Biomedical subjects

Francesca Bernassola

Publications and source records attributed to Francesca Bernassola.

9 recordsLinked to original sources

PubMed2006

A cytoplasmic PML mutant inhibits p53 function.

The promyelocytic leukaemia gene (Pml) is a tumor suppressor identified in acute promyelocytic leukaemia (APL), where it is fused to RAR alpha gene as a result of the chromosomal translocation t(15;17). Pml encodes both nuclear and cytoplasmic isoforms. While nuclear PML has been intensively investigated, cytoplasmic PML proteins are less characterized. PML nuclear isoforms (nPML) are the essential components of subnuclear structures referred to as PML nuclear bodies (PML-NB). In response to cellular insults such as DNA damage and oncogenic activation, nPML modulates p53 activity through CBP-mediated acetylation and activates its pro-apoptotic and growth suppressive functions. Two missense mutations resulting in truncated PML cytoplasmic proteins (Mut PML) have been identified in aggressive APL cases. Here we report that cytoplasmic PML is able to induce the relocation of nPML to the cytoplasm, thus reducing the number of PML-NBs. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive, and apoptotic functions, thus suggesting that cytoplasmic expression of PML has an impact on survival through inhibition of nuclear PML. Overall our findings shed new light on the role of PML cytoplasmic proteins in the regulation of p53.

Apoptosis↗

PubMed2006

Cytoplasmic function of mutant promyelocytic leukemia (PML) and PML-retinoic acid receptor-alpha.

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) regulates major apoptotic and growth-suppressive pathways. In APL, PML is involved in a chromosomal translocation generating the PML-retinoic acid receptor-alpha (RARalpha) fusion protein. Two missense mutations in the remaining PML alleles have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis. Here we show that Mut PML associates with the cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacts with PML-RARalpha in PML-CB and potentiates PML-RARalpha-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilizes PML-RARalpha and inhibits differentiation induced by pharmacological doses of RA. A mutant form of PML-RARalpha that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we show that the bcr3 PML-RARalpha form is predominantly cytoplasmic and accumulates in PML-CBs. Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL.

Animals↗

PubMed2005

The promyelocytic leukaemia protein tumour suppressor functions as a transcriptional regulator of p63.

p63 plays unique developmental roles in epidermal morphogenesis, despite its structural similarity with p53. The p63 gene has two distinct promoters, coding for proteins containing an N-terminal transactivation domain (TA isoforms) and for proteins lacking this region (DeltaN isoforms). The full-length transcriptionally active TAp63 isoforms are capable of transactivating the majority of the p53 target promoters thus inducing cell cycle arrest and apoptosis. On the contrary, the DeltaNp63 isoforms seem to counteract the transactivation activities of p53 and TAp63 proteins, thus possibly conferring a proliferative advantage to cancer cells. However, the molecular mechanisms controlling the transcriptional activity of p63 remain largely unclear. Here we present data indicating that (i) the promyelocytic leukaemia protein (PML) physically interacts with p63, (ii) p63 is localized into the PML nuclear-bodies (PML-NBs) in vivo, and (iii) PML regulates p63 transcriptional activity. We show that the interaction of p63 with PML increases the levels of p63 in cultured cells as well as its ability to transactivate the p53-responsive elements of the GADD45, p21 and bax promoters. These data are consistent with a general role for PML as a functional modulator of all the p53 family members. Our findings strengthen the relevance of the cross talk between PML and the p53 family members, imply a new tumour suppressive function of PML and unveil a possible role for PML in epidermal morphogenesis and differentiation.

DNA-Binding Proteins↗

PubMed2005

Regulation of the p73 protein stability and degradation.

p73, a homologue to the tumor suppressor gene p53, is involved in tumorigenesis, though its specific role remains unclear. The gene has two distinct promoters which allow the formation of two protein isoforms with opposite effects: full-length transactivating (TA) p73 shows pro-apoptotic effects, while the shorter DeltaNp73, which lacks the N-terminal transactivating domain, has an evident anti-apoptotic function. Unlike p53, the p73 gene is rarely mutated in human cancers. However, alterations in the relative levels of TA and DeltaNp73 have been shown to correlate with prognosis in several human cancers, suggesting that the fine regulation of these two isoforms is of pivotal importance in controlling proliferation and cell death. Much effort is currently focused on the elucidation of the mechanisms that differentially control TA and DeltaNp73 activity and protein stability, a process complicated by the finding that both proteins are regulated by a similar suite of complex post-translational modifications that include ubiquitination, sequential phosphorylation, prolyl-isomerization, recruitment into the PML-nuclear body (PML-NB), and acetylation. Here we shall consider the main regulatory partners of p73, with particular attention to the recently discovered Itch- and Nedd8-mediated degradation pathways, along with the emerging roles of PML, p38 MAP kinase, Pin1, and p300 in p73 transcriptional activation, and possible mechanisms for the differential regulation of the TAp73 and DeltaNp73 isoforms.

