Role of yessotoxin in calcium and cAMP‐crosstalks in primary and K‐562 human lymphocytes: The effect is mediated by Anchor kinase a mitochondrial proteins

Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3′,5′‐cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca²⁺), whereas the contrary effect has been observed in a Ca²⁺‐free medium. In the present article, the effect of YTX in K‐562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K‐562 cell line are completely opposite than in fresh human lymphocytes, since in K‐562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K‐562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K‐562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca²⁺ increase in fresh human lymphocytes but no effect was observed on Ca²⁺ pools depletion in these cells. However, our results show that, in K‐562 cells, YTX has no effect on cytosolic Ca²⁺ levels in a medium with Ca²⁺ and induces an increase on Ca²⁺ pools depletion followed by a Ca²⁺ influx. As far as Ca²⁺ modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca²⁺ reservoirs affected by YTX are different than thapsigargin‐sensible pools. Furthermore, YTX‐dependent Ca²⁺ pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase‐4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p‐(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca²⁺, YTX and cAMP pathways. Also, results obtain demonstrate that YTX‐dependent Ca²⁺ influx was only abolished by FCCP pre‐treatment, which indicates a link between YTX and mitochondria in K‐562 cell line. Cytosolic expression of A‐kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised. J. Cell. Biochem. 113: 3752–3761, 2012. © 2012 Wiley Periodicals, Inc.     

The C. elegans TIA‐1/TIAR homolog TIAR‐1 is required to induce germ cell apoptosis

In Caenorhabditis elegans, physiological germ cell apoptosis eliminates more than half of the cells in the hermaphrodite gonad to support gamete quality and germline homeostasis by a still unidentified mechanism. External factors can also affect germ cell apoptosis. The BH3‐only protein EGL‐1 induces germ cell apoptosis when animals are exposed to pathogens or agents that produce DNA damage. DNA damage‐induced apoptosis also requires the nematode p53 homolog CEP‐1. Previously, we found that heat shock, oxidative, and osmotic stresses induce germ cell apoptosis through an EGL‐1 and CEP‐1 independent mechanism that requires the MAPKK pathway. However, we observed that starvation increases germ cell apoptosis by an unknown pathway. Searching for proteins that participate in stress‐induced apoptosis, we found the RNA‐binding protein TIAR‐1 (a homolog of the mammalian TIA‐1/TIAR family of proteins). Here, we show that TIAR‐1 in C. elegans is required to induce apoptosis in the germline under several conditions. We also show that TIAR‐1 acts downstream of CED‐9 (a BCL2 homolog) to induce apoptosis under stress conditions, and apparently does not seem to regulate ced‐4 or ced‐3 mRNAs accumulation directly. TIAR‐1 is expressed ubiquitously in the cytoplasm of the soma as well as the germline, where it sometimes associates with P granules. We show that animals lacking TIAR‐1 expression are temperature sensitive sterile due to oogenesis and spermatogenesis defects. Our work shows that TIAR‐1 is required for proper germline function and demonstrates that this protein is important to induce germ cell apoptosis under several conditions. genesis 51:690–707. © 2013 Wiley Periodicals, Inc.     

Quantitative proteomics characterization on the antitumor effects of isodeoxyelephantopin against nasopharyngeal carcinoma

Isolated from Elephantopus scaber L., a Chinese medicinal herb that is widely used to prevent and treat cancers in China, isodeoxyelephantopin (ESI) exerted antitumor effects on several cancer cells. However, its antitumor mechanism is still not clear. In this study, we found that ESI could induce G2/M arrest and subsequently stimulate cell apoptosis in dose‐ and time‐dependent manners. We used SILAC quantitative proteomics to identify ESI‐regulated proteins in cancer cells, and found that 124 proteins were significantly altered in expression. Gene ontology and Ingenuity Pathway Analysis revealed that these proteins were mainly involved in the regulation of oxidative stress and inflammation response. Functional studies demonstrated that ESI induced G2/M arrest and apoptosis by inducing ROS generation, and that antioxidant N‐acetyl‐l ‐cysteine could block the ESI‐induced antitumor effects. Accumulated ROS resulted in DNA breakage, subsequent G2/M arrest and mitochondrial‐mediated apoptosis. ESI upregulated the expression of anticancer inflammation factors IL‐12a, IFN‐α, and IFN‐β through ROS‐dependent and independent pathways. The current work reveals that ESI exerts its antitumor effects through ROS‐dependent DNA damage, mitochondrial‐mediated apoptosis mechanism and antitumor inflammation factor pathway.     

