Lucia Centurione

GATA-1 as a Regulator of Mast Cell Differentiation Revealed by the Phenotype of the GATA-1low Mouse Mutant

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neoΔHS or GATA-1low mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue+ mast cells and apoptotic metachromatic− mast cell precursors in connective tissues and peritoneal lavage and numerous (60–70% of all the progenitors) “unique” trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

HIF-1alpha cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia.

Intermittent hypoxia, followed by reoxygenation, determines the production of reactive oxygen species (ROS), which may lead to accelerated aging and to the appearance of age‐related diseases. The rise in ROS levels might constitute a stress‐stimulus activating specific redox–sensitive signalling pathways, so inducing either damaging or protective functions. Here, we report that in old rat cerebral cortex exposed to hypoxia, the accumulation in the cytoplasm of hypoxic inducible factor 1α (HIF‐1α) – the master regulator of oxygen homeostasis – concomitant with p66Shc activation and reduced IkBα phosphorylation is associated with tissue apoptosis or necrosis. In young cerebral cortex, we hypothesize that the hypoxic damage may be reversible, based on our demonstration of elevated HIF‐1α levels, combined with a low level of IkBα phosphorylation, a decrease in IAP‐1 and a lack of major change in Bcl2 family proteins. These observations are associated with a low level of cell death induced by hypoxia, suggesting that HIF‐1α activation in cortical neurons may produce rescue proteins in response to intermittent hypoxia.

Age-related death-survival balance in myocardium: an immunohistochemical and biochemical study.

During ageing, the occurrence of apoptosis is due to a progressive impairment of normal functions, leading to eliminate redundant, damaged or infected cells. Here we report that also in myocardial tissue, ageing, besides reduction of the number of myocytes and of specialized conduction tissue cells, reduction in Ca(++) transport across the membrane, includes the establishment of apoptosis. In particular, the occurrence of this process, which is less represented than we would have expected, is mediated by the balance between the well known Bcl-2 protein family members, Bad, Bax and Bcl-2, related to the pathway PI-3-kinase/AKT-1, which is known to deliver a survival signal. In fact, aged myocardial cells disclose a suboptimal response, which underlines the possibility that they can become more sensitive to damaging factors or diseases, more frequently occurring during ageing, probably due to an exploited molecular control of apoptosis.

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.

Correlations between protein kinase C ζ signaling and morphological modifications during rat heart development and aging

From birth to aging the heart undergoes functional changes reflecting biochemical and ultrastructural modifications which imply apoptosis. This is a physiological process resulting from genetic programs closely associated with development and aging. During development apoptosis eliminates redundant cells leading to heart remodeling, while during aging it eliminates damaged or exhausted cells. In the present paper we analyze some molecular mechanisms involved with heart morphological modifications, especially in the neonatal heart which displays different features in the subendocardial and myocardial area. The high number of subendocardial apoptotic cells and the inverted ratio of Bcl-2/Bax molecule expression in the two heart compartments led us to hypothesize a different metabolism in the myocardium as compared with subendocardium. Moreover, we propose that PKC zeta may mediate this different response by activating Nf-kB pathway and by maintaining the balance between hypertrophic growth and apoptosis involved with remodeling of neonatal heart. Further, we underline that in the aged heart, where this pathway is not activated, such balance is not maintained.

Phosphatidylinositol-3-kinase activation and atypical protein kinase C ζ phosphorylation characterize the DMSO signalling in erythroleukemia cells

Here we provide evidence for a role of phosphatidylinositol-3-kinase (PI-3-kinase) and for its product phosphatidylinositol-3,4, 5-triphosphate (PI3,4,5P3) in the occurrence of the metabolic differentiation state induced by DMSO in murine Friend erythroleukemia cells. Of note, the activation of PI-3-kinase correlated with the modulation of the activation of another enzyme, the atypical protein kinase C zeta (aPKC zeta). In particular, the expression of PI-3-kinase was substantially unaffected by DMSO treatment while its phosphorylation and the production of PI3,4,5P3 was strongly increased within 24 h of DMSO. Such a result was paralleled by an evident phosphorylation of a PKC zeta. Treatment of the cells with the two unrelated PI-3-kinase inhibitors wortmannin and LY 294002 impaired the recovery of the number of differentiated cells, therefore indicating that PI-3-kinase might be involved in the induction of erythroid differentiation, possibly engaging a protein kinase C zeta as downstream effector.

Stem Cell Ageing and Apoptosis

Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.

Effect of chronic hypoxia on inducible nitric oxide synthase expression in rat myocardial tissue.

The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in Inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (INOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of INOS. Expression of INOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). INOS expression was evaluated through immunohistochemical techniques (Ab anti-INOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of INOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for INOS in the same experimental conditions. In addition, the INOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 ± 0.01 vs 0.02 ± 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating INOS.

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.

NF-κB activation plays an antiapoptotic role in human leukemic K562 cells exposed to ionizing radiation

Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase‐3 activation, we paid attention to the influence exerted by Protein kinase C δ on transcription factor NF‐κB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC δ/NF‐κB pathway activation, probably mediated by novel IKKs and a role for PI‐3‐kinase in activating PKC δ at Thr 505 by PDK‐1 phosphorylation is suggested. In addition, since caspase‐3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF‐κB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF‐κB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings. J. Cell. Biochem. 89: 956–963, 2003.