Apoptosis↗

PubMed2004

Ubiquitin-dependent degradation of p73 is inhibited by PML.

p73 has been identified recently as a structural and functional homologue of the tumor suppressor p53. Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway. Furthermore, we show that the promyelocytic leukemia (PML) protein modulates p73 half-life by inhibiting its degradation in a PML-nuclear body (NB)-dependent manner. p38 mitogen-activated protein kinase-mediated phosphorylation of p73 is required for p73 recruitment into the PML-NB and subsequent PML-dependent p73 stabilization. We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels. As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells. Our findings demonstrate that PML plays a crucial role in modulating p73 function, thus providing further insights on the molecular network for tumor suppression.

Acetylation↗

PubMed2003

p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation.

p73, an important developmental gene, shares a high sequence homology with p53 and induces both G(1) cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human DeltaNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The DeltaNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.

Animals↗

PubMed2002

GADD153 and 12-lipoxygenase mediate fenretinide-induced apoptosis of neuroblastoma.

The synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with chemotherapeutic drugs used to treat neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). In an attempt to characterize the mechanism of action of fenretinide, cDNA array filters were screened to identify apoptotic genes regulated in response to treatment of SH-SY5Y cells with fenretinide. Expression of the stress-induced transcription factor, GADD153, was up-regulated at both the protein and mRNA levels in response to fenretinide. Overexpression of GADD153 increased apoptosis in the presence and absence of fenretinide, whereas reduced expression of GADD153 by expression of antisense DNA abrogated the response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists did not block the induction of GADD153 by fenretinide; conversely, the induction of GADD153 was blocked by antioxidants. Enzyme inhibitors were used to identify pathways mediating the ROS-dependent effects of fenretinide: inhibitors of phospholipase A(2) and lypoxygenases (LOX), and specific inhibitors of 12-LOX, but not 5-LOX or 15-LOX, inhibited the induction of ROS, apoptosis, and GADD153 in response to fenretinide. The inhibition of ROS and apoptosis was reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid (12-HpETE) and 12 (S)-hydroxyeicosatetraenoic acid (12-HETE). Fenretinide did not increase free arachidonic acid levels, but increased LOX activity without a detectable increase in 12-LOX protein. These results suggest that fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterized by a fenretinide-dependent increase in 12-LOX activity, leading to the induction of GADD153. The targeting of 12-LOX and/or GADD153 in neuroblastoma cells may thus present a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.

Antineoplastic Agents↗

PubMed2002

Osmotic resistance of high-density erythrocytes in transglutaminase 2-deficient mice.

Transglutaminase 2 (TGase 2) is a Ca(2+)-dependent enzyme responsible for the posttransttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Elevating the intracellular concentration of Ca(2+)-ions in human erythrocytes leads to the formation of cytoskeletal and cytoplasmic protein polymers. The Ca(2+)-dependent TGase 2-dependent cross-linking activity has been proposed for its involvement in erythrocyte aging, by inducing irreversible modification of their cell shape and deformability. Accordingly, we found that high-density ("old") TGase 2(minus sign/minus sign) red blood cells (RBCs) were more resistant to osmotic stress-induced hemolysis than those from wild type mice. In addition, elevating the intracellular concentration of Ca(2+) by treatment of total RBCs with ionophore A23187 resulted in enhanced resistance of TGase 2-deficient erythrocytes compared to their normal counterpart. These findings indicate that TGase 2 may have a role in regulating structural flexibility of RBCs, possibly affecting their life span in physiopathological conditions, such as erythrocyte senescence, which are accompanied by increases in intracellular Ca(2+) concentration.

Animals↗

PubMed2002

Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient.

Transglutaminase 2 (TGase 2) is a Ca+2-dependent enzyme that catalyzes both intracellular and extracellular cross-linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane-mediated events required for glucose-stimulated insulin release from the pancreatic beta cells. Here we show that targeted disruption of TGase 2 impairs glucose-stimulated insulin secretion. TGase 2-/- mice show glucose intolerance after intraperitoneal glucose loading. TGase 2-/- mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS-2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2-/- mouse phenotype resembles that of the maturity-onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.

Adolescent↗