Amelia Cataldi

Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes.

Apoptosis and necrosis represent two different mechanisms by which cells die. The dynamics of cellular lesions in these two processes differ. In particular we demonstrate that plasma membrane damage, occurring as a primary event during necrosis represents, on the contrary, a delayed but massive phenomenon during apoptosis. In consequence there are different kinetics of propidium iodide incorporation by necrotic and apoptotic thymocytes. This represents the basis for the flow cytometric identification of different cellular subsets. Analysis of these subsets after sorting showed that clearly apoptotic cells, which are not able to exclude propidium iodide for long incubation periods, do not show any morphologically detectable membrane damage. The kinetics of propidium iodide incorporation in vivo in isolated rat thymocytes can therefore be used in flow cytometric analysis. This technique can be used instead of DNA staining of ethanol-treated cells or nick translation to recognize apoptotic cells, and distinguish apoptosis from necrosis, without killing the cell.

PI-3K/Akt and NF-κB/IκBα pathways are activated in Jurkat T cells in response to TRAIL treatment†

The aim of this work was to evaluate the involvement of survival pathways in the response of Jurkat T leukaemic cells sensitive to the cytotoxic action of tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL)/Apo2L. Jurkat T cells express TRAIL‐R2/DR5 and TRAIL‐R4/DcR2 receptors and start to die by apoptosis early (3 h) upon TRAIL administration reaching a dose‐dependent increase in the percentage of dead cells within 48 h (up to 85–90%). This increase in cell death is accompanied by a dose‐dependent significant (P < 0.05) increase in the G0/G1 phase of the cell cycle and reverted by the treatment with a broad inhibitor of caspases, z‐VAD‐fmk. Co‐treatment of the cells with inhibitors of PI‐3 kinase (LY294002) and nuclear factor kappa B (NF‐κB) (SN50) pathways leads to an earlier significantly increased cytotoxicity, respectively in the form of apoptosis and necrosis. Consistently with the data obtained with the pharmacological inhibitors, the activation and nuclear translocation of both PI‐3K and NF‐κB were observed. In summary, our results provide evidence that even in sensitive neoplastic cells TRAIL paradoxically activates pro‐survival pathways, which protect against TRAIL‐mediated death since their inhibition leads to an earlier and increased cytotoxicity.

Cyclin-Dependent Kinase Modulators and Cancer Therapy

The cell cycle of eukaryotic cells varies greatly from species to species and tissue to tissue. Since an erroneous control of the cell cycle can have disastrous consequences for cellular life, there are genetically programmed signals, so-called cell cycle checkpoints, which ensure that all events of each stage are completed before beginning the next phase. Among the numerous molecules involved in this process, the most important are the cyclin-dependent kinases (CDKs), proteins that are activated only when bound to cyclins (regulatory proteins with fluctuating concentrations).In general, more CDKs are overexpressed in cancer cells than in normal cells, which explains why cancer cells divide uncontrollably. Succeeding in modulating CDK activity with pharmacological agents could result in decreasing the abnormal proliferation rate of cancer cells.This review offers an overview of CDK-cyclin complexes in relation to different cell cycle phases, an analysis of CDK activation and inhibition of molecular mechanisms, and an extensive report, including clinical trials, regarding four new drugs acting as CDK modulators: alvocidib, P276-00, SNS-032 and seliciclib.

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.

Involvement of the pathway phosphatidylinositol-3-kinase/AKT-1 in the establishment of the survival response to ionizing radiation.

Ionizing radiation is one of the agents inducing activation of DNA repair, cell cycle arrest, apoptosis and cell death. Here we report evidence for an enhanced activity of DNA polymerase beta, one of the repair enzymes, concomitant to the activation of the pathway phosphatidylinositol-3-kinase/AKT-1 (PI-3-kinase/AKT-1), which delivers a survival signal in Friend erythroleukemia cells exposed to 15 Gy. Significantly, the preincubation of the cellls with PI-3-kinase inhibitors wortmannin and LY 294002, disactivating this pathway, sensitizes the cells to ionizing radiation by further reducing the rate of proliferation without substantial variations of the number of dead cells. Thus, we suggest a role for these enzymes in maintaining survival programs upon exposure to ionizing radiation and in giving to these cells a chance to recover from this stress.

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.

