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...

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+PD) was synthesized and labeled with a 3' rhodamine (+PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (-PT-F) was specifically quenched by addition of the +PD-R. In solution, the -PT-F/+PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.

Direct gene transfer is achieved in Escherichiu coli by use of square wave electric pulsing. As observed by vidco monitoring, the field pulse causes bacteria to orientate parallel to the field lines. Rapid kinetic turbidity changes indicate that this process happens quickly. In these circumstances, and in pulsing conditions prone to inducing transformation, only caps are affected by the field. Considerable cytoplasmic ion leakage occurs during the pulse, affecting the intcrfacial ionic concentration. The pulsing-buffer osmolarity has to be close to that used with protoplasts. Contact between the plasmid and the bacteria can be very short before the pulse but must be present during the pulse. The plasmid remains accessible to externally added DNases up to 5 days after the pulse, suggesting that the transfer step is slow. Electric-field-mediated transfer can be described in two steps: the anchoring process during the pulse, followed by the crossing of the membrane.The organization and properties of cell membranes can be altcred when the membrane-potential difference is brought to a critical value. This was shown by electropulsation for the first time in 1972, when the permeability of chromaffin granules was reversibly increased when vesicles were submitted to short high-intensity electric pulses (so called electropermeabilization) [I], a property which has since been observed in many cell systems. In 1982, gene transfer was mediated in eukaryotic cells by electropulsation (electrotransformation) [2] and gene expression was obtained showing genomic integration of the plasmid. Electrotransformation was successfully applied to many systems of mammalian cells and plant protoplasts [3]. More recently, gene transfer was obtained with walled systems, such as bacteria [4] or yeasts [5], and electrotransformation is, in [act, now routinely used in many bacterial-genetics laboratories. Yiclds as high as 20 -30% have been reported using strains which were known to be casily transformed by the CaC1, method [6 -1 I]. Nevertheless, mechanisms of electrotransformation remain unexplained in the case of bacteria, as with other systems. Optimization of thc procedure remains, in most cases, empirical. A correlation has nevertheless been shown by our group between electropermeabilization and electrotransformation in the case of Escherichiu coli and Salmonella thyphimurium [12].However, a lack of knowledge of the basic processes remains the major handicap to the definition of efficient transformation conditions. Several processes are induced at the cell level upon electropulsation. Thc well-known Joule-heating phenomenon cannot be neglected under electrical conditions inducing electrotransformation. Dipolcs are induced by the electric field and are oriented by a field effect and reorientation of the Corre.pmdence to J. Teissit, Centre de Recherche dc Biochimie et de Gtnetique Ccllulaires du Centre National de la Recherche Scieiitifiquc, 118 route de Narbonne, F-31062 Toulouse. Cedex, France pulsed vesicles may occur. The most dramatic...

Four mycobacterial wall glycolipids were tested for their effects on phospholipidic liposome organization and passive permeability and on oxidative phosphorylation of isolated mitochondria. From fluorescence polarization of diphenylhexatriene performed on liposomes it was concluded that the two trehalose derivatives (dimycoloyltrehalose and polyphthienoyltrehalose) rigidified the fluid state of liposomes, the triglycosyl phenolphthiocerol slightly fluidized the gel state, while the peptidoglycolipid ("apolar" mycoside C) just shifted the phase transition temperature upward. Dimycoloyltrehalose was without effect on liposome passive permeability, as estimated from dicarboxyfluorescein leak rates, and polyphthienoyltrehalose and triglycosyl phenolphthiocerol slightly decreased leaks, while mycoside C dramatically increased leaks. Activity of these lipids on mitochondrial oxidative phosphorylation was examined. The two trehalose derivatives have been tested previously: both had the same type of inhibitory activity, dimycoloyltrehalose being the most active. Triglycosyl phenolphthiocerol was inactive. Mycoside C was very active, with effects resembling those of classical uncouplers: this suggested that its activity on mitochondria was related to its effect on permeability. All these membrane alterations were called nonspecific because it is likely that they result from nonspecific lipid-lipid interactions, and not from recognition between specific molecular structures. Such nonspecific interactions could be at the origin of some of the effects of mycobacteria glycolipids on cells of the immune system observed in the last few years.

Increasing evidence implicates the aryl hydrocarbon receptor (AhR) as a possible regulator of mammary carcinogenesis. This study aims to clarify its prognostic impact in breast cancer (BC). Meta-analyses performed at the mRNA level demonstrated that the predictive value of AhR expression in BC depends on the lymph node (LN) status. AhR expression and sub-cellular location were then analyzed by immunohistochemistry in 302 primary BC samples. AhR was expressed in almost 90% of cases with a predominant nuclear location. Nuclear and cytoplasmic AhR levels were significantly correlated and associated with the expression of RIP140 (receptor-interacting protein of 140 kDa), an AhR transcriptional coregulator and target gene. Interestingly, total and nuclear AhR levels were only significantly correlated with short overall survival in node-negative patients. In this sub-group, total and nuclear AhR expression had an even stronger prognostic impact in patients with low RIP140-expressing tumors. Very interestingly, the total AhR prognostic value was also significant in luminal-like BCs and was an independent prognostic marker for LN-negative patients. Altogether, this study suggests that AhR is a marker of poor prognosis for patients with LN-negative luminal-like BCs, which warrants further evaluation.