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V De Laurenzi

Publications and source records attributed to V De Laurenzi.

At least 19 recordsLinked to original sources
PubMed2006

FLASH is an essential component of Cajal bodies.

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.

Animals↗
PubMed2006

FLASH is required for histone transcription and S-phase progression.

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1beta-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxia-telangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression.

Animals↗
PubMed2006

Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

Epidermal development requires the transcription factor p63, as p63-/- mice are born dead, without skin. The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (deltaNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63alpha and/or deltaNp63alpha under the K5 promoter into p63-/- mice by in vivo genetic complementation. Whereas p63-/- and p63-/-;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63-/-;deltaN mice showed significant epidermal basal layer formation. Double TAp63alpha/deltaNp63alpha complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, deltaNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layer's genes (Ets-1, K1, transglutaminases, involucrin). Therefore, the two p63 isoforms appear to play distinct cooperative roles in epidermal formation.

Animals↗
PubMed2002

'Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease.

By crossing Huntington's disease (HD) R6/1 transgenic mice with 'tissue' transglutaminase (TG2) knock-out mice, we have demonstrated that this multifunctional enzyme plays an important role in the neuronal death characterising this disorder in vivo. In fact, a large reduction in cell death is observed in R6/1, TG2(-/-) compared with R6/1 transgenic mice. In addition, we have shown that the formation of neuronal intranuclear inclusions (NII) is potentiated in absence of the 'tissue' transglutaminase. These phenomena are paralleled by a significant improvement both in motor performances and survival of R6/1, TG2(-/-) versus R6/1 mice. Taken together these findings suggest an important role for tissue transglutaminase in the regulation of neuronal cell death occurring in Huntington's disease.

Animals↗
PubMed2001

Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53.

Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the N-terminal transactivation domain (Delta Np73) was described in mice. In this study, we report the cloning and characterisation of the human Delta Np73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human Delta Np73 lacks the transactivation domain and starts with an alternative exon (exon 3'). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, Delta Np73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the Delta Np73 is strongly up-regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of Delta Np73 is exerted through a p53 responsive element located on the Delta N promoter. Expression of Delta Np73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of Delta Np73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of Delta Np73 in primary tumours would clarify this issue.

Amino Acid Sequence↗
PubMed2001

Gene disruption of tissue transglutaminase.

Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either epsilon-(gamma-glutamyl)lysine or N(1), N(8)-(gamma-glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, we prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis. TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2(-/-) mice. PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity. TGase 2(-/-) mice showed no major developmental abnormalities, and histological examination of the major organs appeared normal. Induction of apoptosis ex vivo in TGase 2(-/-) thymocytes (by CD95, dexamethasone, etoposide, and H(2)O(2)) and in vitro on TGase 2(-/-) mouse embryonal fibroblasts (by retinoids, UV, and H(2)O(2)) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.

Animals↗
PubMed2000

p63 and p73 transactivate differentiation gene promoters in human keratinocytes.

p53 and its two homologues, p73 and p63, share considerable structural similarities, an ability to interact between themselves and to transactivate the same promoters, including for example p21. Furthermore, p73 can induce cell death via its interaction with c-Abl. In contrast, p63 has been demonstrated to be essential for limb and skin formation. We evaluated the expression of p63 and p73 in differentiating human keratinocytes in vitro. Skin biopsy and primary cultures of normal human epidermal keratinocytes (NHEK) express both p73 and p63. NHEK induced to differentiate in vitro by high calcium exposure show induction of p73 delta and downregulation of all isoforms of p63. This latter gene is predominantly expressed in its transcriptionally inactive form, DeltaNp63. We further evaluated the effect of either p73s or p63 transfected in either NHEK or transformed human keratinocytes (HaCat cells). p73 gamma, delta, and p63 were able to transactivate the promoters of loricrin and involucrin in both NHEK and HaCat cells. These results suggest the involvement of both p73 and p63 genes in keratinocyte terminal differentiation.

