Biomedical subjects

Alberto Gulino

Publications and source records attributed to Alberto Gulino.

At least 37 records · Page 2Linked to original sources

PubMed2005

Constitutively active Notch1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways.

Notch signaling plays a key role in cell-fate determination and differentiation in different organisms and cell types. Several reports suggest that Notch signaling may be involved in neoplastic transformation. However, in primary keratinocytes, Notch1 can function as a tumor suppressor. Similarly, in HPV-positive cervical cancer cells, constitutively active Notch1 signaling was found to cause growth suppression. Activated Notch1 in these cells represses viral E6/E7 expression through AP-1 down-modulation, resulting in increased p53 expression and a block of pRb hyperphosphorylation. Here we show that in cervical cancer cell lines in which Notch1 ability to repress AP-1 activity is impaired, Notch1-enforced expression elicits an alternative pathway leading to growth arrest. Indeed, activated Notch1 signaling suppresses activity of the helix-loop-helix transcription factor E47, via ERK1/2 activation, resulting in inhibition of cell cycle progression. Moreover, we found that RBP-Jkappa-dependent Notch signaling is specifically repressed in cervical cancer cells and this repression could provide one such mechanism that needs to be activated for cervical carcinogenesis. Finally, we show that inhibition of endogenous Notch1 signaling, although results in a proliferative advantage, sensitizes cervical cancer cell lines to drug-induced apoptosis. Together, our results provide novel molecular insights into Notch1-dependent growth inhibitory effects, counteracting the transforming potential of HPV.

Cell Line, Tumor↗

PubMed2005

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Animals↗

PubMed2005

TrkAIII. A novel hypoxia-regulated alternative TrkA splice variant of potential physiological and pathological importance.

Nerve growth factor receptor TrkA is critical for development and maturation of central and peripheral nervous systems, regulating proliferation, differentiation and apoptosis. In cancer, TrkA frequently exhibits suppressor activity in nonmutated form and oncogenic activity upon mutation. Our identification of a novel hypoxia-regulated alternative TrkAIII splice variant, expressed by neural crest-derived neuroblastic tumors, that exhibits neuroblastoma tumor promoting activity, adds significantly to our understanding of potential TrkA involvement in cancer. Our observation that hypoxia, which characterizes the tumor micro-environment, stimulates alternative TrkAIII splicing, provides a way by which TrkA tumor suppressing signals may convert to tumor promoting signals during progression and is consistent with conservation and pathological subversion by neural crest-derived neuroblastic tumors of a mechanism of potential physiological importance to normal neural stem/neural crest progenitors.

Alternative Splicing↗

PubMed2005

Alternative TrkAIII splicing: a potential regulated tumor-promoting switch and therapeutic target in neuroblastoma.

An association between elevated tyrosine kinase receptor (Trk)-A expression and better prognosis; the absence of mutation-activated TrkA oncogenes; the induction of apoptosis, growth arrest, morphological differentiation and inhibition of xenograft growth; and angiogenesis by TrkA gene transduction, provide the basis for the current concept of an exclusively tumor-suppressor role for TrkA in the aggressive pediatric tumor, neuroblastoma. This concept, however, has recently been challenged by the discovery of a novel hypoxia-regulated alternative TrkAIII splice variant, initial data for which suggest predominant expression in advanced-stage neuroblastoma. TrkAIII exhibits neuroblastoma xenograft tumor-promoting activity associated with the induction of a more angiogenic and stress-resistant neuroblastoma phenotype and antagonises nerve growth factor/TrkAI antioncogenic signaling. In this short review, the authors integrate this novel information into a modified concept that places alternative TrkA splicing as a potential pivotal regulator of neuroblastoma behavior and identifies the TrkAIII alternative splice variant as a potential biomarker of patient prognosis and novel therapeutic target.

Alternative Splicing↗

PubMed2004

Regulation of iodide uptake and sodium/iodide symporter expression in the mcf-7 human breast cancer cell line.

