Florence Poirier

The cAMP binding protein Epac regulates cardiac myofilament function

In the heart, cAMP is a key regulator of excitation–contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT) reduced Ca2+ transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca2+ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca2+ sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca2+ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca2+ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.

Identification and verification of heat shock protein 60 as a potential serum marker for colorectal cancer

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two‐dimensional difference gel electrophoresis (2D‐DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione‐S‐transferase Pi, α‐enolase, T‐complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5–15%) and sensitive (0.3 ng·mL−1) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver‐operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three‐marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19‐9 reached 0.77. Serum HSP60 appeared to be more specific for late‐stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection.

Proteomic analysis of brain tissue from an Alzheimer's disease mouse model by two-dimensional difference gel electrophoresis

We used a beta-amyloid precursor protein (APP) transgenic (Tg) mouse model that displays some of the typical Alzheimer-associated pathological features to study the brain proteoma associated with amyloid plaque deposition. Two groups (male and female) of 14-month-old Tg mice were compared with their wild type littermates. We used differential 2D electrophoresis coupled with mass spectrometry to generate one of the first complete image of changes in brain protein expression occurring in this well-recognized model of Alzheimers disease (AD). We identified 15 different proteins, which are significantly regulated in this pathology (p<0.05, > or =1.5-fold variation in expression comparing with the wild type samples). These comprise a number of proteins that were already known to be implicated in AD and neurodegeneration, as well as several proteins which relationship with AD had not been shown before. Identified proteins were grouped according to their biological key pathways. Results obtained are discussed in view of existing bibliographic data on human AD transcriptoma and proteoma.

Protein analysis by mass spectrometry and sequence database searching: A proteomic approach to identify human lymphoblastoid cell line proteins

Lymphoblastoid cell lines correspond to in vitro EBV‐immortalized lymphocyte B‐cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B‐cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two‐dimensional electrophoresis (2‐DE) database, containing B‐lymphoblastoid 2‐DE maps. In this work, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc‐imidazole staining. Everything considered, CBB being more confortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in‐gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2‐DE maps in three of the most important well‐documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http://www‐smbh.univ‐paris13.fr/lbtp/index.htm.

Two-dimensional database of a Burkitt lymphoma cell line (DG 75) proteins: Protein pattern changes following treatment with 5′-azycytidine

Hypermethylation is an important mechanism for repression of tumor gene suppressor in cancer. The drug 5′‐azacytidine (AZC) has been used as demethylating agent to induce the expression of previously silencing genes. In the present work, we attempted to determine, using proteomics, the changes in protein expression profiles following a treatment of an Epstein Barr virus (EBV)‐negative Burkitt lymphoma (BL) cell line DG 75. The effects of the treatment in terms of cell viability and growth were first examined. The following observations were made: AZC treatment led to (i) a decrease in cell growth with an arrest of the cell at G0/G1 phase of the cell cycle, (ii) the expression of p16, a tumor‐suppressor gene whose expression was dependent on its promoter demethylation. Proteomic study evidenced that AZC treatment affected protein expression in two different ways. Twenty‐one polypeptides were down‐expressed, while 14 showed an increased expression. Some of the upregulated proteins appeared related to the energy metabolism, to organization of cytoskeletal structures, and to cell viability and protein synthesis. We also established a reference map for proteins in DG 75 cell line, comprising 74 different polypeptides corresponding to 67 proteins. This map will be accessible viaInternet as a resource for proteome analyses of B‐cells. Taken together, the results presented here highlight new insights into lymphoma cell gene regulations following a treatment of lymphoma cells with AZC and illustrate a use of proteomics to evidence the direct and indirect effects of a drug and the pathways it possibly regulates.

Proteomic map and database of lymphoblastoid proteins

Advances in genomics have led to the accumulation of an unprecedented amount of data, giving rise to a new field in biochemistry, proteomics. We used a combination of two dimensional gel electrophoresis, analysis and annotation using third-generation software, and mass spectrometry to establish the proteome maps of lymphoblastoid B-cells, a prerequisite for analysis of drug effects and lymphocyte cell diseases. About 1200 protein spots were detected and characterised in terms of their isoelectric point, molecular mass and expression. The present status of proteomic technologies, as well as a description of the usefulness of human hematopoietic cells proteomic database are discussed.

