Tumor Necrosis Factor Related Apoptosis Inducing Ligand (Trail) in endothelial response to biomechanical and biochemical stresses in arteries.

Shear stress is determined by three physical components described in a famous triad: blood flow, blood viscosity and vessel geometry. Through the direct action on endothelium, shear stress is able to radically interfere with endothelial properties and the physiology of the vascular wall. Endothelial cells (ECs) have also to sustain biochemical stresses represented by chemokines, growth factors, cytokines, complement, hormones, nitric oxide (NO), oxygen and reactive oxygen species (ROS). Many growth factors, cytokines, chemokines, hormones, and chemical substances, like NO, act and regulate endothelium functions and homeostasis. Among these cytokines Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) has been assigned a regulatory role in ECs physiology and physiopathology. Thus, the aim of this review is to provide a general overview of the endothelial response pathways after different types of biomechanical and biochemical stress in in vitro models and to analyze the crucial role of TRAIL under pathological conditions of the cardiocirculatory system like atherosclerosis, coronary artery disease, and diabetes.

Inhibition of P-glycoprotein-mediated transport by S-adenosylmethionine and cynarin in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells.

Multidrug resistance (MDR) to anticancer chemotherapy is often mediated by the overexpression of the plasma membrane drug transporter P-glycoprotein (Pgp) encoded by multidrug resistance gene (MDR1). Various chemosensitizing agents are able to inhibit Pgp activity but their clinical application is limited by their toxicity. Furthermore, hepatotoxicity related to chemotherapy causes delays of treatment in cancer patients and often requires supplementation of anti-tumour therapy with hepatoprotective agents. In this in vitro study, we investigated the effectiveness of an endogenous hepatoprotective agent, S-adenosylmethionine (SAMe), and a natural hepatoprotective compound, Cynarin (Cyn), to inhibit Pgp activity in order to evaluate their potential use as chemosensitizing agents. Human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) expressing high levels of Pgp were treated with two hepatoprotectors at various concentrations (1, 5 and 10 microM) that are clinically achievable, in the presence or absence of three different concentrations of doxo (2, 4 and 8 microM). In order to evaluate the effects of both hepatoprotectors, we measured the intracellular accumulation and cytotoxicity of doxo, the cellular GSH level, ROS production and catalase (CAT) activity. We found that treatment with 2, 4 and 8 microM doxo in the presence of SAMe or Cyn significantly increased the doxo accumulation and cytotoxicity on MES-SA/Dx5 cells, when compared to control cells receiving doxo alone. Moreover, treatment with SAMe or Cyn significantly increased GSH content, greater than 80 percent and 60 percent, respectively) and CAT activity greater than 60 and 150 percent, respectively) in resistant cancer cells, while ROS production was below the values of corresponding untreated control cells. Our in vitro findings provide a rationale for the potential clinical use of these hepatoprotectors both as chemosensitizing agents, to reverse Pgp-mediated MDR, and as antioxidants to protect normal cells from chemotherapy-induced cytotoxixity.

The effect of the plasticizer diethylhexyl phthalate on transport activity and expression of P-glycoprotein in parental and doxo-resistant human sarcoma cell lines.

Multidrug resistance (MDR) to cancer therapy is frequently associated with the over-expression of the multidrug transporter MDR1 gene product P-glycoprotein (Pgp) in several types of human tumours. Various chemosensitizers have been used to inhibit Pgp activity but toxicity limits their clinical application. Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is released from polyvinyl chloride (PVC) medical devices. Therefore, cancer patients undertaking chemotherapy are exposed to a clinically important amount of DEHP through blood and blood component transfusions, apheresis products, intravenous chemotherapy, parenteral nutrition and other medical treatments. The present study was designed to investigate the effects of DEHP on transport activity and expression of Pgp in order to evaluate its potential use as a chemosensitizer in cancer therapy. Human doxorubicin (doxo) resistant sarcoma cells (MES-SA/Dx5) that over-express Pgp were treated with different doses of doxo (2, 4 and 8 µM) in the presence or absence of various concentrations of DEHP (3, 6 and 12 µM) that were clinically achievable in vivo. Our results show that co-treatment with 2, 4 and 8 µM doxo in the presence of the lowest concentration of DEHP (3 µM) enhanced significantly doxo accumulation in MES-SA/Dx5 cells and, consistently increased the sensitivity to doxo, when compared to controls receiving only doxo. In contrast, higher DEHP concentrations (6 and 12 µM) induced MES-SA/Dx5 to extrude doxo decreasing doxo cytotoxicity toward resistant cells below control values. These results are consistent with the increase in Pgp expression levels in parental MES-SA cells treated with 3, 6 and 12 µM DEHP for 24 h and compared to untreated controls. All in all, these findings suggest a potential clinical application of DEHP as a chemosensitizer to improve effectiveness of the antineoplastic drugs in MDR human tumours.

