Expression of the thrombospondin 1 fragment 167-569 in C6 glioma cells stimulates tumorigenicity despite reduced neovascularization.

Gliomas are among the most malignant and most highly vascularized human tumors. We studied the therapeutic action of an angiostatic fragment of human thrombospondin 1 (named TSP1ang) on experimental glioma tumor growth. TSP1ang (enclosing amino acids 167-569) comprised the procollagen-homology domain and the three type I repeats of the original molecule.

Long oligonucleotide arrays on nylon for large-scale gene expression analysis.

To date, most studies of multigenic expression patterns by long DNA array have used DNA fragments as probes. These probes are usually obtained as PCR products, and this represents a time-consuming and error-prone approach, requiring strict quality control. The present study examines the use of 40- and 70-mer synthetic oligonucleotides as probes for DNA array analysis with radioactive labeled targets.

Prognostic assessment of PTK activity in T1-T2, N0-N1, M0 breast cancer: a multicentric retrospective study.

Protein tyrosine kinases (PTKs) play a major role in the transduction of intracellular mitogenic signal. PTKs are also involved in the process of cellular transformation. A number of studies have reported increased PTK activities in cytosolic fractions from human breast carcinoma.

Identification of two novel ACTH-responsive genes encoding manganese-dependent superoxide dismutase (SOD2) and the zinc finger protein TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b].

ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH.

Microarray RNA quantification assay for large-scale nanosamples analysis.

We have developed a convenient and sensitive method for the quantification of RNA in samples from microbiopsies. This procedure is especially suitable for quantitating very small amounts of RNA in large-scale biological samples. This method, using a microarray-spotting facility for the study of multigenic expression, entails the hybridization of a DNA probe with RNA spotted at high density on nylon membrane.

A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit alpha.

Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits generally combined to form alpha(2)beta(2), alphaalpha'beta(2), or alpha'(2)beta(2) heterotetramers. To gain more insight into the role of CK2 in the control of proliferation in mammalian cells, overexpression of isolated CK2 subunits alpha, alpha', or beta was carried out in two fibroblast cell lines: NIH3T3 and CCL39. To interfere with CK2 cellular functions, cells were also transfected with a kinase-inactive mutant of CK2alpha catalytic subunit: CK2alpha-K68A.

Transcriptional regulation of the gene encoding the StAR protein in the human adrenocortical cell line, H295R by cAMP and TGFbeta1.

In the present work, we have analyzed the transcriptional regulation of StAR expression in the human adrenocortical cell line H295R. We observed that StAR mRNA levels rapidly increased in response to forskolin, starting after 2 h of treatment and reaching a maximum by 12 h. Deletion analysis of the human StAR promoter demonstrated that the first 150 bp upstream of the transcription start were sufficient for both basal and cAMP-induced expression of a reporter gene.

Expression of the angiogenesis markers vascular endothelial growth factor-A, thrombospondin-1, and platelet-derived endothelial cell growth factor in human sporadic adrenocortical tumors: correlation with genotypic alterations.

Several studies have supported the hypothesis that adrenocortical tumor formation is the result of a multistep process. The angiogenic switch has been proposed to be a key step in tumor progression from adenoma to carcinoma. In this study we measured the cytosolic concentrations of three proteins involved in angiogenesis [namely platelet-derived endothelial cell growth factor vascular endothelial cell growth factor A (VEGF-A), and thrombospondin-1 (TSP1)] in a series of 43 human sporadic adrenocortical tumors.

Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1.

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Sequencing of the CYP11B1 gene showed two new base substitutions, a conservative 954 G-->C transversion at the last base of exon 5 (T318T), and a IVS8 + 4A-->G transition in intron 8. In addition, two polymorphisms were found in exons 1 and 2.

Adrenocorticotrophic hormone stimulates phosphotyrosine phosphatase SHP2 in bovine adrenocortical cells: phosphorylation and activation by cAMP-dependent protein kinase.

During activation of adrenocortical cells by adrenocorticotrophic hormone (ACTH), tyrosine dephosphorylation of paxillin is stimulated and this correlates with protrusion of filopodial structures and a decreased number of focal adhesions. These effects are inhibited by Na(3)VO(4), a phosphotyrosine phosphatase inhibitor [Vilgrain, Chinn, Gaillard, Chambaz and Feige (1998) Biochem. J.

The disruption of adherens junctions is associated with a decrease of E-cadherin phosphorylation by protein kinase CK2.

The down-regulation of E-cadherin is a common event in carcinogenesis. Phosphorylation/dephosphorylation is one posttranscriptional process which may regulate intercellular junctions. Here we show that in okadaic acid-treated keratinocytes, E-cadherin expression is shifted from the membrane to the cytoplasm, preventing cells from forming aggregates.

Expression and regulation of melanocortin receptor-5 (MC5-R) in the bovine adrenal cortex.

Among the five members of the melanocortin receptor (MC-R) family, MC2 and MC5 are expressed in peripheral tissues. The receptor MC2 (ACTH receptor) almost exclusively expressed in the adrenal cortex whereas MC5-R is expressed in several organs including the adrenal cortex. Both receptors bind ACTH and activate adenylate cyclase.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) expression is strongly induced by ACTH in adrenocortical cells.

