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B Zhivotovsky

Publications and source records attributed to B Zhivotovsky.

At least 55 records · Page 3Linked to original sources
PubMed1999

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis.

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.

Adenylate Kinase↗
PubMed1999

A comparative study of apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to necrosis.

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to various agents. In the present study the HepG2 cell line was used for a comparative study of CD95-mediated apoptosis and menadione-induced necrosis. Apoptosis coincided with the release of cytochrome c from mitochondria, activation of caspases, cleavage of cellular proteins, and also involved nuclear condensation and DNA fragmentation. Necrosis was not accompanied by DNA fragmentation, caspase activation or cleavage of caspase target proteins, despite cytochrome c release from mitochondria. In fact, the addition of menadione to cells undergoing CD95-mediated apoptosis blocked their caspase activity. Inhibition of caspases coincided with an accumulation of reactive oxygen species (ROS) and ATP depletion. In order to determine the predominance of either of these events in the inhibition of caspase, cells were either co-incubated with antioxidant enzymes or their ATP level was manipulated to maintain it at a relatively high level during the experiments. Co-incubation with catalase, but not Cu/Zn superoxide dismutase, substantially reduced the levels of ROS and reversed the inhibitory effect of menadione on caspase activity. In contrast, increasing cellular ATP level had little effect on restoring caspase activity. These data suggest that menadione inhibits caspase activity by the generation of hydrogen peroxide through redox cycling and that caspase inactivation by this mechanism may prevent cell death by apoptosis in this oxidative-stress model.

Apoptosis↗
PubMed1999

Multimeric alpha-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei.

A fraction from human milk containing spf-multimer alpha-lactalbumin (MAL) induces apoptosis in tumor cells and immature cells but spares mature cells. The mechanism of apoptosis induction and the molecular basis for the difference in susceptibility between tumor cells and healthy cells have not been defined. In this study we examined the interaction of MAL with different cellular compartments, using confocal microscopy and subcellular fractionation. MAL was shown to accumulate in the nuclei of sensitive cells rather than in the cytosol, the vesicular fraction, or the ER-Golgi complex. Nuclear uptake occurred rapidly in cells that were susceptible to the apoptosis-inducing effect, but not in nuclei of resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of DNA fragmentation, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from induction of DNA fragmentation. Ca2+ was required for MAL-induced DNA fragmentation but nuclear uptake of MAL was independent of Ca2+. This way MAL differs from most previously described agents in that it crosses the plasma membrane and cytosol, and enters cell nuclei where it induces DNA fragmentation through a direct effect at the nuclear level.

Animals↗
PubMed1999

Two pathways of apoptosis induced with all-trans retinoic acid and etoposide in the myeloid cell line P39.

P39/Tsugane is a myelomonocytoid cell line derived from a patient with myelodysplastic syndrome (MDS). The cells readily undergo apoptosis in response to various agents, and the cell line has been suggested as a useful model to study apoptosis in MDS. The aims of the present study were to assess differentiation and apoptosis induced with all-trans retinoic acid (ATRA) and etoposide, to characterize the mode of apoptosis in these two model systems, and to assess the influence of granulocyte colony-stimulating factor (G-CSF), which in combination with erythropoietin has been shown to inhibit apoptosis in MDS. ATRA induced differentiation and apoptosis in a concentration- and time-dependent manner. Differentiated cells were partially rescued (by 50%) from apoptosis with G-CSF. Etoposide induced apoptosis in a concentration- and time-dependent manner, but no signs of preceding maturation or G-CSF rescue were detected. ATRA- and etoposide-induced apoptosis were both mediated through the caspase pathway and were partially blocked with the general caspase inhibitor zVAD-fmk. Simultaneous treatment with G-CSF and zVAD-fmk additively blocked ATRA-induced apoptosis. However, the two pathways differed in terms of substrate cleavage during apoptosis. ATRA-induced apoptosis caused actin cleavage, which was not affected by G-CSF, and Bcl-2 downregulation. Etoposide induced a caspase-dependent cleavage of Bcl-2, while actin remained intact. The Fas system did not seem to play a major role in any of these apoptotic pathways. Our results may provide new tools to study the mechanisms of apoptosis in MDS.