Sodium/iodide symporter (NIS) expression has recently been described in human breast cancer, with emphasis on its potential exploitation for the treatment of these tumors with radioiodine. In this study, we analyzed the regulation of NIS expression and function in the MCF-7 human breast cancer cell line. Cell exposure to insulin, IGF-I, IGF-II, or prolactin induced significant increases in 125I uptake and the expression of both NIS mRNA and NIS protein. The latter increases were evident after 6 and 12 h of hormonal stimulation, respectively. In immunocytochemistry studies, NIS was detected mainly in the plasma membrane of MCF-7 cells. A low but significant increase in iodide uptake was produced by treatment with activators of the adenylyl cyclase (cAMP) or protein kinase C pathways. Our study demonstrates that: 1) MCF-7 breast cancer cells are capable of active iodide transport that can be stimulated by insulin, IGF-I, IGF-II, or prolactin; 2) both NIS transcript and protein are expressed in these cells, and this expression is also hormonally stimulated; and 3) MCF-7 iodide transport and NIS expression may be influenced by the activation of cAMP or protein kinase C-dependent signaling. These findings increase our understanding of the molecular mechanisms that regulate NIS expression in breast cancer cells, information that is fundamental for future research aimed at the development of targeted radioiodide treatment for this type of cancer.

Breast Neoplasms↗

PubMed2004

Chromosome 17p deletion in human medulloblastoma: a missing checkpoint in the Hedgehog pathway.

Although deregulation of Hedgehog signalling is considered to play a crucial oncogenic role and commonly occurrs in medulloblastoma, genetic lesions in components of this pathway are observed in a minority of cases. The recent identification of a novel putative tumor suppressor (REN(KCTD11)) on chromosome 17p13.2, a region most frequently lost in human medulloblastoma, highlights the role of allelic deletion of the gene in this brain malignancy, leading to the loss of growth inhibitory activity via suppression of Gli-dependent activation of Hedgehog target genes. The presence on 17p13 of another tumor suppressor gene (p53) whose inactivation cooperates with Hedgehog pathway for medulloblastoma formation, suggests that 17p deletion unveils haploinsufficiency conditions leading to abrogation of either direct and indirect checkpoints of Hedgehog signalling in cancer.

Cerebellar Neoplasms↗

PubMed2004

MRE11 expression is impaired in gastric cancer with microsatellite instability.

Gastric carcinomas (GCs) with high-level microsatellite instability (MSI-H) are characterized by widespread mutations at coding and non-coding mononucleotide repeats. Deletions at coding mononucleotide tracts are predicted to cause frameshift mutations and alter normal protein functions. Mutations affecting non-coding mononucleotide repeats may lead to functional consequences if they occur in gene regulatory regions. To investigate whether mutations in non-coding polypyrimidine tracts within cancer-related genes may contribute to the phenotype of MSI-H GCs, we analysed the poly(T)11 tract constituting an accessory splicing signal within the intron 4 of the MRE11 gene. Mutations at the intronic MRE11 poly(T)11 were evaluated by PCR-based assay in 27 MSI-H, 22 MSI-low and 29 MSI-negative GCs derived from a well-characterized series of GCs identified in a high-risk area in Tuscany, Central Italy. Deletion of 2 and 1 bp at the MRE11poly(T)11 were identified in 33 and 48% MSI-H GCs, respectively. Biallelic mutations were frequently observed (77%) in GCs harbouring 2 bp deletions. The presence of MRE11poly(T)11 2 bp deletion was associated with a totally absent or strongly reduced MRE11 immunostaining (P < 0.001) and with a positive GC family history (P = 0.046). Immunoblotting assays confirmed the absence of MRE11 expression in GCs with a 2 bp deletion. The relatively high frequency of the MRE11poly(T)11 mutations, the occurrence of biallelic mutations and the evidence of loss of protein expression indicate MRE11 as novel mutational target in MSI-H GC. Overall, our results indicate that MSI-associated mutations occurring in non-coding repeats may affect protein expression in MSI-H GC.

Aged↗

PubMed2004

REN(KCTD11) is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma.

