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P Bruni

Publications and source records attributed to P Bruni.

At least 37 records · Page 2Linked to original sources
PubMed1999

Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.

Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.

Acetamides↗
PubMed1999

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.

Animals↗
PubMed1999

Modulation of in vivo growth of thyroid tumor-derived cell lines by sense and antisense vascular endothelial growth factor gene.

Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.

Animals↗
PubMed1999

Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells.

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.

ADP Ribose Transferases↗
PubMed1999

Expression and regulation of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase isozymes in white adipose tissue.

The aim of this work was to identify the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isozyme(s) present in white adipose tissue. Ion-exchange chromatography of PFK-2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP-dependent protein kinase, by specific labelling of the preparation with [2-32P]fructose 2,6-bisphosphate and by reaction with antibodies. Characterization of the PFK-2/FBPase-2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6-bisphosphate concentrations without significant inactivation of PFK-2. These results support previous findings showing that fructose 2,6-bisphosphate is not involved in the adrenaline-induced stimulation of glycolysis in white adipose tissue.

Adipose Tissue↗
PubMed1999

Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells.

The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.

CDC2-CDC28 Kinases↗
PubMed1999

Regulation of thymosin beta10 expression by TSH and other mitogenic signals in the thyroid gland and in cultured thyrocytes.

OBJECTIVE: To investigate the expression of thymosin beta10 - a small conserved acidic protein involved in the inhibition of actin polymerization - in human and experimental thyroid goiters as well as the regulation exerted by TSH on thymosin beta10 expression in thyroid follicular cells both in vivo and in vitro. DESIGN: To this aim, we have used 5 bioptic specimens from patients affected by thyroid goiter, a well known experimental model of thyroid goitrogenesis (rat fed with the drug propylthiouracil) and a cultured rat thyroid cell line (PC Cl 3 cells) as a model system. RESULTS: We report that the mRNA expression of thymosin beta10 is markedly enhanced in human goiters compared with normal thyroid. In vivo results showed that the steady-state level of thymosin beta10 mRNA is up-regulated in the thyroid gland of propylthiouracil-fed rats in parallel with follicular cell proliferation: iodide administration to goitrous rats, which induced a marked involution of thyroid hyperplasia, reduced the mRNA level of thymosin beta10. Finally, in vitro studies showed that in cultured rat thyrocytes, the expression of thymosin beta10 mRNA is induced in a time- and dose-dependent manner by the activation of pathways which are mitogenic for thyroid cells (i.e. the protein kinase (PK) A and PKC pathways). CONCLUSION: Taken together, the findings reported here demonstrate that thymosin beta10 expression is regulated by extracellular signals that stimulate growth of thyroid cells both in vitro and in vivo, and suggest a role for this protein in thyroid diseases characterized by proliferation of follicular cells.

Animals↗
PubMed1998

Protein tyrosine phosphatase-eta expression is upregulated by the PKA-dependent and is downregulated by the PKC-dependent pathways in thyroid cells.

We have recently reported the isolation of a rat cDNA encoding a receptor-type tyrosine phosphatase, which appears to be a marker of thyroid differentiation. To elucidate the molecular mechanisms underlying r-PTPeta expression in normal thyroid cells both in vitro and in vivo, we investigated the regulation of r-PTPeta expression in cultured thyrocytes (the rat cell line PC Cl 3) and in an animal model of TSH-dependent thyroid goitrogenesis. In vitro studies showed that mRNA expression of r-PTPeta in thyroid cells is induced in a time- and dose-dependent manner by the activation of growth- and differentiation-linked PKA pathways (TSH and forskolin), whereas it is down-regulated by the activation of the proliferative dedifferentiating PKC-dependent transduction pathway (TPA). However, the regulation of r-PTPeta expression by TSH and TPA, respectively, is observed only in normal thyroid cells, but is lost in transformed thyroid cells. In vivo studies with thiouracil-fed rats demonstrated that increased serum levels of TSH up-regulated r-PTPeta mRNA expression in parallel with the stimulation of thyroid growth and function. The reduction of blood TSH levels due to iodide refeeding to goitrous rats determined a marked down-regulation of r-PTPeta expression, in parallel with involution of thyroid hyperplasia. Taken together these results demonstrate that the phosphatase r-PTPeta is regulated by the two main thyroid regulatory pathways and suggest that it may play an important role in the growth and differentiation of thyroid cells.

Animals↗
PubMed1998

A one-million-year-old Homo cranium from the Danakil (Afar) Depression of Eritrea.

