Domenico Bosco

Role of nuclear PKC δ in mediating caspase-3-upregulation in Jurkat T leukemic cells exposed to ionizing radiation

The response of Jurkat T cells to ionizing radiation (IR) includes cell cycle arrest and DNA damage, which lead to the occurrence of apoptosis. Here, we try to elucidate some of the early intracellular signals which control the induction of such a process upon IR exposure, addressing to examine the specific role of several PKC isoforms (δ, ε, ζ) and their subcellular distribution. Attention has been focused on the connections between nuclear PKC δ activation and the expression of cell death regulators (Bcl‐2 family proteins Bad, Bax and Bcl‐2) and cell death effector caspase‐3 (CPP32) which lead to the cleavage of cytoskeletal and nuclear proteins and induction of apoptosis. Altogether these results let us to conclude that PKC δ, potentiating the pro‐apoptotic effect of caspase 3, plays a key role in the cellular response to IR and thus can be considered a molecular target for therapy.

Molecular and morphological modifications occurring in rat heart exposed to intermittent hypoxia: role for protein kinase C α

Abstract Exposure of rats to intermittent hypoxia determines different responses at tissue and cell level. Heart mainly undergoes the effects of hypoxic injury and its response is determined both by the relationship between oxygen supply and demand and by its functional state. Since molecular mechanisms mediate cells sensing and response to low O2 concentration, here we explore the role played by Protein Kinase C α (PKC α) in the signal transduction mechanisms leading to the occurrence of morphological responses in rat neonatal, young and old heart subjected to intermittent hypoxia. Along with a key role for hypoxia inducible factor and vascular endothelial growth factor in the occurrence of continuous state of dynamic adaptation of vasculature, PKC α presumably phosphorylates IkBα in rat normoxic and hypoxic neonatal hearts, supporting the hypothesis of a rescue strategy carried out against hypoxia, together with an hypertrophic response. In hypoxic young heart PKC α activation, paralleled by sustained Bax homodimerization and caspase-3 activation, along with reduced p-IKBα and Inhibiting Apoptosis Protein (IAP) expression, suggests that the young early and deeply undergoes the effects of lowered oxygen tension. In addition, since no modifications concerning PKC alpha driven signalling system are evidenced in both the experimental conditions, we suggest an oxygen impaired sensing during ageing.

Caspase-3 is dually regulated by apoptogenic factors mitochondrial release and by SAPK/JNK metabolic pathway in leukemic cells exposed to etoposide-ionizing radiation combined treatment. 2004 May-Aug;17(2):181-90

Ionizing radiation induces a series of multiple intracellular events which can lead to activation of caspases, cytoplasmic proteases involved in the occurrence of apoptosis. The response of leukemic cells to ionizing radiation is amplified when they have been pre-treated with the anticancer drug etoposide, therefore the aim of this work has been to establish the lowest etoposide concentration combined with the lowest ionizing radiation dose to obtain the best antineoplastic response. Two leukemic cell lines, HL-60 and Jurkat, employed in this study, demonstrated different sensitivities to ionizing radiation and to etoposide treatment, with Jurkat T cells requiring a higher dose (1 μM) to display cell cycle perturbation and apoptotic DNA damage similar to those seen in HL-60. We hypothesize that this kind of response could be mediated by mitochondrial release of apoptogenic factors and by SAPK/JNK metabolic pathway activation, both leading to caspase-3 cleavage. All in all these results provide insight into the sensitivity or resistance of leukemic cells to antineoplastic agents and identify molecular targets for rational therapeutic intervention strategies.

Immunocytochemical Localization of Phospholipase C Isozymes in Cord Blood and Adult T-lymphocytes

The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)γ 1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCβ during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCβ1 was almost exclusively expressed in cord T-cells, whereas PLC β2 was more strongly represented in the adult. The amount of PLCγ 1 was found to be larger in the adult than in cord cells. No significant differences were found in PLCγ 2 and

Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation.

2 expression. PLCγ 1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis.

Protein kinase C ζ nuclear translocation mediates the occurrence of radioresistance in friend erythroleukemia cells

Abstract An important step in megakaryocyte maturation is the appropriate assembly of at least two distinct subsets of α-granules. The mechanism that sorts the α-granule components into distinct structures and mediates their release in response to specific stimuli is now emerging. P-selectin and von Willebrand factor are two proteins present in the α-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the α-granule contents in response to external stimuli. If P-selectin and von Willebrand factor are localized in the same or different α-granules is not known. To clarify this question, we analyzed by immunoelectron microscopy the localization of von Willebrand factor and P-selectin during the maturation of wild-type and Gata1low megakaryocytes induced in vivo by treating animals with thrombopoietin. Gata1low is a hypomorphic mutation that blocks megakaryocyte maturation, reduces the levels of von Willebrand factor expression and displaces P-selectin on the demarcation membrane system. The maturation block induced by this mutation is partially rescued by treatment in vivo with thrombopoietin. In immature megakaryocytes, both wild-type and Gata1low, the two receptors were co-localized in the same cytoplasmic structures. By contrast, the two proteins were segregated to separate α-granule subsets as the megakaryocytes matured. These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the α-granule content in response to external stimuli.

