstrated to revert the malignant phenotype of Ras-transformed cells in vitro and in vivo without toxic or anti-proliferative effects on normal cells (15, 24, 28, 28a, 29, 32a). These results present some question as to the biological mechanism of action, since Ras and other farnesylated proteins (e.g., lamin B [14]) are critical for normal cell growth and function. Our previous work revealed that the kinetics of reversion induced by one FT inhibitor, L-739,749, were too rapid to be explained easily by suppression of Ras activity. Instead, the inhibitory effects of L-739,749 correlated with effects on the regulation of actin stress fiber formation (39).We tested the hypothesis that a putative stress fibe...