Hedgehog signaling is suggested to be a major oncogenic pathway in medulloblastoma, which arises from aberrant development of cerebellar granule progenitors. Allelic loss of chromosome 17p has also been described as the most frequent genetic defect in this human neoplasia. This observation raises the question of a possible interplay between 17p deletion and the Hedgehog tumorigenic pathway. Here, we identify the human orthologue of mouse REN(KCTD11), previously reported to be expressed in differentiating and low proliferating neuroblasts. Human REN(KCTD11) maps to 17p13.2 and displays allelic deletion as well as significantly reduced expression in medulloblastoma. REN(KCTD11) inhibits medulloblastoma cell proliferation and colony formation in vitro and suppresses xenograft tumor growth in vivo. REN(KCTD11) seems to inhibit medulloblastoma growth by negatively regulating the Hedgehog pathway because it antagonizes the Gli-mediated transactivation of Hedgehog target genes, by affecting Gli1 nuclear transfer, and its growth inhibitory activity is impaired by Gli1 inactivation. Therefore, we identify REN(KCTD11) as a suppressor of Hedgehog signaling and suggest that its inactivation might lead to a deregulation of the tumor-promoting Hedgehog pathway in medulloblastoma.

Alleles↗

PubMed2004

Specific role for p300/CREB-binding protein-associated factor activity in E2F1 stabilization in response to DNA damage.

E2F1, a member of the E2F family of transcription factors, plays a pivotal role in controlling both physiological cell-cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. In response to genotoxic stresses, E2F1 is stabilized by signals that include ATM-dependent phosphorylation. We recently demonstrated that DNA damage induces also E2F1 acetylation, which is required for its recruitment onto apoptotic gene promoters. Here we show that E2F1 is stabilized in response to doxorubicin and cisplatin treatments even in the absence of either ATM-dependent phosphorylation or p53 and cAbl, two major transducers of DNA damage signaling. We found that acetylation of E2F1 is, instead, required to stabilize the protein in response to doxorubicin. Finally, we report that the formation of E2F1-p300/CREB-binding protein-associated factor (P/CAF) complexes is preferentially induced in doxorubicin-treated cells, and that P/CAF acetyltransferase (HAT), but not p300 HAT activity, is required for a significant E2F1 stabilization and accumulation. Our results unveil a differential role of P/CAF and p300 in acetylation-induced stabilization of E2F1, thus supporting a specific role for P/CAF HAT activity in E2F1-dependent apoptosis in response to DNA damage.

Acetylation↗

PubMed2004

Mutations of an intronic repeat induce impaired MRE11 expression in primary human cancer with microsatellite instability.

Frequent mutations of coding nucleotide repeats are thought to contribute significantly to carcinogenesis associated with microsatellite instability (MSI). We have shown that shortening of the poly(T)11 within the polypyrimidine stretch/accessory splicing signal of human MRE11 leads to the reduced expression and functional impairment of the MRE11/NBS1/RAD50 complex. This mutation was selectively found in mismatch repair (MMR) defective cell lines and potentially identifies MRE11 as a novel target for MSI. Here, we examined 70 microsatellite unstable primary human cancers and we report that MRE11 mutations occur in 83.7 and 50% of the colorectal and endometrial cancers, respectively. In the colorectal cancer series, mutated MRE11 is more frequently associated with advanced age at diagnosis and A/B stages. Biallelic mutations were present in 38.8% of the cases and more frequently associated with lower (G1/G2) grade tumors. Impaired MRE11 expression was prevalent in primary colorectal tumors with larger and biallelic shortening of the poly(T)11. Immunohistochemistry confirmed the impaired MRE11 expression and revealed NBS1-defective expression in MRE11 mutated cancers. Together with the observation that perturbation of the MRE11/NBS1/RAD50 complex predisposes to cancer, our work highlights MRE11 as a new common target in the MMR deficient tumorigenesis and suggests its role in colorectal carcinogenesis.

Aged↗

PubMed2004

Ligand-regulated association of ErbB-4 to the transcriptional co-activator YAP65 controls transcription at the nuclear level.

It has been proposed that ligand-dependent Regulated Intramembrane Proteolysis (RIP) of ErbB-4 receptors generates 80 kDa Intra-Cellular Domains (E4.ICDs) that relocate to the nuclear compartments where they implement the signaling abilities of the ErbB-4 receptors. The E4.ICD may directly regulate gene transcription or, in an alternative scenario, the tyrosine kinase activity of E4.ICDs may target proteins involved in transcriptional regulation upon its relocation into the nucleus. We have identified the transcriptional coactivator YAP65, here referred as YAP (Yes Associated Protein), as binding partner of ErbB-4 in a two hybrid screening in yeast. Interaction between YAP and ErbB-4 occurs via the WW domain of YAP and the PPPPY at positions 1297-1301 and the PPPAY at positions 1052-1056 of the amino acid sequence of the Cyt-1 isoform of ErbB-4. Stechiometry of binding is regulated by the ligand-dependent phosphorylation of Tyr 1056 in the PPPAYTPM module that function as "biochemical switch" to decrease the association of YAP to ErbB-4. In principle, this novel interaction highlights new mechanisms of signaling propagation from the ErbB-4 receptors, offering supporting evidences that the E4.ICDs forms released following ligand-receptor engagement may recruit YAP and relocate to the nucleus to implement or regulate transcription.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma.

