Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Bax- and Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells

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.     

Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.     

Improvement of calcium handling and changes in calcium-release properties after mini- or full-length dystrophin forced expression in cultured skeletal myotubes

Dystrophin is a cytoskeletal protein normally expressed underneath the sarcolemma of muscle fibers. The lack of dystrophin in Duchenne muscular Dystrophy (DMD) muscles results in fiber necrosis, which was proposed to be mediated by chronic calcium mishandling. The extensive comparison of dystrophic cells from human or mdx mice with normal muscles have suggested that the lack of dystrophin may alter the resting calcium permeability and steady-state levels of calcium, but this latter observation remains controversial. It is also not clear, whether calcium mishandling is resulting from the dystrophic process or if dystrophin can directly regulate calcium handling in muscle cells. This prompted us to determine if transfection of full-length dystrophin or Becker Muscular Dystrophy (BMD) minidystrophin, a candidate for viral-mediated gene therapy, could change calcium handling properties. We took advantage of specific properties of Sol8 cell line showing the absence of dystrophin expression together with a drastic calcium mishandling. Here, we show that full-length dystrophin allowed the recovery of a low resting intracellular-free calcium concentration together with lower calcium transients. We also show for the first time that stable expression of minidystrophin was able to restore normal calcium handling in Sol8 myotubes through a better control of steady-state levels, calcium transients, and subcellular calcium events. It suggests that dystrophin could play a regulatory role on calcium homeostasis apparatus and that functional links exist between calcium signaling and cytoskeleton.     

Spatial distribution of acoustic and elastic properties of human femoral cortical bone

The purpose of this study is to quantify the spatial distribution of acoustic velocities and elastic properties (elastic constants) on Human femoral cortical bone. Four cross sections (average thickness of 2.09+/-0.27 mm) have been cut transversally between 40% and 70% of the total length and between them parallelepiped samples in each quadrant have been cut. Ultrasonic technique in transmission with immersion focused transducers at 5 MHz and contact transducers 2.25 MHz were used on the cross sections and parallelepiped samples, respectively. The first technique allows relative spatial distribution of velocities to be obtained, meanwhile the second technique allows the direct assessment of elastic constants. For both techniques, bulk velocities were found to be lower at the posterior side with an increase along the length (from 40% to 70% total length) (p < 0.05). Densities and elastic constants show equivalent pattern of variation. These variations are mainly due the cortical porosity related to vascularisation environment. The spatial distribution of velocities exhibits significant radial variation from the endosteal to the periosteal region. This is in agreement with variation of the porosity at that location. Same range of velocities was obtained with both techniques. The range of longitudinal velocities values varies from 3548 to 3967 m/s and between 18.5 and 33.1 GPa for the bulk velocities and axial elastic constants, respectively. Our results are within the range with those found in the literature. However, it must be noted that the range of acoustic and elastic properties variation is concerning the same bone. So, our new results show the ability of the technique to quantify accurately local variation of acoustic and elastic properties (within the section and along the length) of human cortical bone. Furthermore, our immersion technique could be used to assess the spatial distribution of the elastic constants with the knowledge of spatial distribution of densities.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

Calpain involvement in the remodeling of cytoskeletal anchorage complexes

Cells offer different types of cytoskeletal anchorages: transitory structures such as focal contacts and perennial ones such as the sarcomeric cytoskeleton of muscle cells. The turnover of these structures is controlled with different timing by a family of cysteine proteases activated by calcium, the calpains. The large number of potential substrates present in each of these structures imposes fine tuning of the activity of the proteases to avoid excessive action. This phenomenon is thus guaranteed by various types of regulation, ranging from a relatively high calcium concentration necessary for activation, phosphorylation of substrates or the proteases themselves with either a favorable or inhibitory effect, possible intervention of phospholipids, and the presence of a specific inhibitor and its possible degradation before activation. Finally, formation of multiprotein complexes containing calpains offers a new method of regulation.     

Responses of the steroid circadian system to alcohol in humans: Importance of the time and duration of intake

Reports provide conflicting data about the effects of alcohol consumption on the hormonal system. Any study of these effects must control for a number of variables, including sex, alcohol status (alcoholic addiction vs. non-addiction), medical status (malnutrition, liver disease), and conditions of alcohol exposure, including an acute or continuous pattern of intake. The latter appears to be an especially critical factor in interpreting these effects. The authors therefore conducted a trial with a circadian design in which alcohol was administered repeatedly and regularly over a 26 h period for a total dose of 256 g. Because this protocol involves continuous alcohol administration, it is similar to administration among alcoholics and thus sheds new light on alcohol's effect on hormone secretion. Using healthy volunteers rather than alcoholics, however, prevents any confounding due to liver disorders and nutritional deficiencies, and thus makes it possible to focus on the direct role of alcohol in hormonal modifications. In these conditions, the continuous administration of alcohol did not affect cortisol secretion, but serum testosterone levels were significantly higher at all time points during the alcohol session than at the corresponding time points during the control session. These data are not consistent with previously reported findings for the relation between alcohol and both cortisol and testosterone, because in the current experiment the action of ethanol on steroid secretion should involve the circadian clock more than the hormonal system itself.     

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.