Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis

Reactive oxygen species (ROS) are potent inducers of oxidative damage and have been implicated in the regulation of specific cellular functions, including apoptosis. Mitochondrial ROS increase markedly after proapoptotic signals, though the biological significance and the underlying molecular mechanisms remain undetermined. P66Shc is a genetic determinant of life span in mammals, which regulates ROS metabolism and apoptosis. We report here that p66Shc is a redox enzyme that generates mitochondrial ROS (hydrogen peroxide) as signaling molecules for apoptosis. For this function, p66Shc utilizes reducing equivalents of the mitochondrial electron transfer chain through the oxidation of cytochrome c. Redox-defective mutants of p66Shc are unable to induce mitochondrial ROS generation and swelling in vitro or to mediate mitochondrial apoptosis in vivo. These data demonstrate the existence of alternative redox reactions of the mitochondrial electron transfer chain, which evolved to generate proapoptotic ROS in response to specific stress signals.     

Synthesis, solution equilibria and antiproliferative activity of copper(II) aminomethyltriazole and aminomethylthioxotriazoline complexes

The aim of the present study was the synthesis, the determination of formation constants, and the evaluation of the antiproliferative activity of two copper(II) complexes formed with triazole-type ligands. The synthesis of the unsymmetrical triazole ligand 4-amino-3-aminomethyl-5-methyl-1,2,4-triazole (L1), and its copper(II) complex is reported. The ligand was prepared by functionalization of the carboxylate function of tert-butyloxycarbonyl (BOC) protected glycine O-methyl ester. All intermediates and final products were isolated and characterized with IR, 1H NMR, and elemental analysis. X-ray structures of the ligand as a sulfate salt ((H2L1)2SO4.H2O) and the copper(II) complex [CuCl2(L1)(2)] are described. The ligand forms a (N,N) bidentate chelate with the amino group and one triazole nitrogen atom. The tetragonally distorted octahedral coordination of Cu(II) results from two axially coordinated chloride ions. Protonation constants for L1 and speciation of the Cu(II)/L1 system were determined in 0.1 M aqueous KCl solution at 25 degrees C. Complexes formed in solution were also characterized by visible spectrophotometry. Ligand substitution competition between L1 and glycine has also been studied using potentiometric titrations. Antiproliferative activities of ([CuCl2(L1)2]) and [CuCl2(H2L2)]Cl, where HL2 is the 5-thioxo analog of L1, against human tumor cell lines HT1080 and HT29 as well as normal human fibroblasts (HF) are presented along with the antiproliferative activities of L1, CuCl2, and cisplatin. Activity of these two complexes are discussed and compared with the activity of analogous compounds reported in the literature which contain pyridyl groups in place of the aminomethyl group. In particular, it is suggested that a lypophilic residue such as a pyridyl group is important for antiproliferative activity of this class of compounds.     

Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARalpha and PLZF-RARalpha fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARalpha from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.     

A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.     

Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity

BACKGROUND: the inhibition of histone deacetylase (HDAC) has been reported as an effective mechanism on therapy in neoplastic diseases. Among HDAC inhibitors, Trichostatin A (TSA) and Valproic Acid (VPA) prevent the tumorigenesis in rodent and human models. Malformations as neural tube and axial skeletal defects are well-known VPA side effects. Recent hypotheses suggest the HDAC inhibitor activity as the teratogenic mechanism of VPA. The teratogenic potency of TSA is, at the moment, unknown. The aim of the present work is to investigate the HDAC inhibition on embryos exposed in utero to TSA or VPA and to compare the teratogenic potential of these two molecules on the axial skeleton morphogenesis. METHODS: Pregnant CD mice were i.p. treated on day 8 post coitum (9.00 a.m.) with 400 mg/kg VPA or with 0, 2, 4, 8, 16 mg/kg TSA. Embryos explanted 1 hr after the treatment from some females exposed to 400 mg/kg VPA or to 16 mg/kg TSA were processed for Western blotting and immunohistochemical analysis, in order to evaluate the histone hyperacetylation in the total embryo homogenates and to visualize the hyperacetylated tissues. Foetuses at term were processed for skeletal examination. RESULTS: Both VPA and TSA were able to induce hyperacetylation on embryos, specifically at the level of the caudal neural tube and of somites. At term, TSA showed teratogenic effects at the axial skeleton, quite similar to those observed after VPA exposure. CONCLUSIONS: In conclusion, both VPA and TSA are teratogenic in mice. A direct correlation between somite hyperacetylation and axial abnormalities could suggest the HDAC inhibition as the mechanism of the teratogenic effects.     

Bovine prion (PrP) and Doppel (Dpl) proteins expression after in vitro leukocyte activation or Dpl/PrP blocking

It has been postulated that Doppel (Dpl) and Prion (PrP) proteins have yet undetermined interactions, since Dpl is overexpressed in transgenic PrP-deficient mice. In this study we investigated the expression levels of Dpl and PrP on lymphocytes, monocytes and neutrophils (PMNs) isolated from bovine blood and incubated (2 and 18 h) with TNFalpha, IL-1, IL-2, IL-8, C5a, IFNgamma, anti-PrP, and anti-Dpl antibodies by flow cytometry. The isolation procedures yielded cell populations with high purity, viability and recovery rates. After 2 h incubation, expression of PrP or Dpl was altered only in PMNs. These cells overexpressed PrP when incubated with TNFalpha and IFNgamma, and both PrP and Dpl when incubated with C5a; incubation with TNFalpha, IL-8 and IFNgamma led to down-regulation of Dpl. Lymphocytes incubated for 18 h with IL-2 and with IFNgamma overexpressed Dpl. Incubation with the anti-PrP antibody induced down-regulation of Dpl in PMNs after 2 h and overexpression of Dpl in lymphocytes after 18 h. The differences recorded after 2 h were likely due to redistribution of pre-existing PrP or Dpl molecules, while those seen at 18 h were most probably due to increased protein synthesis. The variations seen using the different activators depend on different receptors and/or signaling pathways. These results demonstrate that is possible to alter the expression of Dpl and PrP in blood cells in vitro by incubation with either cytokines or anti-PrP antibodies. This opens an interesting opportunity to study the biology of these proteins using in vitro systems.