Calcium↗
PubMed2000

Induction of neuronal differentiation by p73 in a neuroblastoma cell line.

The p53-related p73 and p63 genes encode proteins that share considerable structural and functional homology with p53. Despite similarities, their deletion in mice has different outcomes, implying that the three genes may play distinct roles in vivo. Here we show that endogenous p73 levels increase in neuroblastoma cells induced to differentiate by retinoic acid and that exogenously expressed p73, but not p53, is sufficient to induce both morphological (neurite outgrowth) and biochemical (expression of neurofilaments and neural cell adhesion molecule (N-CAM); down-regulation of N-MYC and up-regulation of pRB) markers of neuronal differentiation. This activity is shared, to different extents, by all p73 isoforms, whereas the transcriptionally inactive mutants of p73 isoforms are ineffective. Conversely, blockage of endogenous p73 isoforms with a dominant negative p73 results in the abrogation of retinoid-induced N-CAM promoter-driven transcription. Our results indicate that the p73 isoforms activate a pathway that is not shared by p53 and that is required for neuroblastoma cell differentiation in vitro.

Animals↗
PubMed2000

An SRY-negative XX male with Huriez syndrome.

This report studies a 42-year-old 46,XX patient affected by palmoplantar keratoderma. clinically classified as Huriez syndrome. The patient showed a male phenotype with apparently normal male features including testicular development. Cytogenetic and chromosomal painting analysis excluded the presence of translocation of the Y chromosome. PCR analysis of genomic DNA failed to detect the presence of the testis-determining gene, SRY. The presence of other Y-chromosome genes, known to be involved in testicular maturation and spermatogenesis, has also been analyzed. The data suggest that the sex reversal in this 46,XX male patient is due to a defect on a yet unidentified autosomal or X-linked sex-determining gene. The relationship between the sex reversion and the presence of sclerotylosis is discussed.

Adult↗
PubMed2000

A mutation in the V1 domain of K16 is responsible for unilateral palmoplantar verrucous nevus.

Palmoplantar keratodermas are a group of heterogeneous diseases characterized by thickening, and marked hyperkeratosis, of the epidermis of the palms and soles. Palmoplantar keratodermas can be divided into four major classes: diffuse, focal, punctate, and palmoplantar ectodermal dysplasias. All forms are genetic diseases inherited as autosomal dominant disorders. We studied a patient exhibiting a localized thickening of the skin in parts of the right palm and the right sole, following Blaschko's lines, that does not fit into any classes already described. We sequenced the keratin 16 cDNA derived from skin biopsy material from affected and non affected palms. The keratin 16 cDNA sequence from lesional epidermis showed a 12 base pair deletion (309-320del), which deletes codons 104-107. The mutation is predicted to delete four amino acids, GGFA, from the V1 domain of the keratin 16 polypeptide, close to the 1A domain. Full-length keratin 16 cDNA sequence derived from the unaffected palm was completely normal, consistent with a postzygotic mutation as is suggested by the mosaicism observed. We defined this new clinical entity, "unilateral palmoplantar verrucous nevus", rather than localized or focal epidermolytic palmoplantar keratodermas, as the lesions are present only on one side of the body and follow Blaschko's lines. This study is a report of a mosaic mutation in keratin 16 and also the association of a mutation in the V1 domain of a type I keratin associated with a human disease.

Adolescent↗
PubMed2000

Evolution of functions within the p53/p63/p73 family.

