[Development of farsenyl transferase inhibitors as anticancer agents]. / Développement des inhibiteurs de farnésyl transférase comme agents anticancéreux.

Ras proteins belong to the monomeric GTPases familly. They control cell growth, differentiation, proliferation, and survival. Ras mutations are frequently found in human cancers and play a fundamental role in tumorigenesis. Ras requires localization to the plasma membrane to exert its oncogenic effects. This subcelllular localization is dependent of protein farnesylation which is a post translational modification catalysed by the farnesyl transferase enzyme. Farnesyl transferase Inhibitors (FTI) were then designed ten to twelve years ago to inhibit ras processing and consequently the growth of ras mutated tumor. Preclinical data show that FTIs inhibit cell proliferation and survival in vitro and in vivo of a wide range of cancer cell lines, many of which contain wild type ras suggesting that mutated Ras is not the only target of the FTIs effects. Four FTIs went then through clinical trials and three of then are still developed in the clinic. Phase I et II clinical trials confirmed a relevant antitumor activity and a low toxicity. Phase III clinical trials are currently undergoing for both solid and hematologic tumors. The expected results should allow to define the position of FTIs as anticancer drugs, particularly in combination with conventional chemotherapy, hormone therapy, radiotherapy or any other new targeted compound.

Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells.

The central involvement of estrogen in the development of the mammary gland and in the genesis of breast cancer has lent impetus to studies of the links between estrogen action and the cell cycle machinery. Recent studies of the estrogenic regulation of molecules with known roles in the control of G1/S phase progression have resulted in significant advances in understanding these links. Estrogens independently regulate the expression and function of c-Myc and cyclin D1 and the induction of either c-Myc or cyclin D1 is sufficient to recapitulate the effects of estrogen on cell cycle progression. These pathways converge at the activation of cyclin E-Cdk2 complexes. The active cyclin E-Cdk2 complexes are depleted of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) because of estrogen-mediated inhibition of nascent p21(WAF1/CIP1). Insulin and estrogen synergistically stimulate cell cycle progression, and the ability of estrogen to antagonize an insulin-induced increase in p21(WAF1/CIP1) gene expression appears to underlie this effect. Antiestrogen treatment of MCF-7 cells leads to an acute decrease of c-Myc expression, a subsequent decline in cyclin D1, and ultimately arrest of cells in a state with features characteristic of quiescence. An antisense-mediated decrease in c-Myc expression results in decreased cyclin D1 expression and inhibition of DNA synthesis, mimicking the effects of antiestrogen treatment and emphasizing the importance of c-Myc as an estrogen/antiestrogen target. These data identify c-Myc, cyclin D1, p21(WAF1/CIP1) and cyclin E-Cdk2 as central components of estrogen regulation of cell cycle progression and hence as potential downstream targets that contribute to the role of estrogen in oncogenesis.

RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription.

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 anchorage-dependent 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.

SupraMolecular BioVectors (SMBV) improve antisense inhibition of erbB-2 expression.

New therapeutic strategies are now being developed against adenocarcinoma associated with erbB-2 amplification, particularly by inhibiting p185erbB-2 expression. Antisense oligodeoxynucleotides seem promising for this purpose as long as they are efficiently protected against degradation and targeted into the cells. We present antisense oligonucleotide carriers, the supramolecular biovectors (SMBVs), for which we have already demonstrated the ability to improve both cellular uptake and protection of oligodeoxynucleotide. The present work demonstrates that SMBVs elicit a specific and non-toxic action of antisense compounds in a cell model, irrespective of their sensitivity to nucleases. This is a major point, considering the specificity problems associated with the use of nuclease-resistant phosphorothioate oligodeoxynucleotide. SMBVs improve antisense efficiency of oligodeoxynucleotide designed against p185erbB-2, with a complete growth arrest of SK-Br-3, human adenocarcinoma mammary cells that overexpress p185erbB-2 and no effect on MCF-7 cells that normally express p185erbB-2. The comparison of SMBVs with DOTAP reveals the statistically higher efficiency of SMBVs, which allows the antisense inhibition of p185erbB-2 expression in 65-75% of SK-Br-3 cells (P < 0.05). The efficiency and controlled synthesis of SMBVs underline their potentialities as oligodeoxynucleotide carriers for in vivo experiments.

Improved oligonucleotide uptake and stability by a new drug carrier, the SupraMolecular Bio Vector (SMBV).