Actins↗
PubMed1999

DNA-dependent protein kinase content and activity in lung carcinoma cell lines: correlation with intrinsic radiosensitivity.

Intrinsic radiosensitivity and rejoining of radiation-induced DNA double-strand breaks (DNA-dsb) were analysed in five lung carcinoma cell lines: U-1285, U-1906, H-69, H-82 and U-1810. RS correlated with both the initial phase of DNA-dsb rejoining, at 15 min (r2 = 0.818) and the late phase, at 120 min postirradiation (r2 = 0.774), the most sensitive cell line (U-1285) showing least dsb rejoining and the most resistant (U-1810) showing most dsb rejoining of all five cell lines studied. As DNA-PK has been recognised as an important molecular component involved in DNA-dsb repair, we analysed content and activity of this kinase. We found that DNA-PK content and activity correlated with RS (r2 = 0.941 and r2 = 0.944, respectively). The lowest DNA-dependent content/activity was found in the most radiosensitive cells, U-1285 and H-69, whilst the highest content/activity was found in the most radioresistant cells U-1810. These results suggest a correlation between RS and DNA-PK content/activity in lung carcinoma cell lines.

Blotting, Western↗
PubMed1999

Caspases: their intracellular localization and translocation during apoptosis.

The activation of the caspase family of proteases has been detected in numerous cell systems and appears to function as a common pathway through which apoptotic mechanisms may operate. Caspases are synthesized as precursors (pro-caspases) and are converted into mature enzymes by apoptotic signals. The effects of caspases in apoptosis are accomplished by the cleavage of numerous proteins located in different intracellular compartments. In the present study we have addressed the question of the subcellular localization of different pro- and active caspases as well as several other proteins, such as Apaf-1, calpain and DFF, which also play important roles in the apoptotic process. We found that at least three pro-caspases (pro-caspases-2, -3 and -9) were present in both the mitochondrial and cytosolic fractions of untreated Jurkat T lymphocytes. Only pro-caspase-2 was found in the nuclear fraction. Pro-caspases-7 and -8 were found only in the cytosolic fraction. In apoptotic cells, caspases-3, -8 and -9 were present in the cytosolic fraction, whereas caspases-3 and -9 were also found in the mitochondrial fraction and caspase-7 in the microsomal fraction. Caspases-2 and -3 were present in the nuclear fraction. The selective localization of pro-caspases in different subcellular compartments may play an important, but yet unknown, role in their activation. The translocation of active caspases to other subcellular compartments appears to be critical for the development of the apoptotic process.

Apoptosis↗
PubMed1999

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors.

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.

3T3 Cells↗
PubMed1999

Cleavage of Bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells.

The proto-oncogene product Bcl-2 protects a wide variety of cell types from apoptosis via a hitherto unknown mechanism. Bcl-2 has been shown to function upstream of the death proteases (caspases) in some, but not all, occurrences of apoptotic cell death. Using the myeloid leukemic cell line P39 we report the chemotherapy-induced caspase-dependent cleavage of endogenous Bcl-2. Etoposide treatment of these cells triggered a time-dependent activation of type II and type III caspases and cleavage of Bcl-2 yielding a 23 kDa cleavage fragment. The emergence of this cleavage product was blocked by the general caspase inhibitor zVAD-fmk, as well as the type III caspase inhibitor IETD-fmk and the caspase-9-selective inhibitor LEHD-fmk, while the type II caspase inhibitor DEVD-fmk proved considerably less efficient. Bcl-2 cleavage preceded cleavage of the known caspase-3 substrate, poly(ADP-ribose) polymerase (PARP), as well as that of the caspase-6 substrate, lamin B, indicating that Bcl-2 cleavage is a relatively early event in the apoptosis cascade in this experimental model. While evidence for cleavage of Bcl-2 in several subcellular compartments of etoposide-treated cells was obtained, this cleavage was detected predominantly in the mitochondrial fraction, thus providing further support for the central role of mitochondria in apoptosis. Caspase-mediated cleavage following etoposide treatment of these myeloid leukemic cells may represent a means for the attenuation of Bcl-2 function upon apoptosis induction.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Cytochrome c release and caspase-3 activation during colchicine-induced apoptosis of cerebellar granule cells.

