It has recently been reported that Ki-Ras protein can be modified in vitro by farnesylation or geranylgeranylation. However, a previous analysis of Ki-Ras prenylation in vivo found only farnesylated Ki-Ras. In this report it is shown that under normal conditions, Ki-Ras is farnesylated in vivo and when cells are treated with the farnesyl transferase inhibitors B956 or B957, farnesylation is inhibited and Ki-Ras becomes geranylgeranylated in a dose dependent manner. These results have strong implications in the design of anticancer drugs based on inhibition of prenylation.Post-translational modifications of small molecular weight GTP-binding proteins have been the subject of intense studies since the first reports of prenylation appeared (1-3). Many of these proteins, and specifically the Ras proteins, are involved in tumorigenesis. Since prenylation is necessary for the transformation potential of Ras proteins, a number of groups have been dedicated to the design of inhibitors of prenylation as a strategy to inhibit tumor formation (4).Two types of prenyl modifications have been described: farnesylation and geranylgeranylation. The enzymes FTase 1 and GGTase I recognize a tetra peptide sequence at the C terminus of a protein (5, 6). This sequence has been designated the CAAX box, and it determines whether a protein will be prenylated (7). In this sequence, C stands for cysteine and A for any amino acid. The amino acid X specifies whether the protein will be farnesylated (methionine or serine) or geranylgeranylated (leucine or phenylalanine) by the appropriate enzyme. This rule is followed in the majority of cases. However, recent reports have shown that Ki-Ras, whose C terminus CVIM predicts farnesylation, can also be geranylgeranylated in vitro, (8) and that its prenylation in cells is inhibited by a GGTase I inhibitor (9). Since activated Ki-Ras is the type of Ras most frequently found in human cancers, it is very important to determine how it is modified in vivo and whether this modification can be inhibited. Cell labeling with [ 3 H]mevalonate in the presence of inhibitors of hydroxymethylglutaryl-CoA reductase such as lovastatin allows the radioactive labeling of cholesterol and its intermediate isoprenoids. However, a major drawback of this approach is the low level of [ 3 H]mevalonate incorporation into cells. The fortuitous identification of the gene for a mevalonate transport membrane protein (pMev) has provided a tool that makes the identification of isoprenoid and non-steroidal isoprenoid intermediates easier (10, 11). Transfection of cells expressing either Ha-Ras or Ki-Ras oncogenes with the mevalonate transporter and growth of the cells under conditions in which the endogenous pools of farnesyl diphosphate and geranylgeranyl diphosphate are radioactively labeled enabled us to demonstrate that both Ha-Ras and Ki-Ras are farnesylated under normal conditions. In the presence of the farnesyl transferase inhibitor B956, farnesylation of both Ha-Ras and Ki-Ras was inhibited in a dose-dependent manner. Ho...

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E7974 is a synthetic analogue of the marine sponge natural product hemiasterlin. Here, we show that E7974, such as parental hemiasterlin, acts via a tubulin-based antimitotic mechanism. E7974 inhibits polymerization of purified tubulin in vitro with IC 50 values similar to those of vinblastine. In cultured human cancer cells, E7974 induces G 2 -M arrest and marked disruption of mitotic spindle formation characteristic of tubulin-targeted anticancer drugs. Extensive hypodiploid cell populations are seen in E7974-treated cells, indicating initiation of apoptosis after prolonged G 2 -M blockage. Consistent with this observation, E7974 induces caspase-3 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis. Only a short cellular exposure to E7974 is sufficient to induce maximum mitotic arrest, suggesting that E7974's antitumor effects in vivo may persist even after blood levels of the drug decrease after drug administration. Interactions of E7974 with purified tubulin were investigated using two synthetic tritiated photoaffinity analogues incorporating a benzophenone photoaffinity moiety at two different positions of the E7974 scaffold. Both analogues preferentially photolabeled α-tubulin, although minor binding to β-tubulin was also detected. E7974 thus seems to share a unique, predominantly α-tubulin-targeted mechanism with other hemiasterlin-based compounds, suggesting that, unlike many tubulin-targeted natural products and related drugs, the hemiasterlins evolved to mainly target α-tubulin, not β-tubulin subunits.

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