Pierre Roux

The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects.

Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane. The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekic acid (BA) and isobongkrekic acid (isoBA) on the other hand. It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities. The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques. This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning of ADP/ATP transport system. The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level. Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

Loss of p53 promotes RhoA–ROCK-dependent cell migration and invasion in 3D matrices

In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA–ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.

DNA damage checkpoint kinase Chk2 triggers replicative senescence.

Telomere shortening in normal human cells causes replicative senescence, a p53‐dependent growth arrest state, which is thought to represent an innate defence against tumour progression. However, although it has been postulated that critical telomere loss generates a ‘DNA damage’ signal, the signalling pathway(s) that alerts cells to short dysfunctional telomeres remains only partially defined. We show that senescence in human fibroblasts is associated with focal accumulation of γ‐H2AX and phosphorylation of Chk2, known mediators of the ataxia‐telangiectasia mutated regulated signalling pathway activated by DNA double‐strand breaks. Both these responses increased in cells grown beyond senescence through inactivation of p53 and pRb, indicating that they are driven by continued cell division and not a consequence of senescence. γ‐H2AX (though not Chk2) was shown to associate directly with telomeric DNA. Furthermore, inactivation of Chk2 in human fibroblasts led to a fall in p21waf1 expression and an extension of proliferative lifespan, consistent with failure to activate p53. Thus, Chk2 forms an essential component of a common pathway signalling cell cycle arrest in response to both telomere erosion and DNA damage.

Nuclear localization of c-Fos, but not v-Fos proteins, is controlled by extracellular signals

We report here that transport of the protein product of the c-fos proto-oncogene from the cytoplasm, where it is synthesized, into the nucleus, where it operates as part of the AP-1 transcription complex, is not spontaneous but depends on the continuous stimulation of cells by serum factors. A labile protein inhibitor of transport, the effect of which is reversed by cAMP, is responsible for retention of c-Fos protein within the cytoplasm of serum-starved fibroblasts. In contrast, v-Fos proteins transduced by the murine retroviruses FBJ and FBR, which remain nuclear in the absence of serum, evade the translocation control, which therefore appears to contribute to their tumorigenic potential.

Raf-MEK-Erk Cascade in Anoikis Is Controlled by Rac1 and Cdc42 via Akt

ABSTRACT Signals from the extracellular matrix are essential for the survival of many cell types. Dominant-negative mutants of two members of Rho family GTPases, Rac1 and Cdc42, mimic the loss of anchorage in primary mouse fibroblasts and are potent inducers of apoptosis. This pathway of cell death requires the activation of both the p53 tumor suppressor and the extracellular signal-regulated mitogen-activated protein kinases (Erks). Here we characterize the proapoptotic Erk signal and show that it differs from the classically observed survival-promoting one by the intensity of the kinase activation. The disappearance of the GTP-bound forms of Rac1 and Cdc42 gives rise to proapoptotic, moderate activation of the Raf-MEK-Erk cascade via a signaling pathway involving the kinases phosphatidlyinositol 3-kinase and Akt. Moreover, concomitant activation of p53 and inhibition of Akt are both necessary and sufficient to signal anoikis in primary fibroblasts. Our data demonstrate that the GTPases of the Rho family control three major components of cellular signal transduction, namely, p53, Akt, and Erks, which collaborate in the induction of apoptosis due to the loss of anchorage.

The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.

BACKGROUND Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

Control of cell migration: a tumour suppressor function for p53?

Much remains to be learned about how cancer cells acquire the property of migration, a prerequisite for invasiveness and metastasis. Loss of p53 functions is assumed to be a crucial step in the development of many types of cancers, leading to dysregulation of cell cycle checkpoint controls and apoptosis. However, emerging evidence shows that the contribution of the tumour suppressor p53 to the control of tumorigenesis is not restricted to its well‐known anti‐proliferative activities, but is extended to other stages of cancer development, i.e. the modulation of cell migration. This interesting alternative function has been proposed in light of the effect of p53 on specific features of migrating cells, including cell spreading, establishment of cell polarization and the production of protrusions. The effects of p53 on cell motility are largely mediated through the regulation of Rho signalling, thereby controlling actin cytoskeletal organization. These recent studies connect the regulation of proliferation to the control of cell migration and define a new concept of p53 function as a tumour suppressor gene, suggesting that p53 might be involved in tumour invasion and metastasis. This review focuses on emerging data concerning the properties of p53 that contribute to its atypical role in the regulation of cell migration.

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

Regulation of Cdc42-mediated morphological effects: a novel function for p53

The tumour suppressor functions of p53 that are important for its activity depend on its role as a cell cycle arrest mediator and apoptosis inducer. Here we identify a novel function for p53 in regulating cell morphology and movement. We investigated the overall effect of p53 on morphological changes induced by RhoA, Rac1 and Cdc42 GTPases in mouse embryonic fibroblasts (MEFs). Interestingly, p53 exerted a selective effect on Cdc42‐mediated cell functions. (i) Both overexpression of wild‐type p53 and activation of endogenous p53 counteracted Cdc42‐induced filopodia formation. Conversely, p53‐deficient MEFs exhibited constitutive membrane filopodia. Mechanistic studies indicate that p53 prevents the initiating steps of filopodia formation downstream of Cdc42. (ii) Over expression of p53 modulates cell spreading of MEFs on fibronectin. (iii) During cell migration, the reorientation of the Golgi apparatus in the direction of movement is abolished by wild‐type p53 expression, thus preventing cell polarity. Our data demonstrate a previously uncharacterized role for p53 in regulating Cdc42‐dependent cell effects that control actin cytoskeletal dynamics and cell movement. This novel function may contribute to p53 anti‐tumour activity.

Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells

Mutations in the p53 tumour suppressor gene are associated clinically with tumour progression and metastasis. Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumour progression. Here, we show that wild-type p53 induced to adopt a mutant conformation, and hot-spot p53 mutants, which are both transcriptionally inactive, downregulate E-cadherin expression in the colon carcinoma cell line HCT116. Downregulation of E-cadherin occurred concomitantly with the upregulation of Slug and Zeb-1, transcriptional factors known to repress E-cadherin gene expression. In addition, knockdown of Slug and Zeb-1 expression diminished p53-mediated E-cadherin repression. Knocking down endogenous mutant p53 in MDA-MB-231 and SW620 cancer cell lines lacking E-cadherin protein restored the expression of E-cadherin. Complete loss of E-cadherin expression in HCT116 cells induced morphological alterations along with upregulation of vimentin, a mesenchymal marker. These changes characteristic of the EMT phenotype were, however, not sufficient to confer invasiveness in a three-dimensional matrix. Downregulation of E-cadherin by mutant p53 was not required to promote the invasive phenotype induced by inactivation of p53. These findings indicate that independent control of E-cadherin expression and cell motility could be essential molecular events in p53 mutant-induced invasive phenotypes.