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Biomedical subjects

G Schettini

Publications and source records attributed to G Schettini.

At least 37 records · Page 2Linked to original sources
PubMed2001

The activation of the phosphotyrosine phosphatase eta (r-PTP eta) is responsible for the somatostatin inhibition of PC Cl3 thyroid cell proliferation.

The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTP eta whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTP eta in somatostatin's effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTP eta) recovered somatostatin's ability to inhibit cell proliferation, showing dose-dependence and time course similar to those observed in PC Cl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTP eta did not restore the antiproliferative effects of somatostatin. PC mos/PTP eta cells showed a high basal phosphotyrosine phosphatase activity which, similarly to PC Cl3 cells, was further increased after somatostatin treatment. The specificity of the role of r-PTP eta in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTP eta activity in PCCl3 and PC mos/PTP eta (+300%, P < 0.01) but not in PCmos cells. Conversely, no differences in somatostatin-stimulated SHP-2 activity, (approximately +50%, P < 0.05), were observed among all the cell lines. The activation of r-PTP eta by somatostatin caused, acting downstream of MAPK kinase, an inhibition of insulin-induced ERK1/2 activation with the subsequent blockade of the phosphorylation, ubiquitination, and proteasome degradation of the cyclin-dependent kinase inhibitor p27(kip1). Ultimately, high levels of p27(kip1) lead to cell proliferation arrest. In conclusion, somatostatin inhibition of PC Cl3 cell proliferation requires the activation of r-PTP eta which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27(kip1).

Blotting, Northern↗
PubMed2001

[Somatostatin and its receptors. Role in the control of cell proliferation].

A number of studies have been carried out over the past year to characterise the physiological functions and possible therapeutic applications of somatostatin (sst). Somatostatin is one of the most widespread peptides in the body and it fulfils a very wide spectrum of actions in the various organs and tissues. The aim of this review is to make a critical analysis of knowledge of the structure and physiology of sst and its receptors, together with the possible role played by this peptide in cell proliferation in animal models and the treatment of human tumours. In particular, the authors discuss the most recent findings on cell and intracell mechanisms regulated by somatostatin receptors and how this knowledge may encourage the development of more effective and selective new molecules in terms of therapy.

Animals↗
PubMed2001

[Somatostatinergic control of Kaposi's sarcoma growth through the inhibition of angiogenesis].

Somatostatin and its analogs are active in the inhibition of the proliteration of sst receptor positive endocrine neoplasms, however their activity and mechanism in non-endocrine tumors is not clear. Somatostatin effectively inhibited the growth of a Kaposi's sarcoma xenograft in nude mice, yet in vitro the tumor cells did not express any known somatostatin receptors and were not growth inhibited by somatostatin. Histological examination revealed limited vascularization in the somatostatin treated tumors as compared to the controls. Somatostatin was a potent inhibitor of angiogenesis in an in vivo assay. In vitro, somatostatin inhibited endothelial cell growth and invasion. Migration of monocytes, important mediators of the angiogenic cascade, was also inhibited by somatostatin. Both cells types expressed somatostatin receptor mRNAs. These data demonstrate that somatostatin is a potent anti-tumor angiogenesis compound directly affecting both endothelial and monocytic cells. The debated function of somatostatin in tumor treatment and the design of therapeutic protocols should be reexamined considering these data.

Angiogenesis Inhibitors↗
PubMed2001

[The activation of the phosphotyrosine phosphatase eta is responsible for the somatostatin inhibition of PCCl3 thyroid cell proliferation].

BACKGROUND: This study was aimed to identify possible intracellular effectors of the somatostatin (SST) antiproliferative activity, in PCCl3 thyroid cells. METHODS: To prove the involvement of r-PTPeta in SST's effect, we studied th proliferative activity of subclones of PCCl3 cells that do or do not express this PTP. RESULTS: SST inhibited PCCl3 TSH+insulin-dependent cell proliferation through the induction of a phosphotyrosine phosphatase (PTP) activity, detected using the synthetic substrate pNPP (+150%, p<0.01). Conversely, PCCl3 cells stably expressing the v-mos oncogene (PCmos) were completely insensitive to SST antiproliferative effects due to the incapability of SST to increase PTP activity, that correlated with the abolishment of the expression of the receptor-like PTP, r-PTPeta. In the cells in which r-PTPeta was transfected (PCmos/ PTPeta) SST inhibited cell proliferation showing a dose-dependence similar to that observed in PCCl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTPeta did not restore the responsivity to SST. Also in PCmos/PTPeta cells SST, treatment increased membrane PTP activity. CONCLUSIONS: SST inhibition of PCC13 cell proliferation requires the activation of r-PTPeta.