One of the most contentious topics in the study of human evolution is that of the time, place and mode of origin of Homo sapiens. The discovery in the Northern Danakil (Afar) Depression, Eritrea, of a well-preserved Homo cranium with a mixture of characters typical of H. erectus and H. sapiens contributes significantly to this debate. The cranium was found in a succession of fluvio-deltaic and lacustrine deposits and is associated with a rich mammalian fauna of early to early-middle Pleistocene age. A magnetostratigraphic survey indicates two reversed and two normal magnetozones. The layer in which the cranium was found is near the top of the lower normal magnetozone, which is identified as the Jaramillo subchron. Consequently, the human remains can be dated at approximately 1 million years before present.

Animals↗
PubMed1998

A role for phospholipase D activation in the lipid signalling cascade generated by bradykinin and thrombin in C2C12 myoblasts.

In the present study evidence is provided for a rapid activation of lipid signalling pathways induced by thrombin and bradykinin (BK) in C2C12 myoblasts. Both agonists were able to increase [3H]inositol phosphates (InsP), 1,2-[3H]diacylglycerol (DAG) and [3H]phosphatidic acid (PtdOH) levels. In particular [3H]PtdOH values were rapidly increased and maintained at significantly high values at prolonged times of incubation. BK and thrombin were able to activate phospholipase D (PLD) in vivo as demonstrated by the accumulation of [3H]phosphatidylethanol (PtdEtOH) through the transphoshatidylation reaction catalyzed by the enzyme in the presence of ethanol. The observation that ethanol could significantly reduce [3H]PtdOH formation in myoblasts stimulated with BK and thrombin indicates that stimulation of PLD has a major role. The two agonists appear to stimulate PLD activity through a common molecular mechanism, involving the activation of protein kinase C (PKC). In addition, BK and thrombin appear able to activate DAG kinase at early times of incubation and also this pathway may contribute to determine the increase in [3H]PtdOH levels. This is the first report which describes activation of lipid signalling pathways by BK and thrombin in myoblast cells and it is possible that these early signals may have an important role in mediating the biological effects of the two agonists.

Animals↗
PubMed1998

Novel mutation of the PTEN gene in an Italian Cowden's disease kindred.

Cowden disease (CD) is an autosomal dominant multiple hamartoma syndrome with an elevated risk of thyroid and breast cancers. The CD susceptibility gene has recently been identified as the PTEN/MMAC1/TEP1 gene localized at 10q23 and coding for a dual specificity protein phosphatase. We report the mutational analysis of the PTEN gene in one Italian CD kindred. By using the single strand conformation polymorphism technique and subsequent direct DNA sequencing of the polymerase chain reaction product, we identified a novel mutation in the exon 5 of the PTEN gene. A heterozygous germline TGT-TAT transition was detected at the nucleotide 407; this causes the amino acid substitution cys136-tyr136 and the generation of a new NSI I restriction site. This mutation was not detected in the unaffected member of the family thereby indicating that it is causally linked to the disease. We ruled out that this mutation is a polymorphic variant because it was not detected in over 100 chromosomes analyzed. Using reverse trancriptase-polymerase chain reaction, we detected the expression of the mutant allele in lymphocytes and pathological tissues from an affected member of the family.

Adolescent↗
PubMed1998

Management of intracranial aneurysms: "state of the art".

The authors provide an overview of the past, present and future of intracranial aneurysms management. Excellent results achieved today by neurovascular surgeons are to be ascribed not only to refinements in microsurgical technique but also to an always more aggressive and effective treatment of vasospasm, the most feared complication of aneurysmal SAH. "Triple H" therapy, calcium channel blockers, rTPA and balloon angioplasty represent the corner-stones of arterial spasm treatment. Hopefully new agents such as lazaroids, endothelins inhibitors and nitric oxyde modulators will become available for clinical use in the next future. Although clipping of aneurysmal neck is still considered the "gold standard", obliteration of the aneurysmal sac can now be safely achieved by coil embolization: the exact role and respective indications of each technique are yet to be defined. Further prognostic improvement will be possible through an early diagnosis, i.e. before the occurrence of SAH, as shown by negligible morbidity and mortality associated with the treatment of "incidental" and "unruptured" aneurysms.

Embolization, Therapeutic↗
PubMed1997

Glutathione transport system in human small intestine epithelial cells.