Antisense oligodeoxynucleotides are potential therapeutic agents, but their development is still limited by both a poor cellular uptake and a high degradation rate in biological media. The strategy that we propose to face these problems is to use small synthetic carriers, around 30 nm diameter, the SupraMolecular Bio Vectors (SMBV). We used positively charged SMBV and settled the ionic incorporation of negatively charged oligonucleotides into these carriers. A minimal leakage of 10% of total incorporated oligonucleotides was then measured during two months. Both protection and uptake of oligonucleotides were then analyzed. On the one hand, we showed that the incorporation of oligonucleotides into the selected SMBV allows to significantly increase, 8 times, their half-life, in cell growth medium. On the other hand, the internalization of the SMBV, into cells, by an endosomal pathway has been characterized. The essential point is that the SMBV uptake elicits the simultaneous oligonucleotide uptake. The oligonucleotide amount that goes through cells within 5 h can be up to 30 times higher than for free oligonucleotides and the fraction of oligonucleotides that is present in the cytosol is increased up to 10 fold after incorporation into the SMBV. This study demonstrates the ability of SMBV to improve oligonucleotide cellular behaviour.

Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes.

Electropulsation allowed us to incorporate glycophorin A, an integral membrane protein, into mammalian nucleated cell membranes (Chinese hamster ovary cells). The induction of stable protein association is effective only when the field intensity is higher than its threshold value, creating membrane permeabilization to small molecules. Under controlled conditions, cell viability was only slightly altered by this treatment. Pulse number and duration controlled both the number of modified cells and incorporated molecules. The phenomena was temperature dependent. An average of 5 x 10(4) molecules/cell was bound. About 80% of cells in the pulsed population were observed to incorporate glycophorin. The protein incorporation was shown to be stable 48 h after electroassociation. Electrically bound proteins were shared between the cells after each division. As enhanced binding is detected if glycophorin is added after the pulses, it is the long-lived alteration of the membrane mediated by the pulses which supports the association.

Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET).

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.

Fast kinetics studies of Escherichia coli electrotransformation.

Direct gene transfer is achieved in Escherichia coli by use of square wave electric pulsing. As observed by video 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 interfacial 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.

In vivo targeting of inflamed areas by electroloaded neutrophils.

Due to their spontaneous accumulation in inflamed or infected areas, blood phagocytes are potent drug vectors with specific targeting. Drug like molecule loading was obtained by use of cell electropermeabilization in which the impermeability of their plasma membrane is transiently impaired. Electrical conditions were used which allow electroloading of a drug like molecule (propidium iodide) in 70% of leukocytes in a whole blood sample while preserving in vitro functional properties. Slow release of entrapped hydrophilic molecules was observed with a half lifetime longer than 4 hours at 4 degrees C and at 37 degrees C. With an in vivo assay, using a rat model of inflammation, we showed that, as for non-pulsed cells, pulsed neutrophils accumulate 10 times more in an inflamed area than they do in control areas. Phagocyte electropermeabilization is therefore a very efficient way of drug targeting. Accumulation of electropulsed neutrophils in an area of inflammation gives targeted release of the electroloaded drug.

Optimized conditions for electrotransformation of bacteria are related to the extent of electropermeabilization.

Electropulsation is a simple and efficient way to introduce cloned genes into a variety of cell types, even with walled species. In the case of bacteria, we observed no direct correlation between survival rate and transformation yield. In the present work, we show that the yield of transformation is directly related to the level of the electric-field induced level of cell permeabilization. From experiments on Escherichia coli, it was confirmed that the extent of associated ATP leakage was a reliable assay. This approach was extended to other strains, such as Salmonella typhimurium, which to date had not been electrotransformed by plasmids.

Specific electropermeabilization of leucocytes in a blood sample and application to large volumes of cells.

Electropermeabilization is obtained when the membrane potential difference reaches a critical threshold. This is performed by submitting cells to an external electric field pulse. The field modulates the endogenous potential difference in a cell-size-dependent way. Computer simulations predict that large cells would be specifically permeabilized in a mixture with smaller cells. This was examined on a mixture of Chinese hamster ovary (CHO) cells and erythrocytes. CHO cells were permeabilized to Trypan blue without any occurrence of haemolysis. A similar 'size' specificity was observed on blood samples. This agreement between prediction and experimental observation indicates that induction of electropermeabilization is mainly under the control of the size of the target cell. Its physiology plays only a minor role, if any. Treating blood with 10 square wave pulses lasting 100 microseconds of an intensity of 1.6 kV/cm induced the permeabilization of 70% of the leucocytes (polymorphs and monocytes) but did not affect erythrocytes. No washing of the sample was needed in a procedure in which cells were pulsed in the plasma. A flow electropulsing process allows the treatment of large blood volumes under conditions where cells are kept viable. These results show that electropermeabilization could be used as an effective way to obtain immunocompatible drug vehicles.

Mycobacteria glycolipids as potential pathogenicity effectors: alteration of model and natural membranes.

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.