Biomedical subjects

G Benvenuto

Publications and source records attributed to G Benvenuto.

At least 19 recordsLinked to original sources

PubMed2001

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth.

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.

Binding Sites↗

PubMed2000

The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.

We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and hamartin is known to bind tuberin, these results suggested that hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to hamartin is not ubiquitinated. Co-expression of tuberin stabilized hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize hamartin. We conclude that hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.

Animals↗

PubMed2000

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains.

Amino Acid Sequence↗

PubMed2000

Transient detection of plasma HIV-1 RNA during postexposure prophylaxis.

Transient plasma human immunodeficiency virus (HIV) copies were detected by nucleic-acid sequence-based amplification during combination antiretroviral prophylaxis in a healthcare worker who reported a percutaneous injury from a stylet and who remained HIV-antibody-negative. An HIV-specific T-helper response, assessed by interleukin-2 production, was observed when tested at 13 months following the exposure.

Adult↗

PubMed2000

Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.

We have found that EGF-R expression is associated with the development of the Schwann cell-derived tumors characteristic of neurofibromatosis type 1 (NF1) and in animal models of this disease. This is surprising, because Schwann cells normally lack EGF-R and respond to ligands other than EGF. Nevertheless, immunoblotting, Northern analysis, and immunohistochemistry revealed that each of 3 malignant peripheral nerve sheath tumor (MPNST) cell lines from NF1 patients expressed the EGF-R, as did 7 of 7 other primary MPNSTs, a non-NF1 MPNST cell line, and the S100(+) cells from each of 9 benign neurofibromas. Furthermore, transformed derivatives of Schwann cells from NF1(-/-) mouse embryos also expressed the EGF-R. All of the cells or cell lines expressing EGF-R responded to EGF by activation of downstream signaling pathways. Thus, EGF-R expression may play an important role in NF1 tumorigenesis and Schwann cell transformation. Consistent with this hypothesis, growth of NF1 MPNST lines and the transformed NF1(-/-) mouse embryo Schwann cells was greatly stimulated by EGF in vitro and could be blocked by agents that antagonize EGF-R function.

Animals↗

PubMed2000

Galectin-1 gene expression and methylation state in human T leukemia cell lines.

Galectin-1 has been demonstrated to be a mediator of T-cell apoptosis acting on activated T-cells and, in a selective manner, on different T leukemia cell lines. Here we show that the sensitivity to galectin-1 is associated with repression of the endogenous galectin-1 gene whereas non-sensitive cells express high levels of galectin-1. Repression of galectin-1 gene in sensitive cells is associated with hyper-methylation of the promoter region. Transient treatment of non-expressing cells with the demethylating agent 5-azacytidine led to irreversible demethylation and subsequent reactivation of galectin-1 gene.

Antimetabolites, Antineoplastic↗

PubMed1999

galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas.

Galectin-1 and galectin-3 are galactoside-binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional-cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin-1 mRNA levels were highly increased in most high-grade tumors compared with normal bladder or low-grade tumors. Western blot and immuno-histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin-1 in tumors. Galectin-3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade.

Adult↗

PubMed1999

Control of galectin gene expression.

In this review we summarize the available information on the expression of mammalian galectins in normal and transformed cells. From all these studies it is apparent that each cell might express most of galectins; yet, during development or in various differentiation stages or under different physiological or pathological conditions, one or more galectins are preferentially expressed in each cell type. This implies a fine control of gene expression and suggests that such control should be coordinated. Nevertheless, to date very few studies have been performed on the mechanisms responsible for the regulation of galectin genes. We review the current knowledge on galectin promoter function. We believe that this area of galectin research will expand rapidly in the near future.

Animals↗

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↗

PubMed1997

Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility.

The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.

3T3 Cells↗

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

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↗

PubMed1995

Expression of galectin-1 in normal human thyroid gland and in differentiated and poorly differentiated thyroid tumors.