The present study characterizes for the first time a GSH specific transporter in a human intestinal epithelial cell line (I407). GSH metabolism is very important for the antioxidant and detoxifying action of intestine and for the maintenance of the luminal thiol-disulfide ratio involved in regulation mechanisms of the protein activity of epithelial cells. GSH level decreases have been related to physio-pathological alterations either of intestine or other organs. GSH specific transport systems have been identified in membranes of various cell types of rat, mice and rabbit. The presence of a Na+-independent transport system of GSH is confirmed by the similar behaviour of GSH uptake time-courses when Na+ in extracellular uptake medium was replaced with choline+ or K+ as well as by kinetic saturation and by the trans-stimulation effect on GSH uptake in GSH preloaded cells. Moreover, this transporter is activated when cations are present in extracellular medium and it is affected by membrane potential changes with an increase in GSH uptake values when membrane depolarization occurs. The present results also show a remarkable affinity and specificity of this transporter for GSH; in fact, Km value is very low (90 +/- 20 microM) and only compounds strictly related to GSH structure, such as GSH S-conjugates and GSH-ethyl ester, inhibit GSH uptake in 1407 cells. Finally, a possible hormonal control and modulation by the thiol-disulfide status of GSH transporter activity is suggested.

Animals↗
PubMed1997

Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Animals↗
PubMed1997

Conformational analysis of Amphotericin B.

Within a theoretical approach to the problem of antifungal action of Amphotericin B (AmB), a conformational analysis of the neutral and zwitterionic form of this antibiotic in vacuo was performed by the MM2P and AM1 methods. The analysis was carried out with regard to the mutual orientation of the macrolidic and glycosidic fragments of the molecule, which is defined by the phi and psi steric angles. This orientation defines the overall shape of the molecule and is postulated to be important for the antifungal action of the drug. As a result of the MM2P calculations, phi, psi steric energy and population maps were prepared. Several conformers were found on these maps but only two of them (one each for the zwitterionic and the neutral forms of the antibiotic) were previously observed experimentally for isolated molecules. Our other calculated conformers were not observed experimentally but we propose that they may also appear in the AmB channel structure. The results of our conformational analysis were compared with experimental NMR data (nuclear Overhauser effects between selected hydrogen atoms) obtained previously. New structural information obtained for AmB in the present work will be useful for building a molecular model of AmB-target interactions as well as for designing new derivatives of AmB.

Journal Article↗
PubMed1997

Sphingomyelinase increases 2-deoxyglucose uptake and glucose metabolism of human platelets.

Recent studies have shown that the sphingomyelinase (SMase) catalyzed hydrolysis of sphingomyelin (SM) represents an important cell signalling pathway. Control of SMase activity appears to be crucial for the regulation of multiple biological events in different cell systems; in particular, SMase activity appears to be involved in the control of vascular functions and in atherogenic events. Here we report that SMase treatment of human platelets significantly increases 2-deoxyglucose uptake by decreasing K(m) value of sugar transport and increasing sugar diffusion. In addition SMase treatment enhances basal glycolytic flux of platelets as well as the stimulation of the flux induced by suboptimal concentration of thrombin. The present study demonstrates that exposure of platelets to SMase, which may take place in vivo in physiological and/or in pathological conditions, modifies biochemical parameters of resting and stimulated platelets which are essential for cell physiological role.

Biological Transport↗
PubMed1996

Activation of phospholipase D in human fibroblasts by ceramide and sphingosine: evaluation of their modulatory role in bradykinin stimulation of phospholipase D.

In the present study the modulatory action of exogenous short-chain ceramide and sphingosine on phospholipase D (PLD) activity in young and old human fibroblasts was examined. Sphingosine and also ceramide, thus far described as a negative modulator of PLD, were able to activate PLD. The stimulatory action of the exogenous lipid molecules was mimicked by cell treatment with S.aureus sphingomyelinase (SMase). Similar response was elicited by the sphingoid molecules in young and old cells. Altered levels of sphingosine and ceramide were detected in old fibroblasts confirming that a defect in sphingolipid metabolism occurs in cellular senescence. The modulatory role of sphingoid molecules on the action of bradykinin (BK) in PLD activation was then evaluated in young and old fibroblasts. C6-ceramide or SMase treatment did not affect the action of BK on PLD either in young or in old cells, whereas sphingosine further increased PLD activity stimulated by BK in young but not in old cells. In addition, preincubation with N-oleoylethanolamine, a specific inhibitor of ceramidase, did not affect BK action on PLD in young fibroblasts but significantly decreased the effect of the peptide in old fibroblasts. These results suggest that a specific alteration of BK segnalatory pathway occurs in old fibroblasts, likely involving sphingosine formation which may account for the potentiated PLD activity induced by the peptide in these cells.

Bradykinin↗