The importance of post-translational geranylgeranylation of the GTPase RhoA for its ability to induce cellular proliferation and malignant transformation is not well understood. In this manuscript we demonstrate that geranylgeranylation is required for the proper cellular localization of V14RhoA and for its ability to induce actin stress fiber and focal adhesion formation. Furthermore, V14RhoA geranylgeranylation was also required for suppressing p21 WAF transcription, promoting cell cycle progression and cellular proliferation. The ability of V14RhoA to induce focus formation and enhance plating efficiency and oncogenic Ras anchoragedependent growth was also dependent on its geranylgeranylation. The only biological activity of V14RhoA that was not dependent on its prenylation was its ability to induce serum response element transcriptional activity. Furthermore, we demonstrate that a farnesylated form of V14RhoA was also able to bind RhoGDI-1, was able to induce cytoskeleton organization, proliferation, and transformation, and was just as potent as geranylgeranylated V14RhoA at suppressing p21 WAF transcriptional activity. These results demonstrate that RhoA geranylgeranylation is required for its biological activity and that the nature of the lipid modification is not critical.Small G proteins of the Ras superfamily are regulatory proteins whose activity is controlled by a GDP/GTP cycle. Several members of the Ras superfamily are regulators of signaling pathways that control cell growth, differentiation, and oncogenic transformation as well as actin cytoskeletal organization (1). The Rho protein branch of this superfamily includes at least eight distinct Rho families (RhoA, B, C, D, and G, Rac1 and 2, TC10, Cdc42, and Rnd1, 2, and 3) (2) that are regulated by Rho-GTPase activating proteins and a large family of guanine nucleotide exchange factors of the Dbl family proteins. Moreover Rho guanine nucleotide dissociation inhibitors (RhoGDIs) 1 stabilize the inactive GDP-bound form of the Rho proteins. Rho proteins notably regulate signal transduction from cell surface receptors to intracellular molecules and are involved in a variety of cellular processes including cell morphology (3), motility (4), cytokinesis (5, 6), cell proliferation (7, 8), and tumor progression (9 -11).Ras and Rho proteins are post-translationally modified by the isoprenoid lipids, farnesyl, and geranylgeranyl (12). Two prenyltransferases, farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I), catalyze the covalent attachment of the farnesyl and geranylgeranyl groups, respectively, to the carboxyl-terminal cysteine of proteins ending in a CAAX motif (C is a cysteine, A usually aliphatic amino acid, and X any amino acid). FTase prefers CAAX sequences where X is a serine, methionine, cysteine, alanine, or glutamine, as in Ras or in nuclear lamins (13-15). When X is a leucine or isoleucine the protein, as in the Rho/Rac family of proteins, is geranylgeranylated by GGTase I (16, 17). Protein prenylation is important in target...

This study aimed to assess the effect of cetuximab (C225, Erbitux, a chimeric anti-epidermal growth factor receptor (EGFR) monoclonal antibody) in combination with oxaliplatin in vitro and in vivo on four colon cancer cell lines (HCT-8; HT-29, SW620, HCT-116) expressing different levels of EGFR. In vitro, cetuximab combined with oxaliplatin significantly decreased the IC50 values of oxaliplatin in HCT-8 (EGF-R moderate) and HT-29 (EGF-R weak) cell lines, while SW620 (EGF-R negative) and HCT-116 (EGFR strong) cell lines remained unresponsive. This combination was synergistic in HCT-8 and HT-29 cell lines while cetuximab induced no major modification of the IC50 of oxaliplatin in HCT-116 or SW620 cell lines. We then determined the effect of cetuximab on the EGF-induced EGFR phosphorylation and we highlight a correlation between the basal level of phospho-EGFR and the response to the combination. In vivo, the combination of cetuximab plus oxaliplatin significantly inhibited tumor growth of HCT-8 and HT-29 (tumor delay or Td = 21.6+/-2.9 and 18.0+/-2.9 days respectively, synergistic effect) compared to either oxaliplatin (Td=12.6+/-2.3 and 14.4+/-3.2 days respectively) or cetuximab (Td=13.4+/-2.9 and 14.5+/-2.4 days, respectively) alone in xenograft models. The combination had no effect on HCT-116 and SW-620 cell lines. The observed responses are strictly dependent on the cell type, and are not correlated with the level of EGFR expression but related to the basal level of phospho-EGFR. This study provides promising preclinical results for a possible clinical investigation of the combination of oxaliplatin plus cetuximab in chemorefractory colorectal tumors.

The microenvironment is known to play a dominant role in cancer progression. Cells closely associated with tumoral cells, named hospicells, have been recently isolated from the ascites of ovarian cancer patients. Whilst these cells present no specific markers from known cell lineages, they do share some homology with bone marrow‐derived or adipose tissue‐derived human mesenchymal stem cells (CD9, CD10, CD29, CD146, CD166, HLA‐1). We studied the role of hospicells in ovarian carcinoma progression. In vitro, these cells had no effect on the growth of human ovarian carcinoma cell lines OVCAR‐3, SKOV‐1 and IGROV‐1. In vivo, their co‐injection with adenocarcinoma cells enhanced tumor growth whatever the tumor model used (subcutaneous and intraperitoneally established xenografts in athymic mice). In addition, their injection increased the development of ascites in tumor‐bearing mice. Fluorescent macroscopy revealed an association between hospicells and ovarian adenocarcinoma cells within the tumor mass. Tumors obtained by coinjection of hospicells and human ovarian adenocarcinoma cells presented an increased microvascularization indicating that the hospicells could promote tumorigenicity of ovarian tumor cells in vivovia their action on angiogenesis. This effect on angiogenesis could be attributed to the increased HIF1α and VEGF expression associated with the presence of the hospicells. Collectively, these data indicate a role for these ascite‐derived stromal cells in promoting tumor growth by increasing angiogenesis.

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxylterminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirusinfected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation. P rotein isoprenylation is a posttranslational modification by lipid recently discovered (1-3) that affects about 0.5% of cellular proteins and is essential for protein biological activity (4). Two prenyltransferases catalyze, through thioether bonds, the covalent attachment of prenyl groups from prenylpyrophosphates to the carboxyl-terminal cysteine of the protein included in either a CA 1 A 2 X motif (C is a cysteine, A 1 and A 2 usually aliphatic amino acids) or a CC, CCXX, or CXC motif (5). The CAAX protein family, which contains numerous proteins such as members of the Ras small G protein family (6-8), the nuclear lamins (9), or the ␥ subunit of trimeric G proteins (10-13), are prenylated by the farnesyltransferase (FTase) or geranylgeranyltransferase I (GGTase I), which transfer a 15-carbon farnesyl or a 20-carbon geranylgeranyl from the corresponding prenyl-pyrophosphate to the sulfhydryl group of the carboxyl-terminal cysteine, respectively (5). Mammalian FTase and GGTase I, which both are zinc metalloenzymes, initially were identified and purified from rat brain cytosol as ␣͞␤ heterodimers (14-16). The 48-kDa ␣ subunit is shared by both enzymes, whereas the 46-kDa ␤ subunit of FTase is 30% identical with the 43-kDa of the GGTase I (17, 18).It was concluded from earlier studies that the nature of the carbo...