Expression and phosphorylation of protein kinase C isoforms in Abeta(1-42) activated T lymphocytes from Alzheimers disease.

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction. There is evidence demonstrating altered activity of some PKC isoforms (PKC-alpha, PKC-delta and PKC-zeta) in the neurons of brains of Alzheimers Disease (AD) sufferers, but little is known about their involvement in the intracellular machinery of amyloid beta protein-reactive T lymphocytes in AD. By applying a modified, split-well culture system, for Abeta(1-42) reactivity, we carried out flow cytometry analysis and biochemical investigations on the possible involvement of PKC-alpha, PKC-delta and PKC-zeta in the signalling system activated in Abeta-reactive T cells purified from peripheral blood mononucleate cells (PBMC) from healthy subjects and patients with AD. Flow cytometry analysis of Abeta(1-42) activated T lymphocytes in the majority of AD patients highlighted a distinct cellular cluster highly expressing phospho-PKC-delta (P-PKC-delta), while most full-blown AD patients highly expressed two distinct P-PKC-delta and phospho-PKC-zeta (P-PKC-zeta) bright sub-populations. The same investigation performed in freshly purified peripheral T lymphocytes, did not highlight any subpopulation, suggesting that the detection of P-PKC-delta and P-PKC-zeta bright subpopulations is specifically linked to Abeta(1-42) activated T lymphocytes. The data presented here, therefore, suggest possible novel hallmarks to discriminate between healthy elderly subjects and beginning or full-blown Alzheimers Disease patients.

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.

Novel evidence of PLC delta2 involvement in the regulation of the differential evolution of human aneurysms.

The biological and molecular mechanisms which are responsible for the formation and possible evolution of human aneurysms are unknown. Previous investigations have pointed to the possible involvement of inositol specific-phospholipase C (PLC) in the mechanisms related to the formation or evolution of intracranial aneurysms, but, thus far, a relationship of one or more PLC isoforms with the biological signals influencing the fate of this lesion has not been demonstrated. The aim of this study was to investigate the expression, activity and possible modification of PLC isoforms in intracranial aneurysms in patients undergoing elective surgical repair after casual identification of unruptured aneurysms, or during emergency surgical repair of ruptured aneurysms. PLC and proliferating cell nuclear antigen (PCNA) expressions were detected by immunohistochemical analysis; PLC activity was obtained by measuring its hydrolytic activity on labelled PIP(2); PKC activity was measured by total kinase activity assay. Results indicated no substantial differences between controls and aneurysms, with the only exception being PLC delta2 which was nearly absent in controls and ruptured aneurysms, while strongly expressed and functionally active in almost all unruptured aneurysms. In addition, its expression always correlated with the proliferation cell marker PCNA, while its specific activity always correlated to PKC activity. PLC delta2 distribution, regulation and role in human tissues are still unknown Therefore, although preliminary, these data provide a novel insight into the signalling machinery influencing the aneurismal progression.

Novel shift of Jak/Stat signalling characterizes the protective effect of aurintricarboxylic acid (ATA) from tumor necrosis factor-alpha toxicity in human B lymphocytes.

Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-alpha was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors Stat3 and Stat5b. In this study we demonstrate that the balance of survival signals in the presence of TNF-alpha was altered by the addition of a salicylate compound, the endonuclease inhibitor aurintricarboxylic acid (ATA). Apoptosis effected by TNF-alpha alone was suppressed by ATA and this event was paralleled by phosphorylation and nuclear translocation of Jak2, Stat2, Stat4 and NF-kB, along with inhibition of caspase activation. These results confirm that among the different cellular responses evoked by TNF-alpha in human B cells, recruitment of Jak/Stat proteins and possible related gene modulation represent contributing factors and address the issue of the development of potential therapeutic strategies aimed at the control of systemic or local effects produced by TNF-alpha.

Effects of TNF-alpha/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes.

Tumour necrosis factor alpha (TNF-alpha) kills Daudi cells (Human Burkitt Lymphoma), inducing either necrosis or apoptosis without DNA fragmentation. Therefore, we were interested in studying the molecular and ultrastructural events occurring when the nucleus is more accessible and cells are blocked in mitosis, following colchicine treatment. In fact, as early as after 1 h treatment a typical ladder pattern was shown by means of DNA gel electrophoresis. In parallel the quantitative analysis of the different morphological patterns observed gave evidence of an increased percentage of primary necrosis after 6 h treatment, and a higher incidence of cells in late apoptosis as well as in secondary necrosis after 24 h treatment. Our findings show that Daudi cells respond to the combined treatment with an increased formation of micronuclei and nuclear alterations which follow a number of early mitochondrial changes and result in enhanced cell death. These data imply that TNF-alpha-induced apoptosis of Daudi cells can be triggered by mitochondrial changes and is somehow related to microtubule organization.

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.

Sensitivity of Daudi cells to tumor necrosis factor alpha: early changes in polyphosphoinositide metabolism.

