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Biomedical subjects

C B Bruni

Publications and source records attributed to C B Bruni.

At least 37 records · Page 2Linked to original sources
PubMed1998

Control of Rab5 and Rab7 expression by the isoprenoid pathway.

Rab proteins are small molecular mass GTP-ases involved in the regulation of vescicular transport. The ability of rab proteins to carry out their role in intracellular membrane traffic requires the post-translational attachment to their C-terminus of a geranylgeranyl group, an isoprenoid lipid moiety derived from mevalonate. Here we report that depletion of intracellular mevalonate by lovastatin in FRTL-5 thyroid cells specifically resulted in a four-fold increase of Rab5 and Rab7 protein levels. This increase was reversed within 4 h upon addition of mevalonate. Similarly lovastatin also induced, at same extent, mRNA levels. Lovastatin effect was not common to other prenylated proteins. Moreover incubation with cycloheximide abolished the observed increase in lovastatin treated cells, suggesting that the effect is mediated by newly synthesized protein. These findings demonstrate that Rab5 and Rab7 expression are regulated by the isoprenoid pathway.

Animals↗
PubMed1998

Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells.

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.

Adenocarcinoma↗
PubMed1998

Identification and characterization of a novel RING-finger gene (RNF4) mapping at 4p16.3.

We have isolated a new human RING-finger gene (RNF4) that encodes a 190-amino-acid protein. RNF4, in addition to the carboxyl-terminally located RING-finger motif, contains two putative nuclear localization signals and stretches of acidic amino acids that are similar to the activation domains of some transcription factors. RNF4 was expressed at low levels in all human tissues examined, with the notable exception of very high expression in the testis. The mouse homolog of RNF4 was abundantly expressed in embryonic tissues from the earliest days postgestation and exhibited a ubiquitous pattern of expression as assessed by in situ hybridization. We have mapped RNF4 to 4p16.3, a chromosome region associated with several genetic and neoplastic diseases. RNF4 spans 47 kb, is composed of eight exons, and maps immediately proximal to the anonymous locus D4S183, between the huntingtin (HD) and the fibroblast growth factor receptor 3 (FGFR3) genes.

Amino Acid Sequence↗
PubMed1998

High resolution methylation analysis of the galectin-1 gene promoter region in expressing and nonexpressing tissues.

We conducted by bisulfite genomic sequencing a high resolution study of the methylation of the galectin-1 gene in expressing and nonexpressing tissues. We show that: (i) hypomethylation of galectin-1 promoter correlates with expression; (ii) differences in methylation occur in a small region, which include a CpG cluster; (iii) the density of methyl-CpGs rather than site-specific methylation distinguishes the nonexpressing from the expressing alleles; (iv) the modification profiles in nonexpressing tissues are highly heterogeneous; (v) a single CpG within 1300 bp is always methylated both in expressing and nonexpressing tissues; (vi) these features are conserved in rat and mouse.

3T3 Cells↗
PubMed1998

Genetic mapping of the mouse Rab7 gene and pseudogene and of the human RAB7 homolog.

Rab proteins are small GTP-ases localized to distinct membrane compartments in eukaryotic cells and regulating specific steps of intracellular vesicular membrane traffic. The Rab7 protein is localized to the late endosomal compartment and controls late steps of endocytosis. We have isolated, by library screening, the 5' region, including the promoter, of the mouse Rab7 gene and a Rab7 pseudogene. We have mapped, by genetic linkage analysis, the mouse Rab7 gene on Chromosome (Chr) 6 and the Rab7-ps1 pseudogene on Chr 9, where the Rab7 gene has been previously reported to map. By radiation hybrid mapping, we have located the human RAB7 gene on Chr 3, in a region homologous to the mouse Chr 6, where the Rab7 gene maps.

Animals↗
PubMed1998

Effects of bicyclomycin on RNA- and ATP-binding activities of transcription termination factor Rho.

Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

Adenosine Triphosphate↗
PubMed1997

Relaxation of insulin-like growth factor-2 imprinting in rat cultured cells.

The parental-specific expression of the insulin-like growth factor-2 (Igf-2) and H19 genes was studied in rat fibroblast cells derived from a 3 day-old first-generation hybrid animal obtained by crossing Fisher and Wistar strains (F x W cells). Results showed that the reciprocal imprinting of the Igf-2 and H19 genes was conserved in the rat tissues and in the derived F x W cells when cultured with frequent transfer. Igf-2 and H19 gene expression was coordinately up-regulated upon reaching confluence, but Igf-2 RNA levels were further increased in a time-dependent manner and the repressed state of the maternal Igf-2 allele was progressively relaxed in cultures held in the confluent state and in the presence of low serum for more than 3 days. The active expression and relaxed imprinting status of the Igf-2 gene persisted over cell generations when the growth-constraining conditions were released by trypsinization and dilution. On the contrary, the imprinting of the H19 gene appeared to be unaffected by changes in growth conditions and its expression was down-regulated when the confluent cells were passaged. Methylation of the H19 promoter and Igf-2 coding regions was increased in the F x W cells extensively held under confluence and in the derived 'post-confluent' cultures. The heritable changes in the expression, and imprinting status of the Igf-2 and H19 genes observed in the F x W cells closely resembles events described in human embryonal cancers and cancer-predisposing syndromes. The occurrence of imprinting relaxation under strong growth-inhibitory conditions supports the hypothesis that it is an epigenetic change.