Epigallocatechin‐3‐gallate suppresses the lipid deposition through the apoptosis during differentiation in bovine bone marrow mesenchymal stem cells

Epigallocatechin gallate (EGCG), a major component of tea, has known effects on obesity, fatty liver, and obesity‐related cancer. We explored the effects of EGCG on the differentiation of bovine mesenchymal stem cells (BMSCs, which are multipotent) in a dose‐ and time‐dependent manner. Differentiating BMSCs were exposed to various concentrations of EGCG (0, 10, 50, 100, and 200 µM) for 2, 4, and 6 days. BMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/high‐glucose medium with adipogenic inducers for 6 days, and the expression levels of various genes involved in adipogenesis were measured using real‐time polymerase chain reaction (PCR) and Western blotting. We assessed apoptosis by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining of control and EGCG‐exposed cells. We found that EGCG significantly suppressed fat deposition and cell viability (P < 0.05). The mRNA and protein levels of various adipogenic factors were measured. Expression of the genes encoding peroxisome proliferator‐activated receptor gamma (PPARG), CCAAT/enhancer‐binding protein alpha (CEBPA), fatty acid‐binding protein 4 (FABP4), and stearoyl‐CoA desaturase (SCD) were diminished by EGCG during adipogenic differentiation (P < 0.05). We also found that EGCG lowered the expression levels of the adipogenic proteins encoded by these genes (P < 0.05). EGCG induced apoptosis during adipogenic differentiation (P < 0.05). Thus, exposure to EGCG potentially inhibits adipogenesis by triggering apoptosis; the data suggest that EGCG inhibits adipogenic differentiation in BMSCs.     

NDV‐D90 inhibits 17β‐estradiol‐mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV‐D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β‐estradiol (E2). We found that NDV‐D90 significantly killed the tumor cell lines MCF‐7 and BT549 in a time‐ and dose‐dependent manner. We also found that NDV‐D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV‐D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF‐7 cells (ER‐positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis‐related proteins through ERα‐and GPER‐independent processes. NDV‐D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER‐negative cells), possibly by impairing E2‐mediated GPER expression. Furthermore, NDV‐D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV‐D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER‐positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Increased oxidative-modifications of cytosolic proteins in 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-exposed rat liver

It is well established that 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA‐exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2‐DE analysis showed markedly increased levels of biotin‐N‐methylimide‐labeled oxidized cytosolic proteins in MDMA‐exposed rats compared to vehicle‐treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti‐oxidant defensive enzymes, a calcium‐binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c‐Jun N‐terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti‐apoptotic Bcl‐2 protein, their activation may promote apoptosis in MDMA‐exposed tissues. Our results show for the first time that MDMA induces oxidative‐modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.     

The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing

In plants RNA silencing is a host defense mechanism against viral infection, in which double‐strand RNA is processed into 21–24‐nt short interfering RNA (siRNA). Silencing spreads from cell to cell and systemically through a sequence‐specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according to the isolates that they originated from. Here, we show that P1 proteins from two RYMV isolates displaying contrasting suppression strength reduced local silencing induced by single‐strand and double‐strand RNA in Nicotiana benthamiana leaves. This suppression was associated with a slight and a severe reduction in 21‐ and 24‐nt siRNA accumulation, respectively. Unexpectedly, cell‐to‐cell movement and systemic propagation of silencing were enhanced in P1‐expressing Nicotiana plants. When transgenically expressed in rice, P1 proteins induced specific deregulation of DCL4‐dependent endogenous siRNA pathways, whereas the other endogenous pathways were not affected. As DCL4‐dependent pathways play a key role in rice development, the expression of P1 viral proteins was associated with the same severe developmental defects in spikelets as in dcl4 mutants. Overall, our results demonstrate that a single viral protein displays multiple effects on both endogenous and exogenous silencing, not only in a suppressive but also in an enhancive manner. This suggests that P1 proteins play a key role in maintaining a subtle equilibrium between defense and counter‐defense mechanisms, to insure efficient virus multiplication and the preservation of host integrity.     

