Biomedical subjects

Alberto Gulino

Publications and source records attributed to Alberto Gulino.

53 records · Page 3Linked to original sources

PubMed2003

New mutations and protein variants of NBS1 are identified in cancer cell lines.

Alterations of the NBS1 gene are responsible for Nijmegen breakage syndrome (NBS), which is characterized by chromosomal instability, radiosensitivity, and cancer predisposition. NBS1 protein (Nibrin) is part of a molecular complex (NBS1- MRE11A-RAD50) that is functionally involved in DNA double-strand-break repair. Defects in recombination or in repair mechanisms at the level of DNA breakage can lead to chromosomal aberrations, genetic instability, as well as cancer predisposition syndromes (i.e., NBS, ataxia-telangiectasia, Bloom syndrome). In this study, we examined 20 cancer cell lines to evaluate the potential involvement of NBS1 in tumoral pathogenesis. Three different mutations, generating truncated or aberrant NBS1 transcripts, were identified at the level of NBS1 mRNA. In addition, two shorter NBS1 protein variants were detected in two cell lines. These data suggest a possible involvement of NBS1 in tumor development.

Adenocarcinoma↗

PubMed2003

EGF- and cell-cycle-regulated STAG1/PMEPA1/ERG1.2 belongs to a conserved gene family and is overexpressed and amplified in breast and ovarian cancer.

The abnormal activation of the epidermal growth factor (EGF) pathway is one of the most common findings in human cancer, and a number of molecular devices of laboratory and clinical relevance have been designed to block this transduction pathway. Because of the large number of cellular events that might be regulated through the activation of the four EGF receptor family members, it is possible that screening methodologies for the identification of new molecular targets working downstream of these pathways may provide new tools for cancer diagnosis and potentially prevention and therapy. In searching for EGF target genes, we have identified ERG1.2, the mouse homolog of the solid tumor-associated gene STAG1. Both in humans and in mice, it belongs to a new gene family that can give origin to several protein isoforms through alternative splicing and/or multiple translation starts. Sequence analysis and experimental data suggest that ERG1.2 is likely to function as a membrane-bound protein interacting with downstream signaling molecules through WW- and SH3-binding domains. ERG1.2 is a cell-cycle-regulated gene, and both ERG1.2 and STAG1 are induced by EGF and other growth factors at the transcript and protein levels. Finally, we have demonstrated that, besides prostate cancer and renal cell carcinoma, STAG1 was also overexpressed in breast and ovarian cancer cell lines and in breast primary tumors. Although in most cases STAG1 overexpression is probably due to the abnormal activation of the EGF pathway, we have also demonstrated genetic amplification and rearrangement of its locus in one breast cancer cell line and one primary ovarian cancer, suggesting that STAG1 might be a direct molecular target in the carcinogenetic process. Thus its overexpression might be regarded not only as a tumor marker but also as a potentially pathogenetic event.

Amino Acid Sequence↗

PubMed2003

Notch3, another Notch in T cell development.

Different members of the Notch family have been described to play a critical role in T cell lineage commitment and T cell development and functions. Nevertheless, whether they act as redundant molecules, by affecting the same molecular mechanisms, or play distinct roles in T cell differentiation and/or functions is not clear. Altered Notch3 signaling impairs the developmentally regulated interplay between pre-TCR and NF-kappaB signaling and allows the disruption of early thymocyte differentiation and the development of T cell leukemia, thus identifying the crucial role of Notch3 receptor in the coordination of T cell differentiation and growth control.

Animals↗

PubMed2003

Notch signalling at the crossroads of T cell development and leukemogenesis.

Members of the Notch family (e.g. Notch1 and Notch3) have been recently described to play a critical role in T cell development and their constitutive activation has been related to T cell leukaemia in both animal models and human disease. Nevertheless, whether they act as redundant molecules, by affecting the same molecular mechanisms, or play distinct roles in T cell differentiation and/or leukemogenesis is not clear. Altered Notch signalling impairs the developmentally-regulated interplay between pre-TCR signalling, NFkappaB and E2A activities, thus identifying the crucial role of Notch receptors at the cross-roads of disrupted lymphoid differentiation and neoplastic transformation.

Animals↗

PubMed2003

Notch, a unifying target in T-cell acute lymphoblastic leukemia?

The expression of both Notch3 and pre-T-cell-receptor (pre-TCR) invariant chain appears to be a common feature of all T-cell acute lymphoblastic leukemias (T-ALL). Notch genes, and other genes that are dysregulated in some T-ALL subgroups, encode factors that play a crucial role in both T-cell development and leukemogenesis. A complex network of signals, involving Notchs, pre-TCR, nuclear factor kappaB and E2A, appears to be responsible for the leukemogenesis process. Thus, T-ALL is a paradigm for developmental pathways that underlie the pathogenesis of this disease.