Animals↗
PubMed1997

Expression and parental imprinting of the H19 gene in human rhabdomyosarcoma.

The expression of Insulin-like Growth Factor 2 (IGF-2) and H19, two genes located on human chromosome 11p15 and provided with cell growth modulating activity, is regulated by parental imprinting, in that the activity of their alleles is dependent on the parental origin. Parental bias in the genetic alterations of chromosome 11p15 observed in several pediatric cancers suggests the involvement of imprinted genes in tumor development. We have previously reported that the number of functional IGF-2 alleles is frequently increased in rhabdomyosarcoma (RMS), as a consequence of either relaxation of imprinting (LOI) or gene duplication. Here we show that the expression of the H19 gene is significantly suppressed with respect to normal muscle tissue in 13 out of 15 rhabdomyosarcomas with embryonal histology (ERMS) and in three out of 11 rhabdomyosarcomas classified as alveolar subtype (ARMS). Since a growth-inhibitory activity has been found associated with the H19 gene, the extinction of its expression can contribute to RMS development. Parental imprinting of the H19 gene was found conserved in all informative RMSs, including those whose ICF-2 imprinting was relaxed, indicating that LOI is a gene-specific event. Seven ERMSs and one ARMS displaying low H19 RNA levels showed an underrepresentation of the expressed allele in their genotype. This result is consistent with the paternal imprinting of the H19 gene and with the preferential loss of the maternal 11p15 alleles in these neoplasms. Low H19 expression was also found in four out of eight RMSs retaining the heterozygosity at 11p15, but showing IGF-2 LOI. These findings suggest that the genetic and epigenetic alterations affecting chromosome 11p15 in a high number of RMSs cause deregulation of more than one imprinted gene, possibly affecting tumor growth, including the extinction of H19 expression and an increase in the number of active IGF-2 alleles.

Alleles↗
PubMed1997

Role of the small GTPase Rab7 in the late endocytic pathway.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

Animals↗
PubMed1996

Molecular cloning and expression analysis of the human Rab7 GTP-ase complementary deoxyribonucleic acid.

Rab7 is a small GTP-ase localized on late endosomes, which regulates late endocytic membrane traffic in mammalian cells. Moreover it has been shown that this protein has a fundamental role in the cellular vacuolation induced by the cytotoxin VacA of Helicobacter pylori. We report here for the first time the isolation of a cDNA encoding human Rab7 from a placenta cDNA library. The open reading frame for human Rab7 encodes a protein of 207 amino acids which exhibits high homology with the mouse, rat, and dog counterparts. Northern blot analysis of total RNAs isolated from different cell lines with a cDNA probe containing the entire open reading frame revealed two mRNA transcripts of 2.5 and 1.8 kilobases. The isolation of human Rab7 cDNA will allow further characterization of its function in normal and pathological states.

Amino Acid Sequence↗
PubMed1996

Constitutive insulin-like growth factor-II expression interferes with the enterocyte-like differentiation of CaCo-2 cells.

In this study we have examined the role of insulin-like growth factor-II (IGF-II) in the differentiation of the CaCo-2 human colon carcinoma cell line. We have shown previously that IGF-II is an autocrine growth factor for CaCo-2 cells. IGF-II expression is high in proliferating, undifferentiated CaCo-2 cells and markedly decreases when cells become confluent and start to differentiate. To evaluate whether differentiation of CaCo-2 cells depends on an IGF-II related pathway, we treated cells with a blocking antibody to the IGF-I receptor that mediates most IGF-II biological effects. Treatment of preconfluent CaCo-2 cells with this antibody decreased by 40% autonomous cell proliferation and induced differentiation as shown by an increase in sucrase isomaltase activity and apolipoprotein A-I (apoA-I) mRNA levels. To examine the significance of autocrine IGF-II production in CaCo-2 cell differentiation, we generated stable CaCo-2 cell lines that constitutively express rat IGF-II under the control of a Rous sarcoma virus promoter. Sustained expression of IGF-II resulted in: (a) increased proliferative rate; (b) high IGF-I receptor number, even after reaching confluence; (c) increased capability of anchorage-independent growth; (d) inhibition of the expression of apoA-I and SI mRNAs. Analysis of several independent IGF-II-transfected clones showed an inverse correlation between IGF-II mRNA levels and expression of the differentiation markers, the cells expressing the higher levels of the transfected IGF-II being the less differentiated ones. Our data suggest that perturbation of IGF-II-mediated cell proliferation interferes with the enterocyte-like differentiation pathway of CaCo-2 cells.

