p73, an important developmental gene, shares a high sequence homology with p53 and induces both G1 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 ΔNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The ΔNp73 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.
Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity ...of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)−25 in FH. IL‐25 expression was evaluated in patients with FH and in livers of mice with FH induced by D‐galactosamine (D‐Gal) and lipopolysaccharide (LPS). Mice were treated with IL‐25 before D‐Gal/LPS‐induced FH and before or after concanavalin A (ConA)‐induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL‐25 and analyzed for GR1+CD11b+ cells. CFSE‐labeled T cells were cocultured with GR1+CD11b+ cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti‐GR1 antibody before IL‐25 and D‐Gal/LPS administration. IL‐25 was constitutively expressed in mouse and human liver and down‐regulated during FH. IL‐25 prevented D‐Gal/LPS‐induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1+CD11b+ cells isolated from mice given IL‐25 inhibited T‐cell proliferation. Consistently, in vivo depletion of GR1+ cells abrogated the protective effect of IL‐25 in experimental D‐Gal/LPS‐induced FH. IL‐25 was both preventive and therapeutic in ConA‐induced FH. Conclusions: IL‐25 expression is markedly reduced during human and experimental FH. IL‐25 promotes liver accumulation of GR1+CD11b+cells with immunoregulatory properties. (Hepatology 2013;58:1436–1450)
The ability to form biofilms is a recognized trait of
, but the extent of its clinical relevance is still unclear. The present multicenter prospective study (ANSELM) aims at investigating the ...association between biofilm formation and clinical outcomes of
infections. One hundred and nine isolates were collected from various geographical origins and stratified according to their clinical relevance. Biofilm formation was evaluated by the microtiter plate assay and correlated with microbiological and clinical data from the associated strains. Antibiotic susceptibility of the planktonic cells was tested by the disk diffusion technique, while antibiotic activity against mature biofilms was spectrophotometrically assessed. Most strains (91.7%) were able to form biofilm, although bloodborne strains produced biofilm amounts significantly higher than strains causing hospital- rather than community-acquired infections, and those recognized as "definite" pathogens. Biofilm formation efficiency was positively correlated with mechanical ventilation (
= 0.032), whereas a negative relationship was found with antibiotic resistance (
= 0.107;
< 0.001), specifically in the case of the pathogenic strains. Mature
biofilms were markedly more resistant (up to 128 times) to cotrimoxazole and levofloxacin compared with their planktonic counterparts, especially in the case of bloodborne strains. Our findings indicate that biofilm formation by
is obviously a contributing factor in the pathogenesis of infections, especially in deep ones, thus warranting additional studies with larger cohort of patients and isolates.
Trastuzumab emtansine (T-DM1) is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugated to the microtubule-targeting agent emtansine (DM1). T-DM1 is an effective agent in ...the treatment of patients with HER2-positive breast cancer whose disease has progressed on the first-line trastuzumab containing chemotherapy. However, both primary and acquired tumour resistance limit its efficacy. Increased levels of the phosphorylated form of Translationally Controlled Tumour Protein (phospho-TCTP) have been shown to be associated with a poor clinical response to trastuzumab therapy in HER2-positive breast cancer. Here we show that phospho-TCTP is essential for correct mitosis in human mammary epithelial cells. Reduction of phospho-TCTP levels by dihydroartemisinin (DHA) causes mitotic aberration and increases microtubule density in the trastuzumab-resistant breast cancer cells HCC1954 and HCC1569. Combinatorial studies show that T-DM1 when combined with DHA is more effective in killing breast cells compared to the effect induced by any single agent. In an orthotopic breast cancer xenograft model (HCC1954), the growth of the tumour cells resumes after having achieved a complete response to T-DM1 treatment. Conversely, DHA and T-DM1 treatment induces a severe and irreversible cytotoxic effect, even after treatment interruption, thus, improving the long-term efficacy of T-DM1. These results suggest that DHA increases the effect of T-DM1 as poison for microtubules and supports the clinical development of the combination of DHA and T-DM1 for the treatment of aggressive HER2-overexpressing breast cancer.
