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Biomedical subjects

B Zhivotovsky

Publications and source records attributed to B Zhivotovsky.

At least 19 recordsLinked to original sources
PubMed2006

Functional connection between p53 and caspase-2 is essential for apoptosis induced by DNA damage.

Recent findings have established caspase-2 as an important apical regulator in apoptotic pathways leading from DNA damage to release of mitochondrial cytochrome c and subsequent activation of effector caspases. Yet, the molecular map connecting the embarking stimuli of genotoxic stress with caspase-2 activation remains to be elucidated. Here, we address the question of potential caspase-2 regulators by examining 5-fluorouracil (5-FU)-induced apoptosis in wild-type and p53-deficient human colon carcinoma cells. Apoptosis was observed only in p53(+/+) cells and was preceded by caspase-2 activation. Hence, although no direct interaction between p53 and caspase-2 was observed in the cell system used, our data clearly demonstrate that a functional connection between these two proteins is essential for initiation of the 5-FU-induced apoptotic process. Proposed mediators of caspase-2 activation include PIDDosome complex proteins PIDD and RAIDD. Surprisingly, the presence of a complex encompassing at least RAIDD, PIDD and caspase-2 was verified in both p53(+/+) and p53(-/-) cells, also in the absence of 5-FU treatment. Thus, our results confirm the participation of PIDD and RAIDD in PIDDosome complex formation but question their role as sole mediators of caspase-2 activation. This assumption was further supported by siRNA transfections targeting PIDD or RAIDD. In conclusion, our findings support the hypothesis of p53 as an upstream regulator of caspase activity and provide data concerning caspase-2 processing mechanisms. As suppression of caspase-2 expression in 5-FU-treated cells also affects the level of the p53 protein, possibilities of a reciprocal interaction between these proteins are discussed.

Apoptosis↗
PubMed2006

Early mitochondrial alterations in ATRA-induced cell death.

All-trans retinoic acid (ATRA) induces differentiation and subsequent apoptosis in a variety of cell lines. Using the myeloid cell line P39, we show that ATRA disturbs mitochondrial functional activity long before any detectable signs of apoptosis occur. These early changes include diminished mitochondrial oxygen consumption, decreased calcium uptake by mitochondria and as a result, a lower mitochondrial matrix calcium concentration. Granulocyte colony-stimulating factor (G-CSF) increases mitochondrial respiration and calcium accumulation capacity and subsequently blocks ATRA-induced apoptosis. Nifedipine, a plasma membrane calcium channel blocker, inhibits apoptosis-related changes, such as the loss of the mitochondrial membrane potential and activation of caspases. Thus, the properties of ATRA and G-CSF to modulate mitochondrial respiration and intracellular calcium control are novel findings, which give insight into their precise molecular mode of action.

Animals↗
PubMed2006

The role of p73 in hematological malignancies.

The P73 gene is a homologue of the P53 tumor suppressor. Owing to its structural similarity with p53, p73 was originally considered to have tumor suppressor function. However, the discovery of N-terminal truncated isoforms with oncogenic properties showed a 'two in one' structure of its product, p73 protein. The full-length variants are strong inducers of apoptosis, whereas the truncated isoforms inhibit proapoptotic activity of p53 and the full-length p73. Thus, p73 is involved in the regulation of cell cycle, cell death and development. Moreover, it plays a role in carcinogenesis and controls tumor sensitivity to treatment. p73 is commonly expressed in tumor cells in hematological malignancies. Overexpression of p73 protein and aberrant expression of its particular isoforms, with very low frequency of P73 hypermethylation or mutations, were found in malignant myeloproliferations, including acute myeloblastic leukemia. In contrast, hypermethylation and subsequent inactivation of the P73 gene are the most common findings in malignant lymphoproliferative disorders, especially acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas. Assessment of P73 methylation may provide important prognostic information, as was confirmed in patients with ALL. This review summarizes some aspects of p73 biology with particular reference to its possible pathogenetic role and prognostic significance in hematological malignancies.