The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal populations including cerebellar granule cells (CGCs). In this study we investigated the involvement of cytochrome c release and caspase-3 activation during colchicine-induced CGC apoptosis. Treatment of rat CGCs with 1 micrometer colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II caspases (which includes caspase-3) was demonstrated by the proteolytic degradation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchicine. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alpha-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which was blocked with a specific, peptide-based, aldehyde inhibitor of group II caspases, i. e. DEVD-CHO. We also observed a time-dependent proteolysis of caspase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptosis may be mediated by cytochrome c since there was a close correlation between the time courses of cytochrome c release from the mitochondria and of caspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Androgen treatment of neonatal rats decreases susceptibility of cerebellar granule neurons to oxidative stress in vitro.

Oxidative stress has been implicated in various neurodegenerative diseases. There is substantial evidence indicating that gonadal hormones can affect neuronal cell survival via both a genomic as well as a non-genomic mode of action. In the present study, the potential protective activity of testosterone on neuronal cells was investigated by using an in vitro/ex vivo model. Cerebellar granule cells (CGC) were prepared from 7-day-old rats which had been treated with a single dose of oil or testosterone propionate on postnatal day 3. After 7 days in culture, cells were exposed to oxidative challenges, including hydrogen peroxide and the nitric oxide donor S-nitrosocysteine (SNOC), which can induce CGC death via apoptosis. Colchicine, which causes apoptosis via a different mechanism, was also used. The cells were monitored for apoptotic morphology by propidium iodide and TUNEL staining. Additionally, the presence of chromatin fragmentation was determined. CGC obtained from testosterone-treated rats were found to be more resistant to hydrogen peroxide and nitric oxide toxicity, as shown by a 75 and 45% decrease in apoptotic cells, respectively. In contrast, the susceptibility to colchicine was not modified. As CGC from testosterone-treated pups were selectively protected from oxidative stress, different components of the antioxidant defence systems were analysed. A twofold increase in the activity of catalase and superoxide dismutase was found in the CGC prepared from testosterone-treated rats. These results suggest that in vivo treatment with androgens render CGC less vulnerable to oxidative stress-induced apoptosis by potentiating antioxidant defences.

Animals↗
PubMed1999

Epstein-Barr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) obtained from unaffected healthy individuals and ataxia telangectasia (A-T) patients to undergo apoptosis after X-ray exposure. MATERIAL AND METHODS: The LCL were exposed to 1-4 Gy X-rays at a dose-rate of 1.36 Gy/min. At various post-irradiation times (0, 24, 48 and 72 h) the induction of apoptosis was analysed by: (1) monitoring the formation of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescence staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, was investigated in these cells. RESULTS: The LCL obtained from the A-T homozygotes were resistant to undergoing radiation-induced apoptosis during the observation time used. On the contrary, LCL from unaffected healthy controls displayed significant radiation-induced chromatin fragmentation seen at 48 h and 72 h after irradiation. In these cells, radiation-induced G -arrest (24h post-irradiation) preceded chromatin cleavage. In A-T LCL, the defective G1-arrest was not followed by apoptosis. CONCLUSIONS: In spite of a defective cell-cycle control, EBV-transformed LCL of A-T patients compared with unaffected healthy controls do not undergo X-ray-induced apoptosis, at least during their first post-irradiation cell cycle.

Apoptosis↗
PubMed1999

All along the watchtower: on the regulation of apoptosis regulators.

