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Biomedical subjects

F Cimino

Publications and source records attributed to F Cimino.

At least 19 recordsLinked to original sources
PubMed2003

Protein carbonyl group content in patients affected by familiar chronic nail candidiasis.

Familiar chronic nail candidiasis (FCNC) is a rare disorder characterized by early-onset infections caused by different species of Candida, restricted to the nail of the hands and feet, and associated with a low serum concentration of intercellular adhesion molecule 1. Host defense mechanisms against candidiasis require the cooperation of many immune cells through several candidacidal mechanisms, including oxygen-dependent killing mechanisms, mediated by a superoxide anion radical myeloperoxidase--H2O2--halide system, and reactive nitrogen intermediates. We analyzed protein carbonyl groups (considered a useful marker of oxidative stress) in the serum of patients belonging to a five-generation Italian family with an isolated form of FCNC. Serum protein carbonyl groups in FCNC patients were significantly lower than those measured in healthy donors. Also, if this hypothesis is merely speculative, we could suggest that the decreased circulating level of protein carbonyl groups in these patients is not a marker of a lower oxidative stress condition, but might be linked to a lower protease activity.

Adolescent↗
PubMed2002

Transcription factor NF-Y regulates differentiation of CaCo-2 cells.

The CaCo-2 cell line is used to study the molecular mechanisms underlying differentiation of intestinal epithelial cells. These cells undergo a gradual differentiation process that is growth-related and depends on cellular density. CaCo-2 cells acquire a morphological polarity and express such markers of mature enterocytes as sucrase-isomaltase, apolipoproteins, alkaline phosphatase, and H-ferritin. Because the NF-Y transcription factor is required for H-ferritin gene expression, we investigated whether it is involved in the expression of the other CaCo-2 differentiation markers. We observed that subunit NF-YA increases during CaCo-2 differentiation and that the constitutive expression of NF-YA, obtained in stably transfected CaCo-2 cells, results in the expression of differentiation markers. In fact, sucrase-isomaltase, apolipoprotein A1, and H-ferritin were constitutively expressed in NF-YA-transfected cells and their levels did not increase during prolonged culture, while these markers were not expressed in mock-transfected CaCo-2 cells or transfected with an inactive NF-YA expression vector until the onset of differentiation.

Apolipoprotein A-I↗
PubMed2001

Regulation of p21waf1/cip1 expression by intracellular redox conditions.

Reactive oxygen species (ROS) have been considered for a long time only as molecules for inducing oxidative damage to proteins, lipids, and nucleic acids. However, in the last few years some physiological effects of ROS have been hypothesized, consisting of the redox regulation of several biological processes, including the transduction of mitogenic signals. This means that intracellular generation of ROS could be necessary to maintain homeostasis, as well as that their formation/scavenging should be controlled processes. We developed an experimental procedure that causes redox perturbations in intact cells, based on the exposure of living cells to diethylmaleate (DEM), a GSH-depleting agent. By this procedure we demonstrated that ROS generated following DEM treatment induces a G1 arrest, that is accompanied by several redox-dependent changes in cell cycle-related proteins. One of these is the p53-independent accumulation of p21waf1/cip1, which requires the integrity of the ras-MAPK pathway. Accordingly, DEM treatment strongly activates ERK2. On the other hand, redox perturbations provoked by DEM induce several early phenomena, including p21waf1/cip1 and Rb dephosphorylation.

Animals↗
PubMed2000

Retinoblastoma protein dephosphorylation is an early event of cellular response to prooxidant conditions.

The modification of intracellular redox conditions with diethylmaleate (DEM), a glutathione-depleting agent, induces a p53-independent growth arrest mediated by the accumulation of p21(waf1) mRNA and protein. The same treatment also induces the retinoblastoma protein (pRb) dephosphorylation. This dephosphorylation (i) is very fast, being observed already 5 min after the exposure of the cells to DEM, (ii) is dependent on the prooxidant effects of DEM, being prevented by the treatment with N-acetylcysteine and (iii) is completely reversible, since the rephosphorylation of pRb is promptly obtained upon the removal of the glutathione-depleting agent from the culture medium. The dephosphorylation of pRb is independent of the accumulation of p21(waf1) induced by DEM; in fact, p21(waf1) levels start to increase much later after DEM treatment and accordingly cyclin-dependent kinase activities are not yet induced when pRb is already dephosphorylated following DEM treatment. Finally, pRb dephosphorylation is catalyzed by phosphatases activated by DEM treatment.

Acetylcysteine↗
PubMed1999

The B subunit of the CAAT-binding factor NFY binds the central segment of the Co-activator p300.

