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The biogenesis of cytochrome c oxidase requires coordination between the nucleus and mitochondria because both these compartments provide the structural subunits of this enzyme. In addition, synthesis, membrane insertion and assembly of the mitochondrially encoded subunits are controlled in a concerted way by numerous nuclear-encoded factors, including Oxa1 and Cox18, which play successive roles in Cox2 assembly in Saccharomyces cerevisiae . These two factors share a weak structural similarity and define two sub-branches of the Oxa1/YidC/Alb3 gene family, whose members facilitate the membrane insertion of various hydrophobic proteins into diverse biological membranes. In this study, we have analyzed a second human and a third fission yeast member of the family. We show, by deletion in the fission yeast genome, as well as expression and functional complementation experiments in both yeasts, that these new genes belong to the COX18 rather than to the OXA1 sub-branch. So far, the fission yeast gene cox18Sp+ is the smallest functional member of this gene family. COX18Hs gives rise to various mRNAs with different coding capacities, and we show that cox18Sp+ and COX18Hs are expressed at a low level and appear to be stringently regulated. This transcriptional control contrasts with the constitutive abundance of the OXA1 mRNAs and might reflect major functional differences between these nevertheless structurally related genes.

International audience; Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.

Apoptotic cell death has been involved as a cytopathologic mechanism in response to dengue (DEN) virus infection. Little information exists about how DEN virus replication triggers apoptosis in infected cells. We reported that a nine-residue sequence of the DEN M protein referred to as ApoptoM has proapoptotic properties in transformed and tumor cells of various origins. The aim of the present study was to investigate whether ApoptoM-induced apoptosis is associated to mitochondrial dysfunction and requires caspase activation. Intracellular expression of ApoptoM provokes the disruption of the mitochondrial transmembrane potential without subsequent generation of reactive oxygen species. We showed that ApoptoM-induced apoptosis involves the activation of a caspase-like protease pathway. Caspase-3 like activity was detected in ApoptoM-expressing cells. However, there was no role for caspase-9 in ApoptoM-mediated cell death. Our data suggest that a particular mitochondrion-dependent apoptotic pathway may be involved in induction of apoptosis by ApoptoM.

International audience; Transient peaks of quisqualate (QA)-, but not 1S,3R-1-amino-3-cyclopentane dicarboxylate (1S,3R-ACPD)- and carbachol-induced inositol phosphate formation occur between 2 and 5 days in vitro (DIV) in hippocampal neurons in culture. In order to elucidate the putative origin of such developmental activity differences, the effect of PKC on metabotropic glutamate receptor (mGluR) and muscarinic receptor responses was investigated at 3 and 10 DIV. (i) Stimulation of PKC by phorbol-12,13-dibutyrate inhibited QA, 1S,3R-ACPD and carbachol responses at 3 DIV. At 10 DIV, only 1S,3R-ACPD response was still inhibited by phorbol esters. (ii) Inhibition of PKC by staurosporine at 3 DIV potentiated 1S,3R-ACPD-induced inositol phosphate formation, but had no effect on QA and carbachol responses. At 10 DIV, all responses were potentiated by staurosporine. These data strongly suggest that PKC differently modulates 1S,3R-ACPD- and QA-induced inositol phosphate accumulations during in vitro development. The specific activity of mGluRs during development, vs that of muscarinic receptor, and the peculiar modes of regulation by PKC of these two mGluR activities further suggest their particular involvement in the maturation of neuronal culture.

Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/M checkpoint and are inherently chemoresistant. To understand the mechanism of this chemoresistance, we compared the ability of immature CD34+ versus mature CD34- AML cell lines (KG1a and U937, respectively) to recover from a DNA damage-induced cell cycle checkpoint in G2. Here, we report that KG1a cells have a more stringent G2/M checkpoint response than U937 cells. We show that in both cell types, the CDC25B phosphatase participates in the G2/M checkpoint recovery and that its expression is upregulated. Furthermore, we show that CHK1 inhibition by UCN-01 in immature KG1a cells allows checkpoint exit and induces sensitivity to genotoxic agents. Similarly, UCN-01 treatment potentializes genotoxic-induced inhibition of colony formation efficiency of primary leukemic cells from AML patients. Altogether, our results demonstrate that checkpoint stringency varies during the maturation process and indicate that targeting checkpoint mechanisms might represent an attractive therapeutic opportunity for chemoresistant immature AML cells.

The presenilin-dependent gamma-secretase activity, which is responsible for the generation of amyloid beta-peptide, is a high molecular weight complex composed of at least four components, namely, presenilin-1 (or presenilin-2), nicastrin, Aph-1, and Pen-2. Previous data indicated that presenilins, which are thought to harbor the catalytic core of the complex, also control p53-dependent cell death. Whether the other components of the gamma-secretase complex could also modulate the cell death process in mammalian neurons remained to be established. Here, we examined the putative contribution of Aph-1 and Pen-2 in the control of apoptosis in TSM1 cells from a neuronal origin. We show by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA fragmentation analyses that the overexpression of Aph-1a, Aph-1b, or Pen-2 drastically lowered staurosporine-induced cellular toxicity. In support of an apoptosis rather than necrosis process, Aph-1 and Pen-2 also lower staurosporine- and etoposide-induced caspase-3 expression and diminished caspase-3 activity and poly(ADP-ribose) polymerase inactivation. The Aph-1 and Pen-2 anti-apoptotic phenotype was associated with a drastic reduction of p53 expression and activity and lowered p53 mRNA transcription. Furthermore, the Aph-1- and Pen-2-associated reduction of staurosporine-induced caspase-3 activation was fully abolished by p53 deficiency. Conversely, Aph-1a, Aph-1b, and Pen-2 gene inactivation increases both caspase-3 activity and p53 mRNA levels. Finally, we show that Aph-1 and Pen-2 did not trigger an anti-apoptotic response in cells devoid of presenilins or nicastrin, whereas the protective response was still observed in fibroblasts devoid of beta-amyloid precursor protein and amyloid precursor protein like-protein 2. Furthermore, Aph-1- and Pen-2-associated protection against staurosporine-induced caspase-3 activation was not affected by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and difluoromethylketone. Altogether, our study indicates that Aph-1 and Pen-2 trigger an anti-apoptotic response by lowering p53-dependent control of caspase-3. Our work also demonstrates that this phenotype is strictly dependent on the molecular integrity of the gamma-secretase complex but remains independent of the gamma-secretase catalytic activity.