Regulation of CDK4.

Cyclin-dependent kinase (CDK)4 is a master integrator that couples mitogenic and antimitogenic extracellular signals with the cell cycle. It is also crucial for many oncogenic transformation processes. In this overview, we address various molecular features of CDK4 activation that are critical but remain poorly known or debated, including the regulation of its association with D-type cyclins, its subcellular location, its activating Thr172-phosphorylation and the roles of Cip/Kip CDK "inhibitors" in these processes.

Relationships between cystic fibrosis transmembrane conductance regulator, extracellular nucleotides and cystic fibrosis.

Cystic fibrosis (CF) is one of the most common lethal autosomal recessive genetic diseases in the Caucasian population, with a frequency of about 1 in 3000 livebirths. CF is due to a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a cyclic adenosine 5'-monophosphate (cAMP)-regulated chloride channel localized in the apical membrane of epithelial cells. CFTR is a multifunctional protein which, in addition to be a Cl-channel, is also a regulator of multiple ion channels and other proteins.

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity.

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P(3) 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer.

G1/S Cyclins interact with regulatory subunit of PKA via A-kinase anchoring protein, AKAP95.

The mechanisms by which cyclins promote mammalian cell cycle progression have been a topic of intense investigation over the last decade. We previously described an interaction between D-type cyclins and A-kinase anchoring protein, AKAP95. Here, we demonstrate that AKAP95 can also bind cyclin E1.

Extracellular adenine nucleotides inhibit the release of major monocyte recruiters by human monocyte-derived dendritic cells.

Extracellular ATP is known to affect the maturation of monocyte-derived dendritic cells mainly by regulation of cytokines and costimulatory molecules. The present study describes the inhibition of MCP-1 (CCL2) and MIP-1alpha (CCL3) release by human monocyte-derived dendritic cells in response to adenine nucleotides. Our pharmacological data support the involvement of P2Y11 and P2Y1 purinergic receptors in the downregulation of these major monocyte recruiters.

Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis.

The cAMP signaling pathway regulates growth of many cell types, including somatotrophs, thyrocytes, melanocytes, ovarian follicular granulosa cells, adrenocortical cells, and keratinocytes. Mutations of partners from the cAMP signaling cascade are involved in tumor formation. Thyroid-stimulating hormone (TSH) receptor and Gsalpha activating mutations have been detected in thyroid autonomous adenomas, Gsalpha mutations in growth hormone-secreting pituitary adenomas, and PKAR1A mutations in Carney complex, a multiple neoplasia syndrome.

Identification of differentially expressed genes in thyrotropin stimulated dog thyroid cells by the cDNA-AFLP technique.

In dog thyrocytes in primary culture, thyrotropin (TSH), through cAMP, positively controls proliferation and differentiation. As until now, the key events and the genes involved in the action of TSH remain largely uncharacterized, our goal was to identify new differentially expressed genes in TSH-induced thyroid proliferation. Using cDNA-AFLP, we visualized 105 different transcripts showing significant differential expression during the stimulation of dog thyrocytes with TSH for different times, in the presence of insulin.

Hyperosmotic stress stimulates inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate formation independently of bis-diphosphoinositol tetrakisphosphate modulation.

Hyperosmotic stress induces water diffusion out of the cell, resulting in cell shrinkage, and leading to DNA damage, cell cycle arrest, and cytoskeletal reorganization. A previous report showed that low concentrations of sorbitol (200mM) could increase up to 25-fold the concentration of InsP(8) in animal cells. Here, we investigate the effect of sorbitol (200mM) on the inositol 1,4,5-trisphosphate (InsP(3)) and inositol 1,3,4,5-tetrakisphosphate (InsP(4)) pathway.

Thrombospondin-1 and indoleamine 2,3-dioxygenase are major targets of extracellular ATP in human dendritic cells.