Inducible nitric oxide synthase-activated mitochondrial apoptotic pathway in hypoxic and aged rat hearts.

BACKGROUND: Hypoxia and aging determine on mammalian cells a stress response which implies modified production of oxidants, reactive oxygen species or reactive nitrogen species at the mitochondrial level, interfering with cell-signaling proteins and inducing mitochondrial damage, apoptosis occurrence and functional consequences. OBJECTIVE: Here we report the effects of hypoxia on the in vivo morphological and biochemical response of young and aged Wistar rat hearts. METHODS: Left ventricles were excised from each experimental point and processed. Investigations of vascular endothelial growth factor (VEGF) expression and apoptotic events, mitochondrial damage, were performed by light and electron microscopy, respectively; endothelial, inducible and neuronal NOS, PKCα, pPKCα, caspase-3 expression and Apaf-1/cytochrome c complex formation were assessed by Western blotting and co-immunoprecipitation analyses, respectively. RESULTS: Besides morphological modifications, which confirm mitochondrial suffering upon hypoxia exposure in both young and aged hearts, the role played by PKCα in controlling nitric oxide synthase (NOS) protein level was investigated. Downstream PKCα activation, a dramatic iNOS expression increase, concomitant to enhanced apoptotic cell percentage and Apaf-1/cytochrome c co-immunoprecipitation, is evident in the hypoxic young, suggesting iNOS-mediated activation of the mitochondrial apoptotic pathway. CONCLUSIONS: Moreover, overexpression of iNOS and VEGF in the hypoxic young rat hearts suggests that an increased VEGF level may allow coordinated development of the lymphatic and blood vasculature, necessary for fluid homeostasis and to counteract oxidative stress. Thus the inhibition of such growth factor proposes new therapeutic possibilities for diseases associated to vascular function and for solid tumors which show pathological angiogenesis and lymphoangiogenesis.

Morphometric and ultrastructural analysis of human granulosa cells after gonadotrophin-releasing hormone agonist or antagonist.

Morphological features of granulosa cells can reflect their functional status. The present study was aimed at comparing possible differences in the fine structure of human granulosa cells exposed to gonadotrophin-releasing hormone (GnRH) agonist or antagonist treatment during ovarian stimulation. Cells were obtained from follicular aspirates of 21 women treated with recombinant follicle-stimulating hormone (rFSH) plus either a GnRH agonist or a GnRH antagonist. Conventional light microscopy procedures and computerized image analysis systems were used to identify different cell type morphological patterns and to quantify different cells distribution. Two morphologically distinct granulosa cell populations, defined as large/pale and small/dark cells, were identified and a different distribution in the two groups of women under investigation was found: a significantly higher percentage in large/pale cells was detected in the agonist-treated women (P<0.05), whereas the percentage of small/dark cells was significantly higher in the antagonist-treated group (P<0.05). Ultrastructural observations showed the presence in both cell populations of typical hallmarks of steroidogenic cells, highlighting signs of functional activity in the large/pale cell population. Further investigations are needed to define the possible clinical significance of these morphological findings.

Balance between hypertrophic and hypoxic stimulus in caspase-3 activation during rat heart development.