Ibuprofen and Lipoic Acid Diamides as Potential Codrugs with Neuroprotective Activity

Current evidences support the hypothesis that non‐steroidal anti‐inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimers disease (AD). In the present work, our attention was focused on ibuprofen (IBU) used in clinical trails to prevent Alzheimers disease, and (R)‐α‐lipoic acid (LA) considered as a potential neuroprotective agent in AD therapy. In particular, we investigated a series of lipophilic molecular combinations obtained by joining (R)‐α‐lipoic acid and ibuprofen via an amide bond. These new entities might allow targeted delivery of the parent drugs to neurons, where cellular oxidative stress and inflammation seem related to Alzheimers disease. Our study included the synthesis of conjugates 1–3 and the evaluation of their physicochemical and in‐vitro antioxidant properties. The new compounds are extremely stable in aqueous buffer solutions (pH = 1.3 and 7.4), and in rat and human plasma they showed a slow bioconversion to ibuprofen and (R)‐α‐lipoic acid. Codrugs 1–3 displayed in vitro free radical scavenging activity and were hydrolyzed more rapidly in brain tissue than in rat serum indicating that these new entities might allow targeted delivery of the parent drugs to neurons. The immunohistochemical analysis of Aβ (1‐40) protein showed that Aβ‐injected cerebral cortices treated with ibuprofen or compound 1 showed few plaques within capillary vessels and, in particular, Aβ (1‐40) protein was less expressed in codrug‐1‐treated than in ibuprofen‐treated cerebral cortex.

PI3-K/Akt-dependent activation of cAMP-response element-binding (CREB) protein in Jurkat T leukemia cells treated with TRAIL

We recently demonstrated the activation of phosphatidylinositol 3‐kinase (PI3‐K/Akt) survival pathway in Jurkat T leukemia cells known for their sensitivity to the tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL)/Apo2L cytotoxic action. The present investigation was done to elucidate the role of cAMP‐response element‐binding (CREB) protein in this system. Jurkat T cells were treated with 100–1,000 ng/ml TRAIL for time intervals up to 24 h in the presence or absence of selective pharmacologic inhibitors of PI3‐K/Akt (LY294002) or p38 MAPK (SB253580) pathways. Upon TRAIL treatment, a dose‐dependent increase in the percentage of apoptotic cells as well as in caspase‐3 activity was observed. A further enhancement of apoptotic cell death was obtained with the use of CREB1 siRNA technology, as demonstrated by flow cytometry. Western blot analysis showed a high constitutive level of CREB phosphorylation at Ser133 in Jurkat T cells under normal serum culture conditions. Under low serum culture conditions, an early (within 1 h) and transient increase in CREB phosphorylation was detected in response to both TRAIL doses and reduced upon pre‐treatment with LY294002 or SB253580, demonstrating the PI3‐K/Akt‐ and p38 MAPK‐dependency of this effect. The parallel analysis in immune fluorescence demonstrated the nuclear translocation of the phosphorylated form upon treatment with 100 ng/ml TRAIL, whereas the immune labeling was mainly detectable in the cytoplasm compartment upon the higher more cytotoxic dose. These results let us hypothesize that CREB activation can be an important player in the complex cross‐talk among pro‐ and anti‐apoptotic pathways in this peculiar cell model. J. Cell. Physiol. 214:192–200, 2008.

Transient shift of diacylglycerol and inositol lipids induced by interferon in Daudi cells Evidence for a different pattern between nuclei and intact cells

The effect of human recombinant DNA interferon‐α type A on inositol lipid and diacylglycerol metabolism was investigated in Daudi lymphoma whole cells and isolated nuclei. In isolated nuclei after 90 min of interferon treatment an enhanced rate of PIP2 phosphorylation and an increase of DAG mass were observed. In whole cells, after 1 min of interferon treatment, there was a rapid and transient shift of DAG mass apparently not related to inositol lipid modifications, thus indicating the presence in nuclear and cytoplasmic compartments of inositol lipid fractions with different metabolic features in response to interferon‐α.

Ibuprofen and Glutathione Conjugate as a Potential Therapeutic Agent for Treating Alzheimer's Disease

Non‐steroidal anti‐inflammatory drugs (NSAIDs) and antioxidant therapy might protect against the development of Alzheimers disease (AD). In the present work, we synthesized a molecular combination of glutathione (GSH) and ibuprofen (IBU) via an amide bond and investigated its potential for targeted delivery of the parent drugs to neurons, where cellular oxidative stress and inflammation are related to AD. Evaluation of its physicochemical and in‐vitro antioxidant properties indicated that compound 1 exhibits good stability toward human plasma enzymatic activity, and, like GSH, displays in‐vitro free radical scavenging activity in a time and concentration‐dependent manner. The new compound was also assessed by infusion in a rat model for Alzheimers disease for its potential to antagonize the deleterious structural and cognitive effects of β‐amyloid(1‐40). In behavioral tests of long‐term spatial memory, animals treated with codrug 1 performed significantly better than those treated with β‐amyloid (Aβ) peptide. Histochemical findings confirmed the behavioral data, revealing that Aβ protein was less expressed in cerebral cortex treated with 1 than that treated with IBU. Taken together, the present findings suggest that conjugate 1 treatment may protect against the oxidative stress generated by reactive oxygen species (ROS) and the cognitive dysfunction induced by intracerebroventricular (i.c.v.) infusion of Aβ(1‐40) in rats, and thus that codrug 1 could prove useful as a tool for controlling AD induced cerebral amyloid deposits and behavioral deterioration.