We report that the heterotrimeric transcription factor NFY or "CAAT-binding factor" binds the -60 region of the human H ferritin promoter, the B site. DNA binding analysis with specific antibodies demonstrates that NFY/B/C subunits tightly bind this site and that NFY/C subunit is masked in vivo by binding with other protein(s). NFY binds the co-activator p300. Specifically, the NFY/B subunit interacts with the central segment of p300 in vivo and in vitro. cAMP substantially increases the formation of the NFY.p300 complex. Taken together these data provide a general model of cAMP induction of non-CRE-containing promoters and suggest that the NFY-B.p300 complex is located at the 5' end of the promoter and the NFY-B.C. TFIIB on the 3' end toward the transcription start site.

Binding Sites↗
PubMed1998

P/CAF/p300 complex binds the promoter for the heavy subunit of ferritin and contributes to its tissue-specific expression.

We analysed the role of the nuclear protein P/CAF in regulating the transcription of the gene for human heavy (H) ferritin in given cell types. P/CAF is a histone acetylase, recruited to specific promoters via interaction with the co-activator molecule p300/CREB-binding protein (CBP). Histone acetylation promoted by P/CAF destabilizes the nucleosome structure, thus contributing to activation of transcription. The transcription of the H ferritin gene is regulated by the transcription factor B-box-binding factor (Bbf), which bridges RNA polymerase II via p300/CBP. Northern blot analyses of RNA species from various human tissues and cell lines demonstrate that the H ferritin gene is expressed at high levels in cells containing high levels of the P/CAF transcript. Moreover, transient overexpression of P/CAF in cells constitutively expressing low levels of this protein activates transcription driven by the region of the H promoter interacting with Bbf. The involvement of p300/CBP in the possible P/CAF-mediated regulation of H promoter was also explored by evaluating the phenomenon in the presence of the oncoprotein E1A. The results of these experiments demonstrate that P/CAF activates the H promoter also in the presence of limited amounts of p300/CBP. We argue that P/CAF is a component of the basal transcription apparatus of the H ferritin gene and that the relative amounts of the P/CAF protein in different cell types could account for the cell-specific control of the H ferritin gene transcription.

Acetyltransferases↗
PubMed1998

A new p21waf1/cip1 isoform is an early event of cell response to oxidative stress.

p21waf1/cip1 mRNA and protein accumulate in intact cells exposed to oxidizing agents through a p53-independent, MAPK-dependent mechanism. Treatment with oxidizing agents also yields a second form of this protein (FM p21), characterized by a faster migration on SDS-PAGE. This phenomenon depends on the modification of intracellular redox conditions induced by diethylmaleate, a glutathione-depleting agent, being prevented by the pretreatment with the glutathione precursor N-acetylcysteine. The appearance of this FM p21 form is very early, being observed 5 min after exposure to diethylmaleate, long before the already observed accumulation of p21 induced by oxidative stress. Furthermore, experiments with dominant negative mutants of MEK demonstrate that, in contrast with that observed for the oxidative stress-induced accumulation of p21 mRNA and protein, the appearance of FM p21 form is not dependent from the activation of the MAPK pathway. It was previously observed (Tchou et al, 1996) that in some lung carcinoma cells long exposure to high doses of phorbol esters also induces the appearance of a faster-migrating p21 electrophoretic band and it was suggested that this could result from a different phosphorylation or from a proteolytic processing at the C-terminus of the protein. The latter is not the case for the diethylmaleate-induced FM p21 whose C-terminus is intact, as demonstrated by the expression of a C-terminus tagged p21 cDNA. On the contrary, the observed migration shift seems to be dependent on the hypophosphorylation of the protein; in fact, a pretreatment of cells with okadaic acid, an inhibitor of (serine/threonine) phosphatases, inhibits the oxidation-dependent appearance of the FM p21 and the block of protein synthesis, caused by cycloeximide, does not affect the appearance of FM p21, that thus could derive from the dephosphorylation of preexisting protein.

Animals↗
PubMed1997

DNA-binding protein Pur alpha and transcription factor YY1 function as transcription activators of the neuron-specific FE65 gene promoter.

