Phosphatidylserine externalization during bone marrow cell death after treatment of mice with WR-2721 and gamma-rays

The cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment were assessed in the bone marrow of adult male Swiss mice exposed to a single 6 Gy dose of 60 Co gamma-rays, and treated intraperitoneally with the aminothiol WR-2721 (Amifostine, S-2-/3-aminopropylamino/ethyl phosphorothioic acid), at a dose of 400 mg/kg body weight, 30 min prior to gamma-irradiation. The bone marrow cells were stained with a combination of fluoresceinated annexin V (annexin V--FITC) and propidium iodide (PI) at 3 h, 7 h, and 24 h after treatment of mice with WR-2721 and 60Co gamma-irradiation. The number of early apoptotic cells (annexin V--FITC positive/PI negative), and late apoptotic and necrotic cells (annexin V--FITC positive/PI positive), was increased at 3 h after exposure of mice to 60Co gamma-rays and thereafter declined with the frequency of apoptotic and necrotic cells remaining lower in WR-2721 pre-treated mice. Using the annexin V--FITC flow cytometric assay, the radioprotective effect of WR-2721 against induction of apoptosis and necrosis in normal cells of the haematopoietic system was shown.     

Radiation-induced DNA single-strand breaks in the intestinal mucosal cells of mice treated with the radioprotectors WR-2721 or 16-16 dimethyl prostaglandin E2

Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandin E2 (dm PGE2) protected the intestinal clonogenic cells to some degree from the effects of 137Cs gamma-irradiation. The D0 was increased from 1.1 +/- 0.12 Gy in controls to 1.55 +/- 0.48 Gy in 16-16 dm PGE2 treated and 2.12 +/- 0.20 Gy in WR-2721 treated mice. Both agents also increased the shoulder of the clonogenic-cell survival curve. Studies were done to measure the effects of these two different radioprotectors on radiation-induction of DNA single-strand breaks in cells comprising the murine intestinal mucosa. The number of DNA single-strand breaks increased with increasing doses of gamma-rays in animals killed immediately following exposure. WR-2721 reduced the number of initial radiation-induced DNA single-strand breaks when given one-half hour before exposure; the time of maximum protection. In contrast, 16-16 dm PGE2 given 1 hour before irradiation (the time required to afford maximum protection from radiation cytotoxicity) did not reduce the number of initial DNA breaks. Both agents impeded the rate of rejoining of DNA breaks with increasing time after irradiation. However, the relationship between these effects on the rate of strand rejoining and cell survival is unknown. These results suggest that either both agents are similarly distributed within the cells but the mechanisms of radioprotection are different, or the mechanisms by which these agents protect are similar, but the two agents affect different subcellular targets, the protection of which contributes to increased cell survival.     

Immunocytochemical analysis of apoptotic bone marrow cells after treatment of mice with WR-2721 and chemotherapeutic drugs

The effects of the aminothiol WR-2721 (Amifostine) and the chemotherapeutic drugs, cyclophosphamide (CP) and cisplatin (CDDP) on induction of apoptosis in bone marrow cells of adult male Swiss mice were studied. The mice received intraperitoneal injections of WR-2721 (400 mg/kg), cyclophosphamide (200 mg/kg), and cisplatin (10 mg/kg). WR-2721 was administered alone, or 30 min before treatment of mice with CP or CDDP. The number of apoptotic bone marrow cells was determined at 7 h and 24 h after the agent(s) administration. The In Situ Cell Death Detection Kit, AP based on TUNEL technique, and Fast Red Substrate System were used for microscopic analysis of immunocytochemically stained apoptotic cells. Application of cyclophosphamide and cisplatin resulted in a distinct increase of the number of apoptotic cells in the mouse bone marrow. After treatment of mice with WR-2721 prior to administration of CP or CDDP, as compared to the chemotherapeutic treatment only, the tendency to a decrease--albeit statistically insignificant--in the number of apoptotic cells was observed. Application of WR-2721 alone, without subsequent administration of the chemotherapeutic agents caused an inconsiderable increase in the number of apoptotic cells. The degree of apoptotic DNA cleavage in cells of the mouse bone marrow varied depending on the agent(s) given and the time interval after the drug administration.     

Application of Biotin,Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20–30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Application of Biotin, Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.     