Apolipoprotein A-I↗
PubMed1996

Activation of fetal promoters of insulinlike growth factors II gene in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Increased prevalence of hepatitis C virus (HCV) infection has been found in patients with hepatocellular carcinoma (HCC). The expression of insulinlike growth factor II (IGF-II) has been linked to hepatocarcinogenesis in the experimental animal and in humans. Since reactivation of fetal IGF-II transcripts has been observed in human HCC, we have analyzed the levels of adult P1 and fetal P3 and P4 IGF-II promoter-derived transcripts in the liver of patients with HCV-related chronic active hepatitis (CAH), cirrhosis, and HCC by means of a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. Transcripts derived from adult P1 promoter were increasingly expressed from normals to patients with CAH and cirrhosis, but were undetectable in the tumorous area of 5 of 7 HCC patients and present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were expressed abundantly in most CAH and all cirrhotic patients. Transcripts from fetal P4 promoter were detected at high levels in 3 of 9 CAH patients and in the majority of cirrhotic patients. Increased expression of fetal promoter-derived transcripts was also found in the liver of HCC patients, although levels were lower than in cirrhosis. Also, the activity of fetal P3 and P4 promoters was higher in the nontumorous than in the tumorous area of the liver of HCC patients. The expression of IGF-II transcripts was correlated with the rate of cell mitotic activity by measuring the expression of the proliferating cell nuclear antigen (PCNA) gene. PCNA messenger RNA (mRNA) levels progressively increased from normals to CAH and to cirrhotic patients, and persisted at a high level in the tumorous and in the nontumorous area of HCC subjects, thus showing that the increase of IGF-II transcripts in CAH and cirrhosis is accompanied by an activation of cell mitosis in these samples. These data suggest that the activation of IGF-II gene expression from adult and fetal promoters may play a role in premalignant proliferation observed in HCV-related chronic liver disease.

Adult↗
PubMed1996

Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA.

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo. Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H. pylori strains or isogenic mutants defective in production of VacA, CagA, or both products. We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H. pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism. These findings demonstrate that, in addition to damaging the gastric mucosa, H. pylori products may also impair physiological processes required for mucosal repair.

Antigens, Bacterial↗
PubMed1996

Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region.

The galectin-1 gene is developmentally regulated gene whose activity is strongly modulated during cell differentiation and transformation. We have previously shown that galectin-1 promoter constructs are highly active when transiently transfected in cells both expressing and not expressing the endogenous gene and that the basal activity is determined by a small region encompassing the transcription start site (from positions -50 to +50). We have now investigated the role of DNA methylation in galectin-1 gene expression. Southern blot analysis with HpaII and MspI endonucleases and sodium bisulfite analysis of genomic DNA from expressing and nonexpressing cell lines and cell hybrids showed a close correlation between gene activity and demethylation of the 5' region of the galectin-1 gene. We found that the galectin-1 promoter region is fully methylated, at every CpG site on both strands, in nonexpressing differentiated rat liver (FAO) and thyroid (PC C13) cells and unmethylated in the expressing undifferentiated liver (BRL3A) and thyroid transformed (PC myc/raf) cell lines. In addition, reactivation of the silent FAO alleles in FAO-human osteosarcoma (143tk-) hybrid cells is accompanied by a complete demethylation of the promoter region. Finally, when galectin-1 chloramphenicol acetyltransferase (CAT) promoter constructs were methylated in vitro by SssI methylase at every cytosine residue of the CpG doublets and transfected into mouse fibroblasts, the transcription of the CAT reporter gene was strongly inhibited.

Animals↗
PubMed1995

Cloning and expression analysis of the murine Rab7 cDNA.

A cDNA clone coding for the Rab7 protein was isolated from an NIH3T3 cell line (mouse fibroblasts) cDNA library. Sequence analysis shows high homology to the rat and dog cDNAs. Northern blot analysis showed the presence of two messenger RNA that differ at the 3' untranslated region.

3T3 Cells↗
PubMed1995

Characterization and functional dissection of the galectin-1 gene promoter.

The galectin-1 gene encodes a beta-galactoside-binding protein whose overexpression is associated with neoplastic transformation and loss of differentiation. Transient transfection assays of a series of deletions constructs (pGAT) showed that the galectin-1 promoter is highly active in cells both expressing and non-expressing the endogenous gene, and that the basal activity is determined by sequences encompassing the transcription start site (-50/+50). Both an upstream (-50/-26) and a downstream position-dependent (+10/+50) cis-elements are necessary for efficient transcriptional activity and are able to bind nuclear proteins.

3T3 Cells↗