BACKGROUND: The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines. RESULTS: Low levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082). Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1-2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus. CONCLUSIONS: Suppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene.

Journal Article↗

PubMed2004

TrkA alternative splicing: a regulated tumor-promoting switch in human neuroblastoma.

We identify a novel alternative TrkA splice variant, TrkAIII, with deletion of exons 6, 7, and 9 and functional extracellular IG-C1 and N-glycosylation domains, that exhibits expression restricted to undifferentiated early neural progenitors, human neuroblastomas (NBs), and a subset of other neural crest-derived tumors. This NGF-unresponsive isoform is oncogenic in NIH3T3 cells and promotes tumorigenic NB cell behavior in vitro and in vivo (cell survival, xenograft growth, angiogenesis) resulting from spontaneous tyrosine kinase activity and IP3K/Akt/NF-kappaB but not Ras/MAPK signaling. TrkAIII antagonizes NGF/TrkAI signaling, which is responsible for NB growth arrest and differentiation through Ras/MAPK, and its expression is promoted by hypoxia at the expense of NGF-responsive receptors, providing a mechanism for converting NGF/TrkA/Ras/MAPK antioncogenic signals to TrkAIII/IP3K/Akt/NF-kappaB tumor-promoting signals during tumor progression.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Inhibition of mitochondrial complex II alters striatal expression of genes involved in glutamatergic and dopaminergic signaling: possible implications for Huntington's disease.

Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by motor abnormalities and cognitive impairment. The irreversible succinate dehydrogenase (SD) inhibitor 3-nitropropionic acid (3NP) causes neurodegeration in the striatum resembling HD when administered to rodents or primates. Using corticostriatal brain slice preparations, we analyzed the pattern of gene expression following 3NP application utilizing cDNA microarrays. Acute 3NP treatment modulates the expression of several genes involved in dopaminergic and glutamatergic signaling in corticostriatal brain slices, and unbalances the downstream serine/threonine protein kinase and phosphatase network affecting the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Our data provide new information about the molecular events possibly underlying neurodegeneration induced by this mitochondrial toxin.

Animals↗

PubMed2003

Thioredoxin inhibits microvascular endothelial capillary tubule formation.

Thioredoxin (Trx) inhibited human HMEC-1 dermal microvascular endothelial cell capillary tubule forming capacity in a Matrigel based assay in vitro. Inhibition of capillary tubule formation was Trx catalytic site and thioredoxin reductase (TrxR) dependent, mediated at the Matrigel matrix level, and associated with a shift from morphological differentiation to continuous proliferation, with enhanced cell spreading resulting in eventual monolayer formation. Soluble complex carbohydrates, which inhibited capillary tubule formation on Matrigel without induction of cell spreading or monolayer formation, failed to impair Trx promotion of cell spreading and mono-layer formation, suggesting a shift away from carbohydrate-mediated cell/matrix adhesive interactions. Laminin peptides YIGRS and SIKVAV, which impaired tubule formation on Matrigel without inducing cell spreading or monolayer formation, partially impaired cell spreading upon Trx-treated Matrigel without restoring tubule formation, consistent with a potential role for laminin in Trx-mediated effects. Trx reduced laminin and destabilised laminin/galectin-3 complexes within Matrigel. Native purified EHS Laminin (also containing galectin-3), but not recombinant galectin-3, restored HMEC-1 capillary tubule formation on Trx-treated Matrigel. These data highlight a novel deregulatory effect of extracellular Trx upon morphological capillary differentiation that appears to depend upon the reduction of laminin and destabilisation of its interaction with galectin-3, possibly leading to galectin-3 neutralisation that shifts cell/matrix adhesive interactions away from being carbohydrate mediated and results in loss of proliferation-inhibiting and differentiation promoting cues from this tumor basement membrane matrix.