During heart development, cell hyperplasia and hypertrophy are the main mechanisms by which cardiac mass grows. Both these processes along with programmed cell death lead to complete growth and function. In addition, since the establishment of cardiac function depends on the relationship between oxygen supply and demand, we investigated some of the molecular mechanisms at the basis of rat myocardial cell response to hypoxic stress at different times of neonatal life. In particular, the role played by hypertrophic and survival factors like NF-kB and IAP-1 (Inhibiting Apoptosis Protein) and by death factors ASK-1 (Apoptosis Signal Regulating Kinase), JNK/SAPK (Jun-N-Terminal-Kinase/Stress-Activated Protein Kinase) pathways in regulating caspase-3 expression and activity has been evaluated by immunohistochemical and Western blotting analyses, respectively. Level of phosphorylation of IkBalpha and IAP-1 expression were substantial in 8-day-old hypoxic hearts, suggesting the persistence of NF-kB driven hypertrophic signal along with a rescue attempt against hypoxic stress. In contrast, ASK-1 mediated JNK/SAPK activation, regulating Bcl(2) levels, allows Bax homodimerization and caspase-3 activation in the same experimental conditions. Thus, a regulation carried out by NF-kB and JNK/SAPK pathways on caspase-3 activation at day 8 of neonatal life can be suggested as the main factor for the heart 'adaptive' response to hypoxia.

Protein kinase C zeta regulation of hypertrophic and apoptotic events occurring during rat neonatal heart development and growth.

The development and growth of the rat heart implies hyperplasia, which stops at birth, and hypertrophy, allowing cardiac mass to grow in response to programmed genetic events along with to haemodynamic overload. Moreover, hypertrophy is accomplished to apoptosis which controls the final number of myocardial cells, deletes vestigial structures, and takes part in remodelling the organ. Since at the basis of all these processes, which lead to the complete development of the heart, the activation of specific signalling pathways underlies, attention has been addressed to the role played in vivo by Protein Kinase C zeta (PKC zeta) in regulating NF-kB signalling system and intrinsic mitochondrial apoptotic route at days 1, 4, 10 and 22 of rat life. In fact, a role has been assigned to PKC zeta in indirectly phosphorylating IKBa, which peaks between 10 and 22 days, through a IKK determining, in turn, NF-kB activation, concomitantly to cytochrome c/Apaf 1 co-localization in the cytoplasm and caspase-9/caspase-3 activation, which leads to the occurrence of apoptosis. Thus a key role for PKC zeta in regulating the hypertrophic and apoptotic events leading to establishment of complete function in rat neonatal heart is here suggested.

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.

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 microM) 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.

Correlations between protein kinase C zeta 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.

Protein kinase C zeta nuclear translocation mediates the occurrence of radioresistance in friend erythroleukemia 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 zeta and epsilon isoforms, although PKC zeta 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 zeta for consideration as a possible target of pharmacological treatments aimed at amplifying the effect of such a genotoxic agent.

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Histochemical and biochemical analysis of phospholipase C isoforms in normal human gastric mucosa cells.

The expression and activity of PIP2-specific phospholipase C (PLC) in healthy human gastric mucosa cells were investigated by means of Western blotting, immunohistochemistry and in vitro activity assays. The results provide direct evidence for an almost exclusive expression of the PLC beta family and at the same time supply a cellular cartography of each represented isoform of this family. In this context, the putative roles of each isoform in the signaling events regulating the gastric mucosa metabolic machinery are discussed. These data provide a unique map of the specific expression and cellular distribution of the most represented PLC isoforms in healthy human gastric mucosa cells, which may constitute a reference point in future studies aimed at highlighting possible cytochemical and biochemical hallmarks of metaplastic or malignant transformation.

Phosphatidylinositol-3-kinase activation and atypical protein kinase C zeta 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.

Dimethylsulfoxide-induced cell death of murine erythroleukemia cells exposed to ionising radiation.

The present investigation was aimed at studying the effects of dimethylsulfoxide (DMSO) in combination with high dose (15 and 60 Gy) ionising radiation on the growth and differentiation of murine erythroleukemia cells (MEL). The incubation with DMSO was performed for 96 h starting immediately after exposure to radiation and resulted only in a slight inhibition of cell growth and in a high increase in cell death with the induction of both necrosis and apoptosis. The enhancement of radiation cytotoxicity was directly related to dose, time in culture and degree of differentiation as demonstrated by the severe and multiple aberrations observed in light and electron microscopy. Of interest was the observation in induced cells of a marked rearrangement of the plasma membrane architecture as well as that of the nuclear envelope, with a massive translocation and/or decrease in the nuclear pore complexes.