Animals↗
PubMed2000

C60 carboxyfullerene exerts a protective activity against oxidative stress-induced apoptosis in human peripheral blood mononuclear cells.

C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a free-radical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-d-ribose (dRib) or TNF-alpha plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-d-ribose or TNF-alpha plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria.

Adult↗
PubMed2000

Apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in rat cerebellar granule cells treated with the prion protein fragment 106-126.

Prion diseases are neurodegenerative pathologies characterized by the accumulation, in the brain, of altered forms of the prion protein (PrP), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) was reported to maintain the neurodegenerative characteristics of PrP(Sc). We investigated the intracellular mechanisms involved in PrP106-126-dependent degeneration of primary cultures of cerebellar granule neurons. Prolonged exposure of such neurons to PrP106-126 induced apoptotic cell death. The L-type voltage-sensitive calcium channel blocker nicardipine reproduced this effect, suggesting that blockade of Ca(2+) entry through this class of calcium channels may be responsible for the granule cell degeneration. Microfluorometric analysis showed that PrP106-126 caused a reduction in cytosolic calcium levels, elicited by depolarizing K(+) concentrations in these neurons. Electrophysiological studies demonstrated that PrP106-126 and nicardipine selectively reduce the L-type calcium channel current. These data demonstrate that PrP106-126 alters the activity of L-type voltage-sensitive calcium channels in rat cerebellar granule cells and suggest that this phenomenon is related to the cell death induced by the peptide.

Animals↗
PubMed2000

The importance of surgery in supratentorial ependymomas. Long-term survival in a series of 23 cases.

In view of the widely recognized correlation between extent of surgical resection and length of survival of children with intracranial ependymoma and the statement that total resection is more likely to be achieved in supratentorial than infratentorial primaries, we decided to review our experience with supratentorial ependymomas and the pertinent literature to verify the importance of surgery in treating this subgroup of pediatric ependymal neoplasms. Of 23 patients operated on, 12 are still alive without evidence of disease 72-357 months after surgery (mean 227, median 237 months). One girl treated by surgery alone was lost to follow-up after 234 months when she, and 7 other patients in the series, had already passed the end of the period of risk for recurrence according to Collins' law. Six surviving patients (2 with subependymoma and 4 with ependymoma) were treated by surgery alone and only 1, the oldest in the series, had to undergo a second operation for recurrence after 10 years. The idea of treating intracranial ependymoma by surgery alone was favored by eminent neurosurgeons in the past and has recently received renewed attention. This was in part the consequence of recognizing that unlike diffuse astrocytoma, in which neoplastic cells can be found up to several centimeters away from the apparent tumor borders, ependymoma has more or less well-defined margins and grows mainly by expansion. Early experience with the policy of electively deferring adjuvant therapy after radiologically controlled total resection of ependymoma seems encouraging, although postoperative MRI does not yet indicate absolute certainty. Close surveillance is recommended. The majority of ependymomas so far treated by surgery alone, with relatively good success, have been supratentorial. In conclusion, on the basis of our experience and a review of the literature we favor a change in attitude to the management of intracranial ependymomas, especially of the cerebrum, with radiologically controlled radical surgery alone followed by close surveillance with periodic MR imaging until the child passes the period of risk for recurrence according to Collins' law as the initial option. In children less than 3 years old the period of surveillance should be doubled. In case of recurrence, reoperation should be considered first, particularly for supratentorial primaries. Radiotherapy continues to be a major option in malignant ependymoma and unresectable primary or recurrent benign ependymoma.

Adolescent↗
PubMed2000

Immunofluorescence and biochemical techniques to detect nuclear localization of ciliary neurotrophic factor in glial cells.

Ciliary neurotrophic factor (CNTF) promotes the survival of several populations of neurons, including sensory and motor neurons. It is mainly produced by Schwann cells and astrocytes and exerts its biological function via a specific membrane receptor. We recently determined the nuclear localization of CNTF in producing cells, after transfection and in the heterologous system of Xenopus oocytes. In the present paper, we describe in detail the techniques for the detection of CNTF in the nucleus of rat astrocytes, transfected cells, isolated nuclei and injected Xenopus oocytes.

Animals↗
PubMed2000

C3-fullero-tris-methanodicarboxylic acid protects cerebellar granule cells from apoptosis.

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3-fullero-tris-methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration-dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4-induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.

Animals↗
PubMed2000

Carboxyfullerenes protect human keratinocytes from ultraviolet-B-induced apoptosis.

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.

Apoptosis↗
PubMed2000

Generation of an apoptotic intracellular peptide by gamma-secretase cleavage of Alzheimer's amyloid beta protein precursor.