The effects of recombinant Tumor Necrosis Factor alpha (r-TNF alpha) on polyphosphoinositide metabolism were examined in a Burkitt Lymphoma cell line (Daudi cells). After 1 h of in vitro treatment with r-TNF alpha, the incorporation of 32Pi into phosphatidylinositol 4,5-phosphate (PtdInsP2), phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol (PtdIns) was reduced compared with controls, confirming previous findings observed in other cell lines of a specific PtdIns breakdown following r-TNF alpha treatment. The novelty of this study is therefore the demonstration of early changes in polyphosphoinositide metabolism during the antiproliferative response elicited by this cytokine in Daudi cells.

Synergistic regulatory effects of TNF alpha, IL-1 alpha and IFN alpha on the growth and differentiation of Daudi lymphoma cells.

The regulatory effects of the combined treatment of tumour necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interferon alpha (IFN alpha) on the growth and differentiation of Daudi lymphoma cells were investigated. By means of anti-BrdU monoclonal antibodies and [3H-thymidine] incorporation a reduced proliferation rate was shown both through a combination of TNF alpha with either IL-1 alpha or IFN alpha and, above all, through simultaneous treatment with the three cytokines. In parallel, the degree of differentiation was evaluated via morphological criteria and detection of Fc receptors (FcR) and appeared higher after treatment with the three cytokines. Our results provide evidence of the increased sensitivity of this cell line to this combined treatment supporting the existence of a synergistic interaction in inducing the antiproliferative and differentiative effects.

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.

IL-1 alpha antiproliferative and differentiative effects on Daudi lymphoma cells: multiparametric analysis.

Interleukin I alpha (IL-1) is a potent agent that induces a wide range of biological effects. In this study we analysed its effects on cell cycle progression and differentiation of Daudi lymphoma cells. The parallel analysis in light microscopy and cytofluorimetry by means of anti-BrdU monoclonal antibodies showed a reduced rate of proliferation (S phase) with a G1 arrest. These features were confirmed by the lower incorporation of [3H]-thymidine supporting the decrease in the rate of DNA synthesis. In addition this cytokine was able to induce differentiation after 24 hrs of treatment as assessed by the increased expression of Fc receptors (FcR) and morphological criteria. This multiparametric analysis gives evidence to the sensitivity to this cytokine of this peculiar cell line.

Subtraction of autofluorescent dead cells from the lymphocyte flow cytometric binding assay.

Flow cytometry allows the quantitative analysis of lymphocyte-target cell conjugates and the identification of the lymphocyte subset involved in the binding phenomenon. We recently described a methodology to identify the effector cells bound to K562 targets based on target cell autofluorescence coupled with lymphocyte staining by means of fluorescent monoclonal antibodies. Here we describe an implementation of the methodology that allows the subtraction of spontaneously dead targets to which lymphocytes may or may not adhere, thereby preventing the overestimation of the binding phenomenon and limiting its evaluation to living effector-target conjugates, thus preserving the specificity of the phenomenon.

Interferon affects cell growth progression by modulating DNA polymerases activity.

A multiparametric analysis of the effects of human recombinant interferon alpha type A on Daudi cells involving flow cytometry and in vitro analysis of alpha and beta DNA polymerase activities has been performed. Results have disclosed (within 60 min of interferon treatment) a decrease of alpha polymerase driven DNA synthesis persisting to at least 24 h, while beta polymerase was poorly affected. Moreover, after 24 h of interferon treatment, a reduction of BrdUrd incorporation per cell, assessed by flow cytometry, was observed suggesting that DNA synthesis in S phase cells is almost completely abolished. The analysis of the effect of interferon on the distribution of cell cycle phases indicated that the G1/S transition is not inhibited by the treatment. These results support the hypothesis that interferon generates a transient initiating signal which quickly reaches the nucleus and produces a rapid inhibition of alpha polymerase activity, leading finally to the slowing of cell cycle progression.

Nuclear metabolic changes induced by tumor necrosis factor in Daudi lymphoma cells; a multiparametric analysis.

The effects of r-TNF alpha on cell cycle progression and DNA polymerase activity in Daudi lymphoma cells have been analyzed. Cytofluorimetric analysis of the cell cycle after 6 to 24 hr of treatment revealed both a decrease of BrdU incorporation per cell and a light inhibition of S phase as assessed by the analysis of the percentual distribution of cell cycle compartments. The reduction of BrdU incorporation can be related to the early decrease in the rate of DNA synthesis that follows r-TNF alpha treatment. These results suggest that one of the early events induced by r-TNF alpha at nuclear level is the slowering of DNA synthesis leading to a reduced cell cycle progression.

Nuclear lipids in Friend cells. Shifted profile of diacylglycerol during erythroid differentiation induced by DMSO.

Friend cells were labelled for 90 min. with [3H]-Glycerol and the radioactivity in DAG# and TG was measured. The relative percentage of radiolabelled DAG differs in isolated nuclei as compared to intact cells. Moreover the level of newly synthesized DAG decreases in nuclei isolated from cells treated with DMSO for 24 hrs. as well as in nuclei from cells treated for 96 hrs. and terminally differentiated in the erythroid pathway. Since these changes are not seen in intact cells, the results are consistent with the hypothesis that nuclear lipids and namely the products of polyphosphoinositide hydrolysis, such as DAG, are involved in the regulation of the processes leading to cell differentiation.