TNF-alpha-induced apoptosis in Daudi cells: multiparametric analysis.

Tumour necrosis factor alpha (TNF-alpha) is a cytokine that induces physiological and pathophysiological effects in the immune system. In this study we analyzed its action on a human lymphoma cell line (Daudi cells) after 1 h, 6 h and 24 h of incubation. Using vital DNA stains, DNA gel electrophoresis, in situ nick translation, transmission electron microscopy and flow cytometry we showed that as early as after 6 h of treatment, target cells were able to undergo death by apoptosis. This was associated with cell shrinkage, chromatin condensation, apoptotic bodies in the cytoplasm without the typical DNA fragmentation into low molecular weight nucleosomes. Of interest was the observation of a significant number (60%) of cells positive to the nick translation in specimens treated for 6 h, decreasing to 40% in samples treated for 24 h, when most of the cells were in late apoptosis. In addition, no subdiploid peak was evident in flow cytometry regardless of the time of incubation with TNF. Our study on Daudi cells clearly supports the existence of alternative forms of apoptosis in which DNA degradation does not result only in oligonucleosomal fragmentation.

Age- and training-related events in active T subpopulation. Changes in polyphosphoinositide metabolism during mitogenic stimulation.

To examine the effects of age and training on the active T subpopulation we considered elderly amateur cyclists over 65 in comparison with young amateur cyclists and young and aged sedentary healthy controls. Significant differences were observed between trained and sedentary elderly subjects consisting of an increase in the percentage of active E rosettes after 4 and 24 h of in vitro PHA stimulation, and of a decrease in the in vitro phosphorylation of phosphatydylinositol 4,5-bisphosphate (PtdInsP2) and a corresponding increase in phosphatydylinositol 4-phosphate (PtdInsP) in the early steps of the mitotic response. Our findings support the hypothesis of the involvement of inositol lipids in controlling the expression of lymphocyte surface receptors.

Ionizing radiation-induced apoptosis and DNA repair in murine erythroleukemia cells.

A morphological study of DNA repair and apoptotic patterns in relationship with cell cycle events was performed on murine erythroleukemia cells. The presence and distribution of DNA replicon sites were evaluated through the BrdU-anti BrdU immunofluorescence and immunogold techniques in light and electron microscopy. Different patterns of labelling and percentages of BrdU positive cells were observed depending on irradiation dose (up to 60 Gy) and time in post-irradiation culture (up to 24 hours). An enlargement of the S phase of the cell cycle was evidenced 18 hours post-irradiation as determined by flow cytometry analysis. The high resolution approach showed that, in spite of several morphological alterations, BrdU labelling was present even in cells displaying early and late apoptotic features.

Evidence for an early and transient involvement of nuclear inositol lipids in subcellular signalling events related to DNA repair processes.

The involvement of nuclear inositol lipids in the processes related to DNA repair upon ionizing radiation has been investigated in Murine Erythroleukaemia cells. Early changes in the in vitro phosphatidylinositol-bisphosphate phosphorylation in isolated nuclei were found to precede transiently the marked increase in DNA synthesis occurring after irradiation. Such an increase detected by anti-BrdU monoclonal antibodies has been found to be related mainly to DNA polymerase beta activity as revealed by the kinetic analysis of in vitro DNA synthesis. The results here presented allow us to speculate on a possible involvement of nuclear inositol lipids in the cascade of the early events leading to the regulation of DNA repair in the nucleus.

Age-related events in active T lymphocyte subpopulation. A morphological study.

The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of active T lymphocytes from healthy aged donors was evaluated after in vitro PHA stimulation by means of light microscopy, electron microscopy and flow cytometry. The percentage of BrdU-labelled cells differed markedly between aged and young donors after 48 h of PHA stimulation, due to an enlarged early S phase compartment. In contrast, after 72 h the percentage of positive cells was quite similar in both age groups and no significant differences in the distribution within S phase nor changes in the patterns of ultrastructural localization of DNA replicon sites were observed. Our study provides evidence that an altered synchronization and a substantial delay in the in vitro cell proliferation occur in this peculiar T subpopulation as a consequence of the ageing process.