Members of the expanding family of Bcl-2-like proteins have emerged as important regulators of programmed cell death, and recent studies have unearthed numerous mechanisms for regulating the function of these death agonists and antagonists. In addition to the transcriptional control of gene expression, these mechanisms include posttranslational events such as phosphorylation, proteolysis, and the induction of conformational changes, which may either activate or inactivate these molecules. Interaction with homologous and nonhomologous proteins and specific subcellular targeting of Bcl-2-like proteins are other means of fine-tuning the cellular response to noxious stimuli. Recently, considerable attention has turned to the regulation of so-called BH3-only molecules, which appear to act as stress sensors that relay signals to other pro- or antiapoptotic family members. We discuss how the regulation of these apoptosis regulators may control the ultimate fate of the cell.

Apoptosis↗
PubMed1998

Glucose and tolbutamide induce apoptosis in pancreatic beta-cells. A process dependent on intracellular Ca2+ concentration.

High concentrations of glucose are considered to be toxic for the pancreatic beta-cell. However, the mechanisms underlying beta-cell dysfunction and resulting cell death are not fully characterized. In the present study we have demonstrated that incubation of pancreatic islets and beta-cells from ob/ob mice and Wistar rats with glucose induced a process of apoptotic beta-cell death, as shown by DNA laddering, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and by using DNA-staining dye HOECHST 33342. The obtained results show that the percentage of apoptotic cells was dependent on glucose concentration, being minimal at 11 mM glucose. At a concentration of 100 microM, aurintricarboxylic acid, an inhibitor of endonuclease activity, almost completely inhibited apoptosis triggered by 17 mM glucose. We have also shown that long term incubation with 100 microM sulfonylurea, tolbutamide, triggered apoptosis in pancreatic beta-cells. The process of beta-cell death induced by high glucose concentration and tolbutamide were Ca2+-dependent, because introduction to the culture medium of 50 microM D-600 or 200 microM diazoxide, which blocked glucose- and tolbutamide-induced [Ca2+]i increase, inhibited apoptosis. Thus, this study shows for the first time that high glucose concentrations and tolbutamide induce apoptosis in pancreatic beta-cells, and that this process is Ca2+-dependent.

Animals↗
PubMed1998

Detection of pro-caspase-3 in cytosol and mitochondria of various tissues.

Caspases are a family of cysteine proteases of critical importance in the apoptotic cell death process. They are normally present as zymogens (pro-caspases) in the cytoplasm of vertebrate and other organisms. In this study we have shown that pro-caspase-3 is localized to cytosol and mitochondria of various rat tissues (brain, heart, kidney, liver, spleen and thymus). Although the majority of pro-caspase-3 was localized in the cytosol, the amount of mitochondrial pro-caspase-3 was significant. The ratio of cytosolic and mitochondrial pools of pro-caspase-3 appeared to vary between different tissues. The higher amount of mitochondrial pro-caspase-3 was found in thymus and spleen, i.e. tissues in which spontaneous apoptosis plays an important role. Our findings provide further support for mitochondrial localization of pro-caspase-3 and the critical role of this organelle in apoptosis.

Animals↗
PubMed1998

Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts.

The export of cytochrome c from mitochondria to the cytoplasm has been detected during apoptosis. Addition of cytochrome c to cytosolic extracts can activate the caspases, suggesting that this export could be an important intracellular signal for initiating the apoptotic programme. We have investigated the mechanism of caspase activation by cytochrome c. Mitochondrial cytochrome c normally shuttles electrons between complexes III and IV of the electron transport chain. Interaction with these complexes is dependent on electrostatic interactions via a polylysine binding pocket. Cytosolic caspase activation was only observed with intact holocytochrome c, and increasing the ionic composition of the extracts prevented activation, suggesting that stringent allosteric interactions between cytochrome c and other cytoplasmic factors are necessary. Cytochrome c was fully reduced within 5 min of addition to the cytosolic extracts. Potassium ferricyanide could maintain cytochrome c in an oxidized state, but care was taken to use ferricyanide at concentrations where its polyanion effect did not cause interference. The oxidized form of cytochrome c was able to activate the caspases. We conclude that reduced cytochrome c will function in the cytoplasm; however, its reduction is not a critical step, and electron transfer from cytochrome c to its cytoplasmic-binding partner(s) is not necessary in the pathway leading to apoptosis.

Adenosine Triphosphate↗