Capillaries↗

PubMed2003

Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

Thymic-derived dysregulated tolerance has been suggested to occur in type 1 diabetes via impaired generation of CD4(+)CD25(+) T regulatory cells, leading to autoimmune beta cell destruction. In this study, we demonstrate that Notch3 expression is a characteristic feature of CD4(+)CD25(+) cells. Furthermore, streptozotocin-induced autoimmune diabetes fails to develop in transgenic mice carrying the constitutively active intracellular domain of Notch3 in thymocytes and T cells. The failure to develop the disease is associated with an increase of CD4(+)CD25(+) T regulatory cells, accumulating in lymphoid organs, in pancreas infiltrates and paralleled by increased expression of IL-4 and IL-10. Accordingly, CD4(+) T cells from Notch3-transgenic mice inhibit the development of hyperglycemia and insulitis when injected into streptozotocin-treated wild-type mice and display in vitro suppressive activity. These observations, therefore, suggest that Notch3-mediated events regulate the expansion and function of T regulatory cells, leading to protection from experimental autoimmune diabetes and identify the Notch pathway as a potential target for therapeutic intervention in type 1 diabetes.

Animals↗

PubMed2003

Pre-TCR-triggered ERK signalling-dependent downregulation of E2A activity in Notch3-induced T-cell lymphoma.

Notch and basic helix-loop-helix E2A pathways specify cell fate and regulate neoplastic transformation in a variety of cell types. Whereas Notch enhances tumorigenesis, E2A suppresses it. However, whether and how Notch and E2A interact functionally in an integrative mechanism for regulating neoplastic transformation remains to be understood. It has been shown that Notch3-induced T-cell leukaemia is abrogated by the inactivation of pTalpha/pre-T-cell antigen receptor (pre-TCR). We report here that Notch3-induced transcriptional activation of pTalpha/pre-TCR is responsible for the downregulation of E2A DNA binding and transcriptional activity. Further, the E2A messenger RNA and protein levels remain unaltered but the E2A inhibitor Id1 expression is augmented in thymocytes and T lymphoma cells derived from Notch3 transgenic mice. The increase in Id1 expression is achieved by pre-TCR-induced extracellular-signalling-regulated kinase 1/2. These observations support a model in which the upregulation of pre-TCR signalling seems to be the prerequi-site for Notch3-induced inhibition of E2A, thus leading to the development of lymphoma in Notch3 transgenic mice.

Animals↗

PubMed2003

Reconstitution of TIMP-2 expression in SH-SY5Y neuroblastoma cells by 5-azacytidine is mediated transcriptionally by NF-Y through an inverted CCAAT site.

Advanced stage neuroblastomas (NB) exhibit a tissue inhibitor of metalloproteinase (TIMP)-2/matrix metalloproteinase (MMP) imbalance, considered a prerequisite for MMP involvement in tumor progression in vivo. Human SH-SY5Y NB cells exhibit a similar TIMP-2/MMP imbalance that promotes in vitro invasive behavior that is inhibited by exogenous TIMP-2. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) redresses this TIMP-2/MMP imbalance, reconstituting TIMP-2 expression, without effecting that of MMP-2, by stimulating TIMP-2 transcription and inhibiting in vitro invasivity of SH-SY5Y cells. 5-AzaC stimulated transcription from a nonmethylated TIMP-2 promoter reporter gene construct consistent with regulation of a TIMP-2 transactivator. Promoter deletion and point-mutation analysis localized this effect to an inverted CCAAT element at position -73. This element bound specific complexes containing NF-YA and NF-YB proteins in SH-SY5Y nuclear extracts, the binding of which was augmented by 5-AzaC in association with enhanced levels of NF-YB protein and the function of which was confirmed by inhibition using dominant-negative NF-YA. The data highlight a novel indirect methylation-mediated mechanism for regulating the TIMP/MMP equilibrium in NB cells, involving repression of TIMP-2 relative to MMP-2 expression, dependent upon suboptimal NF-Y transcription factor function, which can be reversed by methyltransferase inhibition.

Antimetabolites, Antineoplastic↗