The amyloid beta protein precursor (AbetaPP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide. The biochemical path leading to Abeta formation has been extensively studied since extracellular aggregates of amyloidogenic forms of Abeta peptide (Abeta42) are considered the culprit of Alzheimer's disease. Aside from its pathological relevance, the biological role of AbetaPP proteolysis is unknown. Although never previously described, cleavage of AbetaPP by gamma-secretase should release, together with Abeta, a COOH-terminal AbetaPP Intracellular Domain, herein termed AID. We have now identified AID-like peptides in brain tissue of normal control and patients with sporadic Alzheimer's disease and demonstrate that AID acts as a positive regulator of apoptosis. Thus, overproduction of AID may add to the toxic effect of Abeta42 aggregates and further accelerate neurodegeneration.

Journal Article↗
PubMed1999

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.

We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.

3T3 Cells↗
PubMed1999

C3-fullero-tris-methanodicarboxylic acid protects epithelial cells from radiation-induced anoikia by influencing cell adhesion ability.

Anoikia is a type of apoptotic cell death that occurs in cells that are substrate-restricted in their growth. Buckminsterfullerenes represent a new class of chemical compounds with wide potential pharmacological antioxidant activity. In this report we provide the first demonstration that a water-soluble fullerene derivative, C3-fullero-tris-methanodicarboxylic acid, synthesized in our laboratories, is capable of inducing anoikia resistance in epithelial cells by a mechanism involving a 'trophic' effect on cell spreading-associated cytoskeletal components, i.e. on actin microfilaments.

Antioxidants↗
PubMed1999

Expression of chemokine receptors in the rat brain.

Human immunodeficiency virus (HIV-1) infects the brain and causes a progressive encephalopathy in 20 to 30% of infected children and adults called AIDS dementia complex. Evidence from in vitro and in vivo studies suggests a role for the viral envelope glycoprotein gp120, as a mediator of neurotoxicity. However, the site of interaction of gp120 with neurons and astrocytes to mediate neuronal death is still unknown. Recently the chemokine receptors, CCR5 and CXCR4, have been identified as co-receptors together with CD4 for HIV-1 entry into the target cells, suggesting a possible role for these receptors in the pathogenesis of the HIV-1 infection in the brain. Here we report the expression of CCR5 and CXCR4 in many different rat brain areas. We also found both receptors in cultured type I astrocytes demonstrating that glial cells may represent an important target for chemokines in vivo. Indeed, the functional capacity of CXCR4 receptor in astrocytes was demonstrated showing that SDF 1 alpha induced an increase of intracellular calcium concentration.

Animals↗
PubMed1999

The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells.

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.

A Kinase Anchor Proteins↗
PubMed1999

Nuclear localization of ciliary neurotrophic factor in glial cells.

In this work we studied the subcellular localization of ciliary neurotrophic factor (CNTF) in primary culture of rat cortical type I astrocytes and rat glioma C6 cells, transfected COS-7 cells and in the Xenopus oocytes. In all these models, morphological and biochemical evidence are provided for the nuclear localization of CNTF. In addition the nuclear translocation of CNTF is temperature-sensitive and thus strongly suggestive of a mechanism of facilitated transport.

Animals↗
PubMed1999

New vs old fashioned oestradiol antagonists in mammary carcinoma: 'in vitro' and 'in vivo' pharmacological approaches.

The rationale underlying therapeutic strategies designed to inhibit the action of endogenous sex hormones in malignant breast cells is provided by the demonstration of their involvement in supporting the development and growth of breast carcinoma. The surgical removal of steroid-secreting glands, in order to reduce the level of oestrogens reaching their target tissues, has for years been substituted by the so-called endocrinotherapeutic approach, which is based on the counteraction of the steroid hormone activity by the hormonal receptor blockade with suitable antioestrogenic compounds. Over the past 25 years, the non-steroidal oestrogen antagonist tamoxifen has become the standard endocrine treatment for breast cancer. The triphenylethylene-derivative compound competes efficiently for binding to the oestrogen receptor, but the complex retains some transcriptional activity. Consequently, tamoxifen exhibits, both ' in vitro and in vivo ', a range of biological activity from full oestrogen antagonism to partial agonism. There is also evidence suggesting that the agonist activity of this compound may ultimately stimulate breast tumour growth, thus causing some treatment failures. Moreover, the use of tamoxifen is limited by the possible onset of drug-resistance in many patients. Nevertheless, widely tested tamoxifen has proved to be very helpful for the development of new compounds to be used as long-term adjuvant therapy or as preventive agents. These novel oestrogen antagonists belong to two major classes: tamoxifen analogs and new pure steroidal-like antioestrogens. The search for and development of compounds devoid of tamoxifen cross-resistance, with a safer toxicity profile as well as the lack of oestrogenic effects, provide the bases to improve the current therapeutic applications of antioestrogens.

Animals↗