Alternative Splicing↗
PubMed2004

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation.

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.

Apoptosis↗
PubMed2002

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid.

A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.

Adenylate Kinase↗
PubMed2001

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis.

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 microM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H(2)O(2)) exposure (100 microM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H(2)O(2) preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H(2)O(2) stimulated divergent pathways, with caspases being activated only by H(2)O(2). The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H(2)O(2), suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H(2)O(2). The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.

Apoptosis↗
PubMed2001

Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax.

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating this event remain elusive. In the present study, using isolated liver mitochondria, we demonstrate that cytochrome c release occurs via distinct mechanisms that are either Ca(2+)-dependent or Ca(2+)-independent. An increase in mitochondrial matrix Ca(2+) promotes the opening of the permeability transition (PT) pore and the release of cytochrome c, an effect that is significantly enhanced when these organelles are incubated in a reaction buffer that is based on a physiologically relevant concentration of K(+) (150 mm KCl) versus a buffer composed of mannitol/sucrose/Hepes. Moreover, low concentrations of Ca(2+) are sufficient to induce mitochondrial cytochrome c release without measurable manifestations of PT, though inhibitors of PT effectively prevent this release, indicating that the critical threshold for PT varies among mitochondria within a single population of these organelles. In contrast, Ca(2+)-independent cytochrome c release is induced by oligomeric Bax protein and occurs without mitochondrial swelling or the release of matrix proteins, although our data also indicate that Bax enhances permeability transition-induced cytochrome c release. Taken together, our results suggest that the intramitochondrial Ca(2+) concentration, as well as the reaction buffer composition, are key factors in determining the mode and amount of cytochrome c release. Finally, oligomeric Bax appears to be capable of stimulating cytochrome c release via both Ca(2+)-dependent and Ca(2+)-independent mechanisms.

Animals↗
PubMed2001

Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria.

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.

Animals↗
PubMed2001

Granulocyte colony-stimulating factor inhibits Fas-triggered apoptosis in bone marrow cells isolated from patients with refractory anemia with ringed sideroblasts.

Treatment with granulocyte colony-stimulating factor (G-CSF) plus erythropoietin may synergistically improve hemoglobin levels and reduce bone marrow apoptosis in patients with refractory anemia with ringed sideroblasts (RARS). Fas-induced caspase activity is increased in RARS bone marrow cells. We showed that G-CSF significantly reduced Fas-mediated caspase-8 and caspase-3-like activity and the degree of nuclear apoptotic changes in bone marrow from nine RARS patients. A decrease in mitochondrial membrane potential and an increase in intracellular reactive oxygen species occurred in Fas-treated cells, but became significant only 24 h after changes in caspase activity and decrease in proliferation. G-CSF also reduced the magnitude of these late apoptotic changes. In CD34-selected normal cells, G-CSF induced myeloid colony growth, and an overall small decrease in the number of erythroid colonies. By contrast, G-CSF induced a 33-263% increase of erythroid colony formation in CD34+ cells from four of five RARS patients with severely reduced erythroid growth, while the normal or slightly reduced erythroid growth of three other patients was not influenced by G-CSF. This study suggests that G-CSF may reduce the pathologically increased caspase activity and concomitant apoptotic changes, and promote erythroid growth and differentiation of stem cells from RARS patients. Our data support the clinical benefit of G-CSF in this subgroup of myelodysplastic syndromes.

Aged↗
PubMed2001

Apoptosis in refractory anaemia with ringed sideroblasts is initiated at the stem cell level and associated with increased activation of caspases.

Treatment with granulocyte colony-stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0.05) and caspase activity (P < 0.05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrophage CFU (CFU-GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU-E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.

Aged↗
PubMed2001

A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria.

A human milk fraction containing multimeric alpha-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of alpha-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of alpha-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (Delta Psi(m)), mitochondrial swelling and the release of cytochrome c. These changes were Ca(2+)-dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of Delta Psi(m), indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human alpha-lactalbumin made lethal to tumour cells), which is native alpha-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of alpha-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.

Animals↗