Interconnections between apoptotic,autophagic and necrotic pathways: implications for cancer therapy development

The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer‐ and anti‐ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia‐induced damage. However, initial clinical studies on apoptosis‐modulating drugs led to unexpected results in different clinical conditions and this may have been due to co‐effects on non‐apoptotic interconnected cell death mechanisms and the ‘yin‐yang’ role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2‐family members and p53). We also briefly highlight stress‐induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation‐induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.     

iTRAQ‐based proteomic analysis of dioscin on human HCT‐116 colon cancer cells

Dioscin shows various pharmacological effects. However, its activity on colorectal cancer is still unknown. The present work showed that dioscin significantly inhibited cell proliferation on human HCT‐116 colon cancer cells, and affected Ca²⁺ release and ROS generation. The content of nitric oxide (NO) and its producer inducible NO synthase (iNOS) associated with DNA damage and aberrant cell signaling were assayed using the kits. DNA damage and cell apoptosis caused by dioscin were also analyzed through single‐cell gel electrophoresis and in situ terminal deoxynucleotidyl transferase dUTP nick‐end labeling assays. The results showed that dioscin increased the levels of NO and inducible NO synthase. The comet length in dioscin‐treated groups was much longer than that of the control group, and the number of terminal deoxynucleotidyl transferase dUTP nick‐end labeling positive cells (apoptotic cells) was significantly increased by the compound (p < 0.01). Furthermore, dioscin caused mitochondrial damage and G2/M cell cycle arrest through transmission electron microscopy and flow cytometry analysis, respectively. To study the cytotoxic mechanism of dioscin, an iTRAQ‐based proteomics approach was used. There were 288 significantly different proteins expressed in response to dioscin, which were connected with each other and were involved in different Kyoto Encyclopedia of Genes and Genomes pathways. Then, some differentially expressed proteins involved in oxidative phosphorylation, Wnt, p53, and calcium signaling pathways were validated by Western blotting and quantitative real‐time PCR assays. Our work elucidates the molecular mechanism of dioscin‐induced cytotoxicity in colon cancer cells, and the identified targets may be useful for treatment of colorectal cancer in future.     

Quantitative proteomics analysis of BMS‐214662 effects on CD34 positive cells from chronic myeloid leukaemia patients

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr‐Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS‐214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC‐specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS‐214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34⁺ CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS‐214662‐treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS‐214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug‐induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS‐214662, possibly via Ran proteins as targets.     

Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false‐positive control

Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface‐exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital‐acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface “shaving” technique with either trypsin or proteinase‐K combined with LC‐MS/MS. Trypsin‐derived data were controlled using a “false‐positive” strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin‐shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface‐exposed peptides. Trypsin and proteinase‐K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase‐K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false‐positive strategy improved enrichment of surface‐exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance–surface protein SACOL0050 (pls) and penicillin‐binding protein 2′ (mecA), as well as bifunctional autolysin and the extracellular matrix‐binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface‐exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.     

Enhanced effects of xanthohumol plus honokiol on apoptosis in 3T3-L1 adipocytes

Objective: To study the effects of xanthohumol (XN), a flavonoid found in hops (Humulus lupulus) and honokiol (HK), a lignan isolated from Magnolia officinalis, alone and in combination, on apoptotic signaling in 3T3‐L1 adipocytes. Methods and Procedures: 3T3‐L1 mature adipocytes were incubated with various concentrations of XN and HK alone and in combination. Viability and apoptosis were quantified using an MTS‐based cell viability assay and single‐stranded DNA assay, respectively. Expression of apoptosis related proteins including cleaved poly(ADP‐ribose) polymerase (PARP), cytochrome c, Bcl‐2, caspase‐3/7, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt was analyzed by western blotting. Results: Combinations of XN and HK significantly decreased viability and induced apoptosis in a dose‐dependent manner and more than the additive responses to XN and HK alone. Western blot analysis showed an increase in cleaved PARP and cytochrome c release and decrease in expression of Bcl‐2 protein by XN plus HK, whereas XN and HK individually had no effect. Furthermore, the combination of XN and HK activated PTEN and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. Discussion: We demonstrated that although XN and HK showed little or no effect as individual compounds, in combination (XN plus HK) they showed enhanced activity in inducing apoptosis via the cytochrome c/caspase‐3/PARP and PTEN/Akt pathways in 3T3‐L1 adipocytes.     

Plasma and liver proteomic analysis of 3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one‐induced hepatotoxicity in Wistar rats

3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one (Z24), a synthetic anti‐angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2‐DE and MALDI‐TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP‐nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid β‐oxidation, and oxidative phosphorylation is a potential mechanism of Z24‐induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis‐mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24‐induced hepatotoxicity.     

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G‐protein‐coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl‐Met‐Leu‐Phe) peptide, in a rat basophilic leukaemia RBL‐2H3 cell line stably expressing an HA (haemagglutinin)‐tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist‐induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist‐enhanced cell spreading was inhibited. Analysis of agonist‐stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans‐well migration. The internalization of the formyl‐peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody‐enhanced agonist‐induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant‐induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G‐protein‐coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist‐induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.