Animals↗

PubMed2003

Differential regulation of E2F1 apoptotic target genes in response to DNA damage.

E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3-5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation.

Acetylation↗

PubMed2002

REN: a novel, developmentally regulated gene that promotes neural cell differentiation.

Expansion and fate choice of pluripotent stem cells along the neuroectodermal lineage is regulated by a number of signals, including EGF, retinoic acid, and NGF, which also control the proliferation and differentiation of central nervous system (CNS) and peripheral nervous system (PNS) neural progenitor cells. We report here the identification of a novel gene, REN, upregulated by neurogenic signals (retinoic acid, EGF, and NGF) in pluripotent embryonal stem (ES) cells and neural progenitor cell lines in association with neurotypic differentiation. Consistent with a role in neural promotion, REN overexpression induced neuronal differentiation as well as growth arrest and p27Kip1 expression in CNS and PNS neural progenitor cell lines, and its inhibition impaired retinoic acid induction of neurogenin-1 and NeuroD expression. REN expression is developmentally regulated, initially detected in the neural fold epithelium of the mouse embryo during gastrulation, and subsequently throughout the ventral neural tube, the outer layer of the ventricular encephalic neuroepithelium and in neural crest derivatives including dorsal root ganglia. We propose that REN represents a novel component of the neurogenic signaling cascade induced by retinoic acid, EGF, and NGF, and is both a marker and a regulator of neuronal differentiation.

Amino Acid Sequence↗

PubMed2002

Combined expression of pTalpha and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis.

Notch receptors are conserved regulators of cell fate and have been implicated in the regulation of T cell differentiation and lymphomagenesis. However, neither the generality of Notch involvement in leukemia, nor the molecules with which Notch may interact have been clarified. Recently, we showed that transgenic mice expressing the constitutively active intracellular domain of Notch3 in thymocytes and T cells developed early and aggressive T cell neoplasias. Although primarily splenic, the tumors sustained features of immature thymocytes, including expression of pTalpha, a defining component of the pre T cell receptor, known to be a potent signaling complex provoking thymocyte survival, proliferation, and activation. Thus, enforced expression of Notch3, which is ordinarily down-regulated as thymocytes mature, may sustain pre T cell receptor expression, causing dysregulated hyperplasia. This hypothesis has been successfully tested in this article by the observation that deletion of pTalpha in Notch3 transgenic mice abrogates tumor development, indicating a crucial role for pTalpha in T cell leukemogenesis. Parallel observations were made in humans, in that all T cell acute lymphoblastic leukemias examined showed expression of Notch3 and of the Notch target gene HES-1, as well as of pTalpha a and b transcripts, whereas the expression of all these genes was dramatically reduced or absent in remission. Together, these results suggest that the combined expression of Notch3 and pTalpha sustains T cell leukemogenesis and may represent pathognomonic molecular features of human T-ALL.

Animals↗

PubMed2002

Human MRE11 is inactivated in mismatch repair-deficient cancers.

Mutations of the ATM and NBS1 genes are responsible for the inherited Ataxia-Telangiectasia and Nijmegen Breakage Syndrome, both of which are associated with a predisposition to cancer. A related syndrome, the Ataxia-Telangiectasia-like disorder, is due to mutations of the MRE11 gene. However, the role of this gene in cancer development has not been established. Here we describe an often homozygous mutation of the poly(T)11 repeat within human MRE11 intron 4 that leads to aberrant splicing, impairment of wild-type MRE11 expression and generation of a truncated protein. This mutation is present in mismatch repair-deficient, but not proficient, colorectal cancer cell lines and primary tumours and is associated with reduced expression of the MRE11--NBS1--RAD50 complex, an impaired S-phase checkpoint and abrogation of MRE11 and NBS1 ionizing radiation-induced nuclear foci. Our findings identify MRE11 as a novel and major target for inactivation in mismatch repair-defective cells and suggest its impairment may contribute to the development of colorectal cancer.

Amino Acid Sequence↗

PubMed2002

retSDR1, a short-chain retinol dehydrogenase/reductase, is retinoic acid-inducible and frequently deleted in human neuroblastoma cell lines.