Fe65 is an adaptor protein that interacts with the Alzheimer beta-amyloid precursor protein and is expressed mainly in the neurons of several regions of the nervous system. The FE65 gene has a TATA-less promoter that drives an efficient transcription in cells showing a neuronal phenotype, whereas its efficiency is poor in non-neuronal cells. A short sequence encompassing the transcription start site contains sufficient information to drive the transcription in neuronal cells but not in non-neural cells. Electrophoretic mobility-shift assays performed with rat brain nuclear extracts showed that three major DNA-protein complexes, named BI, BII and BIII, are formed by the FE65 minimal promoter. The proteins present in complexes BI and BII were purified from bovine brain; internal microsequencing of the purified proteins demonstrated that they corresponded to the previously isolated single-stranded-DNA-binding protein Pur alpha, abundantly expressed in the brain. In Chinese hamster ovary (CHO) cells, where the efficiency of FE65 promoter is very low, transient expression of Pur alpha increased the transcription efficiency of the FE65 minimal promoter. By using oligonucleotide competition and a specific antibody we demonstrated that the transcription factor YY1 is responsible for the formation of complex BIII. Also in this case, the transient expression of the YY1 cDNA in CHO cells resulted in an increased transcription from the FE65 minimal promoter. The absence of any co-operative effect when CHO cells were co-transfected with both YY1 and Pur alpha cDNA species suggests that two different transcription regulatory mechanisms could have a role in the regulation of the FE65 gene.

Animals↗
PubMed1997

Okadaic acid stimulates H ferritin transcription in HeLa cells by increasing the interaction between the p300 CO-activator molecule and the transcription factor Bbf.

The transcription of the human H ferritin gene is regulated by a transcription factor, called Bbf, which binds an enhancer element located in the -100/+1 region of the H promoter. To evaluate a possible role of Bbf phosphorylation on the promoter efficiency, we exposed HeLa cells to the phosphatase inhibitor okadaic acid (OA). The okadaic acid treatment increased about 4-fold the transcription driven by the -100/+1 region of the H promoter. However, the DNA binding activity of Bbf was not modified by OA, as assessed by EMSA. Immunoprecipitation experiments demonstrated that the OA-treatment stimulates and/or stabilizes the complex between Bbf and the nuclear protein p300, most probably by inducing the phosphorylation state of the complex. Bbf depends on the p300 molecule to trigger RNA polymerase II and thus transcription of the H ferritin gene.

Cyclic AMP Response Element-Binding Protein A↗
PubMed1997

A common mechanism underlying the E1A repression and the cAMP stimulation of the H ferritin transcription.

Transcription of the H ferritin gene in vivo is stimulated by cAMP and repressed by the E1A oncoprotein. We report here the identification of the cis-element in the human promoter responsive to both cAMP- and E1A-mediated signals. This promoter region is included between positions -62 to -45 and binds a approximate 120-kDa transcription factor called Bbf. Bbf forms a complex in vivo with the coactivator molecules p300 and CBP. Recombinant E1A protein reduces the formation of these complexes. In vivo overexpression of p300 in HeLa cells reverses the E1A-mediated inhibition of the ferritin promoter transcription driven by Bbf. These data suggest the existence of a common mechanism for the cAMP activation and the E1A-mediated repression of H ferritin transcription.

Adenovirus E1A Proteins↗
PubMed1997

Redox-mediated regulation of p21(waf1/cip1) expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway.

p21(waf1/cip1) gene expression is induced by DNA damage in cells with wild-type p53 and contributes to the arrest of cell growth. It was demonstrated that under many experimental conditions, including oxidative stress, p21(waf1/cip1) expression can be induced through p53-independent pathways. Since most of these experimental conditions induce the phosphorylation of mitogen-activated protein kinase (MAPK) and thus its activation, we evaluated p21(waf1/cip1) mRNA levels in cells exposed to an oxidative stress, induced by diethylmaleate (Et2Mal), and in which the MAPK pathway was blocked. The expression of a dominant-negative mutant of MEK, the MAPK kinase that phosphorylates and activates MAPK, and of a dominant-negative [Asn17]Ras mutant prevented the Et2Mal-induced accumulation of p21(waf1/cip1) mRNA. Similarly, the expression of MEK- and of [Asn17]Ras mutants decreased the 12-O-tetradecanoyl-phorbol 13-acetate (TPA)-mediated p21(waf1/cip1) induction. Furthermore, TPA-induced and serum-induced p21(waf1/cip1) mRNA accumulation was blocked by pretreating the cells with the antioxidant compound N-acetylcysteine, suggesting that oxidative stress is involved in these responses. p21(waf1/cip1) mRNA levels reached a maximum within 2 h of adding Et2Mal or TPA; however, the rate of transcription from a p21(waf1/cip1)-promoter construct did not increase during this period. In contrast, cells treated with actinomycin D show an increase of p21(waf1/cip1) mRNA stability after Et2Mal treatment. This result suggests that the increase in p21(waf1/cip1) mRNA at early times results from post-transcriptional regulatory events. Longer exposure to TPA may activate p21(waf1/cip1) gene transcription through an Sp1-dependent mechanism, while Et2Mal treatment gradually inhibits p21(waf1/cip1) gene transcription through oxidative changes that affect Sp1 binding to DNA.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress.