Troponin T as a marker of differentiation revealed by proteomic analysis in renal arterioles

Renovascular hypertension is characterized by stenosis of the renal artery and high plasma renin levels. The renal phenotype is characterized by high levels of renin in the hypoperfused kidney due to the recruitment of renin‐producing cells along the afferent arterioles. This increase in myoepithelioïd cells is due mainly to the differentiation of existing smooth muscle cells with acquisition of a secretory phenotype. To understand the molecular mechanisms involved in this recruitment, we used the established rat model of renovascular hypertension known as the two‐kidney, one‐clip model in the Lewis rat. Renal arterioles were isolated using magnetized iron suspension. Differential proteomic analysis was performed using 2‐D polyacrylamide gel electrophoresis followed by mass spectrometry. Comparative analysis of soluble proteins extracted from afferent arterioles of clipped and contralateral kidneys showed 14 proteins significantly differentially expressed by at least a factor of 2. These proteins were identified by mass spectrometry. The most striking protein revealed by proteomics is troponin T, which is down‐regulated in the afferent arterioles of the clipped kidney. Confocal microscopy showed that troponin T is specific of the smooth muscle phenotype and absent in the myoepithelioïd phenotype. Our data suggest that troponin T is only present in renal smooth muscle cells.

Effect of 5-azacytidine and galectin-1 on growth and differentiation of the human b lymphoma cell line bl36

Background5-AzaCytidine (AzaC) is a DNA demethylating drugs that has been shown to inhibit cell growth and to induce apoptosis in certain cancer cells. Induced expression of the galectin1 (Gal1) protein, a galactoside-binding protein distributed widely in immune cells, has been described in cultured hepatoma-derived cells treated with AzaC and this event may have a role in the effect of the drug. According to this hypothesis, we investigated the effect of AzaC and Gal1 on human lymphoid B cells phenotype.MethodsThe effect of AzaC and Gal1 on cell growth and phenotype was determined on the Burkitt lymphoma cell line BL36. An immunocytochemical analysis for detection of Gal1 protein expression was performed in AzaC-treated cells. To investigate the direct effects of Gal1, recombinant Gal1 was added to cells.ResultsTreatment of lymphoid B cells with AzaC results in: i) a decrease in cell growth with an arrest of the cell cycle at G0/G1 phase, ii) phenotypic changes consistent with a differentiated phenotype, and iii) the expression of p16, a tumor-suppressor gene whose expression was dependent of its promoter demethylation, and of Gal1. A targeting of Gal 1 to the plasma membrane follows its cytosolic expression. To determine which of the effects of AzaC might be secondary to the induction of Gal1, recombinant Gal1 was added to BL36 cells. Treated cells displayed growth inhibition and phenotypic changes consistent with a commitment toward differentiation.ConclusionsAltered cell growth and expression of the cell surface plasma cell antigen, CD138 are detectable in BL36 cells treated by AzaC as well as by Gal1. It seems that AzaC-induced Gal1 expression and consequent binding of Gal1 on its cell membrane receptor may be, in part, involved in AzaC-induced plasmacytic differentiation.

A computer-assisted two-dimensional gel electrophoresis approach for studying the variations in protein expression related to an induced functional repression of NFκB in lymphoblastoid cell lines

Strategies are needed for conclusive interpretation of two‐dimensional gel electrophoresis (2‐D PAGE) maps in order to identify pertinent differences in protein expression during regulation of the transcription of discrete sets of genes. The model used in this study was a human lymphoblastoid cell line in which a functional repression of the transcription factors NFκB was obtained by induction of overexpression of IκBα, a physiological inhibitor of NFκB. The analytical methodology used relies on the comparison of two sets of 2‐D PAGE maps for detecting differences in protein expression between samples overexpressing or not overexpressing IκBα. The analysis was based on a combination of an automatic computerized analysis, constituting an actual aid for deciding, and of an interactive visual validation, corresponding to the interpretation of computer propositions. This strategy is proposed as a rapid way to detect potential variations in protein expression applicable to any biological model. In this study, correspondence analysis data made it possible to discrimate between the samples overexpressing or not overexpressing IκBα, and pointed out some of the potential meaningful spots characterizing the samples in which NFκB was active. Then, after visual validation of the computer data, 53 polypeptides were considered to be different in the two classes of gels. Five polypeptides were specifically found in both samples overexpressing IκBα. The overexpression of IκB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IκBα was not overexpressed and, consequently, in which NFκB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFκB.

Modulation of septin and molecular motor recruitment in the microtubule environment of the Taxol‐resistant human breast cancer cell line MDA‐MB‐231

Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2‐DE coupled with MS to identify changes in the MT environment of Taxol‐resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA‐MB‐231 cells, we verified by Western blotting that in resistant cells, α‐ and β‐tubulins (more specifically the βIII and βIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol‐resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin‐1 heavy chain expression and recruitment on MTs while dynein light chain‐1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane‐resistance could also be valuable to identify new biological markers of resistance.