Vitamin A is required for a number of developmental processes and for the homeostatic maintenance of several adult differentiated tissues and organs. In human neuroblastoma (NB) cells as well as some other tumor types, pharmacological doses of retinoids are able to control growth and induce differentiation in vitro and in vivo. In a search for new genes that are regulated by retinoids and that contribute to the biological effects retinoids have on NB cells, we have isolated five differentially expressed transcripts. Here we report on the characterization of one of them (DD83.1) in NB cell lines. DD83.1 is identical to the human retSDR1, a short chain dehydrogenase/reductase that is thought to regenerate retinol from retinal in the visual cycle. Its expression is strongly, but differently, regulated by retinoids in NB cell lines, and it is widely expressed in human tissues, which suggests that it is involved in a more general retinol metabolic pathway. Both the retinoic acid-dependent and the exogenous expression of retSDR1 in SK-N-AS cells induce the accumulation of retinyl esters, which indicates that it is involved in generating storage forms of retinol in tissues exposed to physiological retinol concentrations. We also show that the human retSDR1 gene, which maps on chromosome 1p36.1, is contained in the DNA fragment deleted in many NB cell lines bearing MYCN amplification but is conserved in a cell line with a small 1p deletion and normal MYCN. Our observations suggest that retSDR1 is a novel regulator of vitamin A metabolism and that its frequent deletion in NB cells bearing MYCN amplification could compromise the sensitivity of those cells to retinol, thereby contributing to cancer development and progression.

Alcohol Oxidoreductases↗

PubMed2002

High sensitivity of detection of TP53 somatic mutations by fluorescence-assisted mismatch analysis.

We analyzed exons 5-9 of the TP53 gene in 41 breast cancers using direct sequencing, PCR-SSCP (single-strand conformation polymorphism), and fluorescence-assisted mismatch analysis (FAMA), to test the level of specificity and sensitivity of each method. A major issue for the correct detection of TP53 somatic mutations in primary tumors is often represented by the large amount of normal DNA, which can cause excessive dilution of the mutant allele, with consequent possible false-negative results. High sensitivity upon dilution of the mutant allele has been demonstrated for FAMA, a method based on the chemical cleavage of a mismatch within heteroduplex DNA molecules. Exons 5-9 of the TP53 gene were analyzed by FAMA using only two long bifluorescent fragments. Differences in sensitivity, accuracy, and specificity were observed among the above-mentioned procedures. Thirteen of the 41 samples (31.7%) revealed TP53 genetic alterations by automated sequencing, 19 samples (46.3%) were positive for SSCP, whereas 14 samples (34%) showed variants detectable by FAMA. Seven samples were positive in SSCP, but negative in both FAMA and sequencing assays; however, 2 SSCP-negative samples revealed evident signals by FAMA, indicating the presence of TP53 mutations. One of the latter samples showed the alteration by sequence analysis, whereas the other failed to reveal the mutation signal even by sequencing, as a consequence of the very small amount of the mutant allele resulting from the excess of contaminating normal DNA. Our results show that FAMA may represent a suitable and accurate assay for the routine diagnosis of TP53 somatic mutations in DNAs from solid tumor biopsies.

Base Pair Mismatch↗

PubMed2002

Prenatal diagnosis of a rhodopsin mutation using chemical cleavage of the mismatch.

OBJECTIVE: Mutations of the rhodopsin gene are responsible for autosomal dominant or recessive retinitis pigmentosa (RP). The present study reports the first prenatal diagnosis performed on chorionic villi biopsy of a pregnant woman affected by a severe form of autosomal dominant transmitted RP, due to the Arg135Trp substitution. METHODS: The rhodopsin gene was analysed by automated direct sequencing and, for the first time, by fluorescence-assisted mismatch analysis (FAMA). The latter is an inexpensive, rapid and particularly sensitive method, based on the chemical cleavage of the mismatch in heteroduplex DNA molecules marked with strand-specific fluorophores. RESULTS: FAMA is a feasible procedure for prenatal molecular diagnosis of rhodopsin mutations. The redundancy of signals obtained by FAMA and its sensitivity make it suitable for identifying exactly the position of the mutation and the nucleotide substitution. CONCLUSIONS: An association is proposed between FAMA and automated direct sequencing procedures, in order to achieve optimal results in terms of reliability for prenatal diagnosis of rhodopsin mutations.

Adult↗

PubMed2002

Experimental parkinsonism modulates multiple genes involved in the transduction of dopaminergic signals in the striatum.

The irreversible loss of the dopamine-mediated control of striatal function is considered the functional substrate of the motor symptoms of Parkinson's disease. This pathological event causes a complex rearrangement of neuronal activity which involves specific dopamine-regulated cellular functions and, secondarily, several other cellular properties and transmitter systems. In the present study, we applied recently developed cDNA microarray technology to investigate the genetic correlates of the alterations produced by 6-hydroxydopamine-induced dopamine denervation in the nucleus striatum. We found that chronic dopamine denervation caused the modulation of 50 different genes involved in several cellular functions. In particular, products of the genes modulated by this experimental manipulation are involved both in the intracellular transduction of dopamine signal and in the regulation of glutamate transmission in striatal neurons, providing some information on the possible neuronal events which lead to the reorganization of glutamate transmission in the striatum of parkinsonian rats.