Extracellular adenosine triphosphate affects the maturation of human monocyte-derived dendritic cells (DCs), mainly by inhibiting T-helper 1 (Th1) cytokines, promoting Th2 cytokines, and modulating the expression of costimulatory molecules. In this study, we report that adenosine triphosphate (ATP) can induce immunosuppression through its action on DCs, defining a new role for extracellular nucleotides. Microarray analysis of ATP-stimulated human DCs revealed inter alia a drastic up-regulation of 2 genes encoding mediators involved in immunosuppression: thrombospondin-1 (TSP-1) and indoleamine 2,3-dioxygenase (IDO).

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues.

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design.

Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells.

Regulation of H2O2 generation in thyroid cells does not involve Rac1 activation.

Objectives: The H2O2 generating system of the thyrocyte and the O2- generating system of macrophages and leukocytes present numerous functional analogies. The main constituent enzymes belong to the NADPH oxidase (NOX) family (Duox/ThOX for the thyroid and NOX2 /gp91phox for the leukocytes and macrophages), and in both cell types, H2O2 generation is activated by the intracellular generation of Ca2+ and diacylglycerol signals. Nevertheless, although the controls involved in these two systems are similar, their mechanisms are different.

Immune characterization of clinical grade-dendritic cells generated from cancer patients and genetically modified by an ALVAC vector carrying MAGE minigenes.

Gene delivery into dendritic cells (DC) is most efficiently achieved by viral vectors. Recombinant canarypox viruses (ALVAC) were validated safe and efficient in humans. We aimed firstly to evaluate DC transduction by ALVAC vectors, then to investigate if such infection induced or not the maturation of the DC, and finally to assess the efficiency of ALVAC-MAGE-transduced DC to activate specific CTL clones.

Identification and subcellular distribution of endogenous Ins(1,4,5)P(3) 3-kinase B in mouse tissues.

Inositol 1,4,5-trisphosphate 3-kinase (IP(3)-3K) catalyses the phosphorylation of inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three mammalian isoforms have been reported and referred to as IP(3)-3KA, IP(3)-3KB, and IP(3)-3KC. IP(3)-3KB is particularly sensitive to proteolysis at the N-terminus, a mechanism known to generate active fragments of lower molecular mass.

Involvement of multiple P2Y receptors and signaling pathways in the action of adenine nucleotides diphosphates on human monocyte-derived dendritic cells.

Adenosine 5'-triphosphate (ATP), which is released from necrotic cells, induces a semimaturation state of dendritic cells (DC), characterized by the up-regulation of costimulatory molecules and the inhibition of proinflammatory cytokines. This action is mediated by cyclic adenosine monophosphate (cAMP) and involves the P2Y11 receptor. As DC express the ecto-enzyme CD39, which converts ATP into adenosine 5'-diphosphate (ADP), the effects of adenine nucleotides diphosphates on molecular signaling [intracellular calcium ([Ca2+]i), cAMP, extracellular signal-regulated kinase 1 (ERK1)], costimulatory molecule expression (CD83), and cytokine production [interleukin (IL)-12, tumor necrosis factor alpha (TNF-alpha), IL-10] were investigated in human monocyte-derived DC.

Assessment of in vivo chemotherapy-induced DNA damage in a p53-mutated rat tumor by micronuclei assay.

Production of DNA damage is the basis of cancer treatments such as chemo- and radiotherapy. Such treatments induce mitotic catastrophe, a form of cell death resulting from abnormal mitosis and leading to the formation of interphase cells with multiple micronuclei. In this study, we compared apoptosis induction and micronuclei formation to assess the DNA damage provoked in vivo by cytotoxic agents in established 9L rat gliosarcoma tumors expressing a mutated p53 gene.

Small amplified RNA-SAGE.

Serial analysis of gene expression (SAGE) is a powerful genome-wide analytic tool to determine expression profiles. Since its description in 1995 by Victor Velculescu et al., SAGE has been widely used.

Small amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA.

Serial analysis of gene expression (SAGE) is a widely used and powerful technique to characterize and compare transcriptomes. Although several modifications have been proposed to the initial protocol with the aim of reducing the amount of starting material, unless additional PCR steps are added, the technique is still limited by the need for at least 1 micro g of total RNA. As extra PCR amplification might introduce representation biases, current SAGE protocols are not fully suitable for the study of small, microdissected tissue samples.