Even though the tumor suppressor gene p53 is highly important in human cancer, as indicated by the fact that it is mutated in about 50% of cases, up to a few years ago no similar proteins had been identified. Recently, two p53 homologues have been identified, p73 and p63, with high amino acid identity suggesting similar functions. Indeed, like p53, p73 as well (i) can bind mdmX, mdm2, p300/CAF and adenovirus E4-orf6 proteins, (ii) can trigger several promoters including p21, bax, mdm2, gadd45, cyclin G, IGFBP3, 14-3-3 sigma, (iii) is able to trigger cell death, (iv) is involved in the DNA damage response, although through a different pathway. Here we analyze the data present in the literature in search of diverging pathways among the p53, p63, p73 family. Both p63 and p73 present two significant structural peculiarities: the presence of an extended non-conserved C-terminus containing a sterile alpha motive (SAM), typical of developmental proteins, and the presence of number of different splicing isoforms differing in the N-terminus or in the absence of the transactivation domain (delta N forms), acting as dominant negative. The mouse knockout of p63 and p73, unlike the ones for p53, shows developmental abnormalities; p63 and p73 are rarely mutated in human cancers; both genes are regulated in different differentiation models. This strongly suggests the involvement of p63 and p73 in development. A picture is emerging showing a gradient of function among p53, p73, p63 ranging from tumor suppression to development.

Animals↗
PubMed2000

The p53/p63/p73 family of transcription factors: overlapping and distinct functions.

The p53 gene is the most frequently mutated gene in human cancer. The identification of two homologues, p63 and p73, revealed that p53 is a member of a family of related transcription factors. Given that they share amino acid sequence identity reaching 63% in the DNA-binding domain, p53, p63 and p73 should have redundant functions in the regulation of gene expression. Indeed, p73 can activate p53-regulated genes and suppress growth or induce apoptosis. Moreover, p53 and p73 are both induced by DNA damage - albeit through distinct mechanisms. Other evidence, however, suggests that p63 and p73 are important for regulation of normal development. An extended C-terminal region, not found in p53, is alternatively spliced in p63 and p73. Within this C-terminal extension is a sterile &agr; motif (SAM) previously found in other proteins that regulate development. The p63-deficient mice showed developmental abnormalities. Interestingly, the human p63 gene is mutated in children who have the disease Ectrodactyly, Ectodermal dysplasia and facial Clefts (EEC) syndrome, and the disease phenotype is similar to the one of p63-deficient mice. The p63 and p73 genes are rarely mutated in human cancer, although p73 loss is observed in neuroblastoma and a subtype of T-cell lymphoma. p53, p63 and p73 appear to have overlapping and distinct functions: p53 regulates the stress response to suppress tumors; p63 is essential for ectoderm development; and p73 might regulate both the stress response and development. Because p53 and p73 are linked to different upstream pathways, this family of transcription factors might regulate a common set of genes in response to different extracellular signals and developmental cues.

Animals↗
PubMed1999

Structure, function and regulation of p63 and p73.

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers.1 p53 is a sequence-specific transcription factor and plays a critical role in activating the expression of genes involved in cell cycle arrest or apoptosis under conditions of genotoxic stress.2,3 For over two decades, p53 was thought to be the only gene of its kind in the vertebrate genomes. This strong conviction, which was widely accepted in the p53 field, has now been proven to be incorrect. Two genes, referred to as p63 and p73, have been found to encode proteins that share a significant amino-acid identity in the transactivation domain, the DNA binding domain, and the oligomerization domain with p53. In the short period since their cloning, a number of investigators have reported on the structure, the function and the regulation of p63 and p73. This review summarizes the current information on the p63 and the p73 genes, with a focus on the differences between the three members in this newly defined p53-gene family.

Alternative Splicing↗
PubMed1998

Two new p73 splice variants, gamma and delta, with different transcriptional activity.

p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, alpha, or a shorter beta mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, gamma (splicing out exon 11) and delta (splicing out exons 11, 12, and 13). Both gamma and delta p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73gamma nor p73delta interact with p53, whereas p73gamma showed strong interactions with all p73 isoforms, and p73delta binds efficiently p73alpha and p73gamma but only weakly p73beta. At the functional level, p73gamma is significantly less efficient in activating transcription of the p21(Waf1/Cip1) promoter than p53 or p73beta, whereas the effect of p73delta is intermediate and comparable to that of p73alpha. The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21(Waf1/Cip1) promoter: p73beta is the most efficient in inhibiting colony formation, whereas p73gamma is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.

Alternative Splicing↗