Animals↗

PubMed2002

Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen.

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.

Angiostatins↗

PubMed2002

Tissue plasminogen activator is required for corticostriatal long-term potentiation.

Several experimental data indicate that tissue plasminogen activator (tPA) is involved in memory formation and synaptic plasticity in different brain areas. In the attempt to highlight the role of this serine protease in striatal neuron activity, mice lacking tPA have been used for electrophysiological, immunohistochemical and Western blot experiments. Disruption of tPA gene prevented corticostriatal long-term potentiation, an NMDA-dependent form of synaptic plasticity requiring the stimulation of both dopamine and acetylcholine receptors. Spontaneous and evoked glutamatergic transmission was intact in the striatum of tPA-deficient mice, as was the nigrostriatal dopamine innervation and the expression of dopamine D1 receptors. Conversely, the sensitivity of striatal cholinergic interneurons to dopamine D1 receptor stimulation was lost in these mutants, suggesting that tPA facilitates long-term potentiation (LTP) induction in the striatum by favouring the D1 receptor-mediated excitation of acetylcholine-producing interneurons. The demonstration that tPA ablation interferes with the induction of corticostriatal LTP and with the dopamine receptor-mediated control of cholinergic interneurons might help to explain the altered striatum-dependent learning deficits observed in tPA-deficient mice and provides new insights into the molecular mechanisms underlying synaptic plasticity in the striatum.

Animals↗

PubMed2002

All-trans-retinoic acid induces nuclear factor kappaB activation and matrix metalloproteinase-9 expression and enhances basement membrane invasivity of differentiation-resistant human SK-N-BE 9N neuroblastoma Cells.

A comparison between retinoic acid (RA) differentiation-resistant and differentiation-sensitive SK-N-BE neuroblastoma (NB) cell lines revealed an association between resistance to differentiation, exhibited by N-myc stable transfected SK-N-BE 9N cells, with sensitivity to RA induction of p50/p65 nuclear factor kappaB (NF-kappaB) transcription factor activity and induction of matrix metalloproteinase (MMP)-9 expression leading to enhanced invasive behavior in vitro. These effects were not observed in differentiation-sensitive parental SK-N-BE or control-transfected SK-N-BE 2N counterparts. RA activated a MMP-9 promoter reporter gene construct in SK-N-BE 9N but not parental SK-N-BE or SK-N-BE 2N cells through a NF-kappaB element (-600) in association with enhanced p50 mRNA expression, reduced cytoplasmic inhibitor of nuclear factor kappaBalpha protein levels, and the induction of nuclear p50/p65 containing MMP-9 NF-kappaB site binding activity. RA activation of the MMP-9 promoter was inhibited by transient overexpression of a dominant-negative inhibitor of nuclear factor kappaBalpha protein and stimulated by transient p50 but not p65 overexpression in the absence of RA. A limited, nonessential function for activator protein 1 (-74), Ets (-540), and SP1 (-560) elements within the MMP-9 promoter was revealed by point mutation but was not associated with changes in the binding or position of complexes constitutive to differentiation-sensitive or -resistant cells. Our data indicates that in this model of NB resistance to differentiation that results from uncoupled RA regulation of N-myc expression, RA stimulates malignant NB cell behavior by inducing nuclear NF-kappaB transcription factor activity, which in turn induces MMP-9 expression and stimulation of basement membrane invasive capacity involving MMP-9 activity.

Antineoplastic Agents↗

PubMedDate not supplied

P16 hypermethylation contributes to the characterization of gene inactivation profiles in primary gastric cancer.

The objective of this study was to investigate the contribution of p16 inactivation in gastric cancer and to compare it with p53. A cohort of 34 primary GCs were analyzed for p16 mutations and transcriptional silencing of the gene due to hypermethylation of the promoter. SSCP analysis and direct sequencing of exons 1 and 2 of the p16 gene were performed to detect any structural alterations. The methylation specific PCR (MSP) assay was applied to reveal hypermethylation of the 'CpG' island in the regulatory region using specific primer pairs for methylated and unmethylated nucleotides after a chemical reaction converting cytosines into uracile when unmethylated. SSCP and direct sequencing analysis did not detect any p16 mutations. The MSP assay showed 4 MSP(+) variants (11.8%). Three MSP(+) were stage III-IV disease and 1 MSP(+) was detected in an early stage disease (IB). All MSP(+) were diffuse type adenocarcinomas. The MSP(+) samples were different from previously reported samples harboring p53 mutations in the same cohort. These data increase the number of gastric cancers showing alterations of either p53 or p16 to 29.4% (10/34). Functional inactivation by hypermethylation of the p16 locus and p53 mutations could play a significant, complementary role in the pathogenesis of sporadic gastric cancer.

Adenocarcinoma↗