Incubating human cells in diethylmaleate (DEM) depletes the intracellular pool of reduced glutathione (GSH) and increases the concentration of oxidative free radicals. We found that DEM-induced oxidative stress reduced the ability of p53 to bind its consensus recognition sequence and to activate transcription of a p53-specific reporter gene. Nevertheless, DEM treatment induced expression of WAF1/CIP1 but not GADD45 mRNA. The fact that N-acetylcysteine, a precursor of GSH that blocks oxidative stress, prevented WAF1/CIP1 induction by DEM suggests that WAF1/CIP1 induction probably was a consequence of the ability of DEM to reduce intracellular GSH levels. DEM induced WAF1/CIP1 expression in Saos-2 and T98G cells, both of which lack functional p53 protein. DEM treatment did not produce an increase in membrane-associated protein kinase C, but ERK2, a mitogen-activated protein kinase, was phosphorylated in a manner consistent with ERK2 activation. DEM treatment also produced a dose-dependent delay in cell cycle progression, which at low concentrations (0.25 mM) consisted of a G2/M arrest and at higher concentrations (1 mM) also involved G1 and S phase delays. Our results indicate that oxidative stress induces WAF1/CIP1 expression and arrests cell cycle progression through a mechanism that is independent of p53. This mechanism may provide for cell cycle checkpoint control under conditions that inactivate p53.

Animals↗
PubMed1995

Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf.

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

Base Sequence↗
PubMed1995

Negative and positive elements in the promoter region of the human apoferritin L gene.

We have characterized the promoter of the human gene coding for the apoferritin L subunit. Transient transfections of 5' and 3' deletion mutants indicate that the efficiency of the L promoter depends on both negative and positive cis-elements, located upstream and downstream of the transcription start point. DNaseI footprinting analysis of this DNA region revealed the presence of five protected segments. The most upstream one (element 1) corresponds to the negative cis-element and is recognized by factor(s) sharing a GC-sequence specificity. Three positive elements are in the region upstream of the start of transcription; a fifth positive cis-element (element 5) is localized in the first exon of the L gene.

Apoferritins↗
PubMed1995

Differentially expressed mRNAs as a consequence of oxidative stress in intact cells.

Intracellular redox conditions influence the activity of several transcription factors leading to a modulation of the expression of the genes controlled by these factors. We examined the changes in cell transcription patterns after oxidative stress induced by diethylmaleate (DEM). Using the differential display technique we identified several differentially expressed sequence tags, four of which are identical or highly homologous to sequences contained in the human cDNAs encoding vimentin, c-fos, cytochrome oxidase IV and ribosomal protein L4; another one corresponds to a transcript of the mitochondrial genome of unknown function. The remaining five cDNAs are not recorded in any sequence data bank. One of these, named Rox3, lights up two mRNA species of approximately 3400 and 3600 bp, significantly increased after treatment with DEM or with other oxidizing agents. This increase appears precociously after exposure to DEM and it is completely prevented by pretreatment with N-acetylcysteine. The Rox3 fragment was used to screen a cDNA library; one fully sequenced clone showed 100% homology with the putative human guanine nucleotide regulatory protein nep1.

Cell Line↗
PubMed1995

DNA binding activity of the glucocorticoid receptor is sensitive to redox changes in intact cells.

The effect of changes of redox conditions on glucocorticoid receptor (GR) activity in intact cells has been studied using two approaches. One was to evaluate the GR-DNA binding in extracts of COS2 cells transiently overexpressing GR and in which reactive oxygen intermediates (ROI) accumulate as a consequence of glutathione (GSH) depletion. GR-DNA binding was significantly decreased in COS2 cells treated with diethylmaleate (DEM), which causes GSH depletion by forming GSH-DEM complexes. A similar effect was observed for Sp1, another Zn-finger transcription factor, whereas no difference was observed for the C/EBP transcription factor, which is known to be unaffected by redox changes in vitro. N-Acetylcysteine (NAC), which counteracts the effects of DEM by increasing GSH biosynthesis, prevents the decrease of GR-DNA binding in cells treated with DEM. The GR-DNA binding efficiency was similarly decreased using extracts from H2O2-treated COS2 cells and from COS2 cells treated with buthionine sulphoximine, which causes GSH depletion via a mechanism different from that of DEM. The other approach was to evaluate the efficiency of a GR-regulated promoter under different redox conditions. In HeLa cells, transfected with a plasmid containing the CAT gene under the control of the glucocorticoid responsive element (GRE) within the mouse mammary tumor virus promoter, and treated with dexamethasone to activate GR, exposure to DEM significantly impaired the activation of CAT gene expression induced by dexamethasone. Also in this case NAC treatment inhibited the effects of DEM.

Base Sequence↗