Besides its acute and chronic effects on corticosteroid synthesis, the pituitary adrenocorticotropic hormone (ACTH) regulates diverse adrenocortical biological functions including the synthesis of a number of mitochondrial, cytoplasmic, and secreted proteins. ACTH-induced secreted proteins are candidates to act as local extracellular relays of the hormone in either an autocrine or a paracrine manner. In the present study, we report that stimulation of primary cultures of bovine adrenocortical (BAC) fasciculata cells with 10 nM ACTH for 24 h results in a mean 8 +/- 4-fold induction of the synthesis of a secreted protein presenting an apparent Mr of 21 kDa.

[Interactions between steroidogenic cells and capillary endothelial cells in the adrenal gland].

Adrenocortical capillary endothelial (ACE) cells showed a two- to three-fold increase in number when they were cocultured with bovine adrenocortical (BAC) cells from the zona fasciculata-reticularis. This effect was detectable within 48 h, persisted throughout the seven-day coculture period, and occurred in the absence of addition of exogenous growth factors. It was similar to that obtained by addition of 1 ng/ml of FGF-2.

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution.

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids).

CK2alpha-protein phosphatase 2A molecular complex: possible interaction with the MAP kinase pathway.

Despite its wide range of known substrates, the signaling function of protein kinase CK2 is still enigmatic. Mounting evidence suggests that CK2alpha, the catalytic subunit of holoenzymic CK2, may exist free of its usual regulatory partner CK2beta, raising the possibility that 'free' CK2alpha has regulation and function distinct from those of the holoenzyme. We previously reported that CK2alpha could bind to the core dimer of protein phosphatase 2A, and indirectly cause down-regulation of the PP2A substrate MEK1, possibly via activation of PP2A and/or targeting of PP2A to some element of the Ras/Raf/MEK pathway.

Searching interaction partners of protein kinase CK2beta subunit by two-hybrid screening.

To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed that the enzyme associates with several intracellular proteins and the formation of such molecular complexes may represent a mechanism for the control of CK2 activity. Using the Interaction Trap system in yeast, with the CK2beta as a bait, we looked for CK2 partners.

Dissecting subdomains involved in multiple functions of the CK2beta subunit.

We have characterized several subdomains of the beta subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators.

Crystallization and preliminary x-ray diffraction analysis of the regulatory subunit of human protein kinase CK2.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. A C-terminal truncated form of the beta subunit has been overproduced in Escherichia coli and purified to homogeneity. Two crystal forms of the truncated protein which diffract to at least 2 A resolution have been obtained.

Smad3 is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on StAR expression.

Transforming growth factor betas (TGFbetas) constitute a family of dimeric proteins that regulate growth and differentiation of many cell types. TGFbeta1 is also a potent autocrine regulator of adrenocortical steroidogenesis. We have recently shown that in primary cultures of bovine fasciculo-reticularis cells, the main target of TGFbeta is the steroidogenic acute relay protein (StAR), a key protein necessary for intramitochondrial cholesterol transport.

Gastric inhibitory polypeptide (GIP) stimulates cortisol secretion, cAMP production and DNA synthesis in an adrenal adenoma responsible for food-dependent Cushing's syndrome.

We studied in vitro an adrenal tumor responsible for food-dependent, ACTH independent, Cushing's's syndrome. Cortisol secretion by isolated tumor cells was stimulated by GIP and ACTH, but not by the gut hormone glucagon-like peptide-1 (GLP-1). Both GIP and ACTH stimulated production of cAMP but not inositol 1,4,5-trisphosphate IP3).

Differential implication of StAR and P450c17 in TGFbeta1-induced decrease of adrenocortical steroidogenesis.

In primary cultures of bovine adrenocortical fasciculata cells, we have previously identified two targets of TGFbeta action: StAR and P450c17. Since it is well known that adrenocortical cells tend to dedifferentiate as soon as they are placed in culture, we investigated the regulation of StAR and P450c17 expression by TGFbeta1 at different times of primary culture. On day 1, TGFbeta1 decreased the expression of basal levels of both genes.

Expression of ACTH receptors (MC2-R and MC5-R) in the glomerulosa and the fasciculata-reticularis zones of bovine adrenal cortex.

The recent cloning of a family of melanocortin receptors (MC-R) has identified five distinct G protein- and adenylate cyclase-coupled receptors. The MC2-receptor (MC2-R) preferentially binds ACTH. It is expressed in the adrenal cortex and is hence considered to be the ACTH receptor.

Cushing's syndrome due to a gastric inhibitory polypeptide-dependent adrenal adenoma: insights into hormonal control of adrenocortical tumorigenesis.

We studied a patient with food-induced, ACTH-independent, Cushing's syndrome and a unilateral adrenocortical adenoma. In vivo cortisol secretion was stimulated by mixed, glucidic, lipidic, or proteic meals. Plasma ACTH levels were undetectable, but iv injection of ACTH stimulated cortisol secretion.