We previously reported that galectin-1 gene expression increases up to 100-fold in oncogene-transformed rat thyroid cells compared with their normal counterparts and that the relative mRNA levels correlate with the degree of malignancy. In the present study we investigated whether galectin-1 is differentially expressed in human thyroid neoplasms, which range from well-differentiated tumors to undifferentiated anaplastic carcinomas. We analyzed 74 human thyroid specimens of neoplastic, hyperproliferative and normal tissues and several tumor cell lines. Galectin-1 mRNA and protein levels were higher in 6 thyroid carcinoma-derived cell lines than in normal thyroid primary cultures and adenoma cells. Galectin-1 mRNA levels increased in 28/40 papillary carcinomas and in 6/7 anaplastic carcinomas compared with normal or hyperplastic thyroid. Conversely, galectin-1 expression was unaffected in follicular carcinomas and benign adenomas. Immunohistochemical analysis of normal thyroid and papillary carcinoma sections revealed a higher content of galectin-1 protein in neoplastic follicular cells than in normal cells.

Base Sequence↗

PubMed1994

Expression of the soluble lectin L-14 gene is induced by TSH in thyroid cells and suppressed by retinoic acid in transformed neural cells.

Expression of the soluble lectin L-14 is low in normal and very high in transformed thyroid cells. We show that L-14 gene expression is transiently induced upon thyrotropin stimulation of normal quiescent FRTL-5 rat thyroid cells. Permanent activation of L-14 gene expression is obtained in the same cells infected with a wild-type and a temperature sensitive mutant of Kirsten murine sarcoma virus, both at the permissive and non permissive temperature for transformation. We also find that L-14 mRNA is undetectable in rat brain but is abundant in rat oligodendrocytes precursors transformed by polyoma middle T oncogene. Retinoic acid treatment of these transformed cells leads to acquisition of a differentiated phenotype accompanied by a 30-fold decrease of L-14 mRNA levels. Removal of retinoic acid restores both the transformed undifferentiated phenotype and high L-14 expression. Taken together these results indicate that growth stimulation and induction of cell differentiation are accompanied by strong modulation of L-14 gene expression.

Animals↗

PubMed1994

Activation of the galectin-1 (L-14-I) gene from nonexpressing differentiated cells by fusion with undifferentiated and tumorigenic cells.

Expression of the galectin-1 (L-14-I) gene, elevated in most differentiated and transformed cell lines, has been studied in cell hybrid systems. Fusion of L-14-I nonproducing rat liver differentiated FAO cells with dedifferentiated rat liver BRL3A cells leads to extinction of liver-specific gene expression while L-14-I mRNA levels remain high. Interspecific hybrids produced by fusion of tumorigenic human osteosarcoma 143TK- with FAO cells show loss of both differentiated functions and tumorigenic phenotype and activation of the FAO L-14-I alleles. Increased expression of rat L-14-I alleles was also observed in human osteosarcoma x rat thyroid cells transient heterokaryons. The data presented here show that expression of the L-14-I gene is subject to dominant positive control and that it correlates with loss of differentiation-specific functions, but it is independent from tumorigenicity. L-14-I activation in FAO cells is achieved by treatment with 5-azacytidine. This result suggests that DNA demethylation is responsible or a prerequisite for L-14-I activation in hybrids.

Alleles↗

PubMed1975

[Diagnosis and early treatment of juvenile delinquency].

Longitudinal research has shown that social failure to adjust can be prevented by early removal of ambiental factors encouraging pathological development of the Ego, and the detection and treatment of psychological tendencies towards the acquisition of deviant stimuli.

Adolescent↗

PubMed1975

[Environmental factors in juvenile delinquency].

Experimental data are cited for the proposition that the complicated aspects of juvenile delinquency can only be understood and explained by adopting a simultaneous psychological and sociological approach. It is also shown that the manifestations of delinquency, though not its actual presence, may be influenced by sex and a depressed city or rural background. Environmental factors serving as stimulating features and hereditary (i.e. predisposing) factors undoubtedly contribute to the formation of the Ego. The former, however, are elaborated by their receipient and are not sufficient to explain a certain type of behaviour. The influence of cultural models should not be underestimated. These, where predominant, tend to render normative what may be considered as deviant.

Adolescent↗