Inositol lipid-mediated intranuclear signalling: A comparative analysis of in vivo labelling in interferon alpha-sensitive and -resistant daudi lymphoma cells

Friend erythroleukemia cells require high doses (15 Gy) of ionizing radiation to display a reduced rate of proliferation and an increased number of dead cells. Since ionizing radiation can activate several signaling pathways at the plasma membrane which can lead to the nuclear translocation of a number of proteins, we looked at the intranuclear signaling system activated by Protein Kinases C, being this family of enzymes involved in the regulation of cell growth and death. Our results show an early and dose‐dependent increased activity of ζ and ε isoforms, although PKC ζ is the only isoform significantly active and translocated into the nuclear compartment upon low (1.5 Gy) and high (15 Gy) radiation doses. These observations are concomitant and consistent with an increase in the anti‐apoptotic protein Bcl‐2 level upon both radiation doses. Our results point at the involvement of the PKC pathway in the survival response to ionizing radiation of this peculiar cell line, offering PKC ζ for consideration as a possible target of pharmacological treatments aimed at amplifying the effect of such a genotoxic agent.

Electrochemically Synthesized Silver Nanoparticles are Active against Planktonic and Biofilm Cells of Pseudomonas aeruginosa and Other Cystic Fibrosis-Associated Bacterial Pathogens

Changes in inositol lipid and diacylglycerol metabolism have been analysed in Daudi lymphoma cells treated up to 24 h with human DNA recombinant interferon alpha. Results showing a different response of nuclear phosphoinositides and diacylglycerol, compared to whole cells, suggest that the intranuclear signalling system activated by interferon in Daudi cells involves nuclear inositol lipid metabolism. A well-characterized clone of Daudi cells selected for resistance to the antiproliferative action of interferon provided controls for the specificity of results.

Thrombotic events in models GATA-1 low myelofibrosis characterized by altered localization of P-selectin during megakaryocyte development

A novel, electrochemically synthesized, silver nanoparticles (AgNPs) formulation was evaluated in vitro against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Staphylococcus aureus strains from cystic fibrosis (CF) patients. AgNPs were particularly active against P. aeruginosa and B. cepacia planktonic cells (median MIC: 1.06 and 2.12 μg/ml, respectively) by a rapid, bactericidal and concentration-dependent effect. AgNPs showed to be particularly effective against P. aeruginosa and S. aureus biofilm causing a viability reduction ranging from 50% (1×MIC) to >99.9% (4×MIC). Electron microscopy showed that AgNPs deconstruct extracellular matrix of P. aeruginosa biofilm, and accumulate at the cell surface causing cell death secondary to membrane damage. Compared to Tobramycin, AgNPs showed comparable, or even better, activity against planktonic and biofilm P. aeruginosa cells. AgNPs at concentrations effective against B. cepacia and P. aeruginosa were not toxic to G. mellonella larvae. Our silver-based formulation might be an alternative to antibiotics in CF patients. Further in vitro and in vivo studies are warranted to confirm this therapeutic potential.

Inhibition of TGF-β1 signaling restores both microenvironmental and stem cells abnormalities in the Gata-1low Mouse Model of Myelofibrosis

Patients with primary myelofibrosis (PMF) have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions.This interactions are mediated by P-selectin and even though the megakaryocytes(Mk)of MF patients express normal levels of P-selectin,it remains abnormally localized to the DMS rather than being assembled into the a-granules in platelets.Mice carrying the hypomorphic Gata1low mutation express the same Mk abnormalities presented by PMF patients,including abnormal P-selectin localization to the DMS and develop with age myelofibrosis,that closely resembles human PMF.The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and,in this case,the role played by P-selectin in the development of the trait.To this aim,Gata1low mice were crossed with P-selnull mice according to standard genetic protocols and Gata1lowP-selWT,Gata1lowP-selnull and Gata1WTP-selnull or Gata1WTP-selWT littermates obtained.Platelet count,hematocrit as well as platelet microparticle levels were determined on all the different mutants.It was shown that platelet counts are reduced in Gata1low mice.Moreover,platelet microparticles are reduced in Gata1low mice and P-selectin positive platelet microparticles were not found.The presence of thrombosis was determined by immunohistological staining of organs.Gata1low mice with or without the P-selectin null trait had a prolonged bleeding time and thrombosis was seen adult and old Gata1low mice,but the Gata1low/P-selnull mice were rescued.Thus,presence of the P-selectin null trait rescued Gata1low mice from the thrombotic phenotype,but did not change level of platelet microparticles.All these data indicate that abnormal localization of P-selectin,induced by the Gata1low mutation,and thus, increased pathological interactions with leucocytes,is responsible for the increased presence of thrombosis seen in these mice.