Stem cells are a centerpiece of regenerative medicine research, and the recent development of adult stem cell-based therapy systems has vigorously expanded the scope and depth of this scientific ...field. The regeneration of damaged and/or degraded bone tissue in orthopedic, dental, or maxillofacial surgery is one of the main areas where stem cells and their regenerative potential could be used successfully, requiring tissue engineering solutions incorporating an ideal stem cell type paired with the correct mechanical support. Our contribution to this ongoing research provides a new model of in vitro osteogenic differentiation using blood-derived stem cells (BDSCs) and rapamycin, visibly expressing typical osteogenic markers within ten days of treatment. In depth imaging studies allowed us to observe the adhesion, proliferation, and differentiation of BDSCs to both titanium and bone scaffolds. We demonstrate that BDSCs can differentiate towards the osteogenic lineage rapidly, while readily adhering to the scaffolds we exposed them to. Our results show that our model can be a valid tool to study the molecular mechanisms of osteogenesis while tailoring tissue engineering solutions to these new insights.
Brain tumors are the most common solid neoplasms of childhood. They are frequently reported in children with Neurofibromatosis type 1 (NF1). The most frequent central nervous system malignancies ...described in NF1 are optic pathway gliomas and brainstem gliomas. Medulloblastoma (MB) in NF1 patients is extremely rare, and to our knowledge, only 10 cases without molecular characterization are described in the literature to date. We report the case of a 14-year-old girl with NF1 that came to our attention for an incidental finding of a lesion arising from cerebellar vermis. The mass was completely resected, revealing a localized classic medulloblastoma (MB), subgroup 4. She was treated as a standard-risk MB with a dose-adapted personalized protocol. The treatment proved to be effective, with minor toxicity. Brain and spine MRI one year after diagnosis confirmed the complete remission of the disease. To our knowledge, this is the only case of MB reported in a patient with NF1 with molecular characterization by the methylation profile. The association between NF1 and MB, although uncommon, may not be an accidental occurrence.
Down syndrome (DS) is the most common chromosome abnormality with a unique cancer predisposition syndrome pattern: a higher risk to develop acute leukemia and a lower incidence of solid tumors. In ...particular, brain tumors are rarely reported in the DS population, and biological behavior and natural history are not well described and identified. We report a case of a 10-year-old child with DS who presented with a medulloblastoma (MB). Histological examination revealed a classic MB with focal anaplasia and the molecular profile showed the presence of a
variant associated with the wingless (WNT) molecular subgroup with a good prognosis in contrast to our case report that has shown an early metastatic relapse. The nearly seven-fold decreased risk of MB in children with DS suggests the presence of protective biological mechanisms. The cerebellum hypoplasia and the reduced volume of cerebellar granule neuron progenitor cells seem to be a possible favorable condition to prevent MB development via inhibition of neuroectodermal differentiation. Moreover, the NOTCH/WNT dysregulation in DS, which is probably associated with an increased risk of leukemia, suggests a pivotal role of this pathway alteration in the pathogenesis of MB; therefore, this condition should be further investigated in future studies by molecular characterizations.
The p53-related p73 andp63 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.
ABSTRACT
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 β 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.—Bernassola, F., Federici, M., Corazzari, M., Terrinoni, A., Hribal, M. L., De Laurenzi, V., Ranalli, M., Massa, O., Sesti, G., Mclean, W. H. I., Citro, G., Barbetti, F., Melino, G. Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB J. 16, 1371–1378 (2002)
Transglutaminases (TGases) are seven enzymes, cross-linking proteins by γ-glutamil-ε-lysine bonds, four of which are expressed in the skin. A new member of the TGase family, TGase 5, has been ...identified recently, and in the present study we evaluated its role in keratinocyte differentiation in vitro. In addition to the previously described isoforms, full-length TGase 5 and Δ3 (deletion of exon 3), we identified two new splicing variants, Δ11 and Δ3Δ11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, Δ3, Δ11, and Δ3Δ11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and Δ11 are active, whereas Δ3 and Δ3Δ11 have very low activity. Expression studies show that full-length TGase 5 is induced during the early stages of keratinocyte differentiation and is differently regulated in comparison with the other epidermal TGases. Kinetic and in vitrocross-linking experiments indicate that full-length TGase 5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). In keratinocyte expression system, TGase 5 isoforms are retained in an intermediate filament-enriched fraction, suggesting its association with insoluble proteins. Indeed, TGase 5 co-localize with vimentin and it is able to cross-link vimentinin vitro.
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