Modulation of cellular defense processes in human lymphocytes in vitro by a 1,4-dihydropyridine derivative

The aim of this pilot study was to assess whether a compound of the β-carbonyl-1,4-dihydropyridine series (AV-153 or sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate), which has high efficiency in stimulating DNA repair, can simultaneously modulate apoptosis in human cells. Peripheral blood lymphocytes of healthy donors were used in this study. DNA strand-break rejoining was assessed with the alkaline comet assay after a 3-h incubation of lymphocytes in the presence of a wide range of concentrations of AV-153 (10⁻¹⁰–10⁻⁵ M). Apoptotic and micronucleated (MN) cells were scored in phytohaemagglutinin-stimulated lymphocytes after a 72-h incubation with AV-153, using the standard cytokinesis-blocked micronucleus test. The study revealed dual effects of AV-153 on cellular defense systems against endogenously generated DNA damage: the compound per se simultaneously reduces DNA strand breaks and stimulates apoptosis, with a maximal efficiency of 76% and 42%, respectively; in contrast, after genotoxic stress (2 Gy of gamma-radiation) AV-153 reduces DNA strand breaks, the number of MN cells and apoptotic cells in a similar dose-dependent manner. A maximal efficiency of 67% was found for reduction of DNA strand breaks, while for MN cells and apoptotic cells the efficiencies were, respectively, 47% and 44%. While limited in number, these preliminary studies show the direct correlation between the efficiency of AV-153 in reduction of radiation-induced DNA breaks and MN cells on one side, and in reduction of apoptosis on the other. It suggests that the major target of the compound's action on genotoxic stress is DNA repair, followed by reduction of the number of damaged cells entering apoptosis.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

Postirradiation glucan administration enhances the radioprotective effects of WR-2721

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucan's ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.     

Splenic lymphocytes of adult Xenopus respond differentially to PMA in vitro by either dying or dividing: significance for cancer resistance in this species

Wild-type populations of amphibians, unlike mammalians, appear to be resistant to spontaneous and chemically induced neoplasms. Few true cancers have been reported for non-isogeneic members of Xenopus laevis, despite their widespread use in laboratories around the world. Injection of even the most powerful direct mammalian oncogens e.g. N-methyl N-nitrosourea, that depleted specific populations of T lymphocytes, did not induce cancer. Phorbol diesters, e.g. PMA, are mitogens and apoptogens in both amphibian, and mammalian immunocytes. In mammalian cells, regulation of the cell cycle and of apoptosis are often intimately linked, however, a disjunction in time between early apoptosis and later cell cycling, has been observed with PMA-treated Xenopus splenocytes. Thus, a particular difference between amphibians and mammals may be the requirement to enter the cell cycle before a progression to death by apoptosis. This hypothesis was tested here using dual staining flow cytometry. Xenopus laevis splenocytes were cultured for 8, 24 and 48 hours with phorbol 12-myristate 13-acetate (PMA), previously shown to be mitogenic and apoptotic with mature Xenopus lymphocytes. The cells were stained with FITC-conjugated Annexin V or with FITC-labeled deoxyuridine triphosphates (FITC-dUTP) to assay for the apoptotic markers phosphotidylserine or DNA strand breaks respectively. Phycoerythrin (PE)-conjugated anti-human proliferating cell nuclear antigen (PE-PCNA) was used as a cell cycle marker that is present during the entire cell cycle. Propidium iodide (PI) binds DNA and was used to assay for late stage apoptosis, as well as to assess DNA content.Significantly higher levels of apoptosis develop rapidly in PMA-exposed splenocytes and are maintained at 24 hours, declining by 48 hours. Cells expressing PCNA or incorporating PI in excess of the normal genomic level were found by 48 hours following PMA exposure. The absence of any significant rise in a small (<5%) dual staining cell population indicates that the apoptotic cell population remained distinct from cells already in the cell cycle from the onset of PMA exposure. Thus, Xenopus splenocytes respond differentially to PMA. Those that undergo apoptosis rapidly were quiescent, non-cycling small lymphocytes. Moreover, the cells that eventually begin division, following PMA exposure, were unaffected by the early apoptois and do not themselves die while in the cell cycle. The rapid apoptotic response of X. laevis cells to PMA may confer a natural cancer resistance in this species, as cells that fail to enter the cell cycle after exposure to cancer promoting reagents cannot express genetic destabilization that might have led to transformation.     

Radiation-induced DNA double-stranded breaks and the dynamics of apoptotic death of human peripheral lymphocytes

DNA double-stranded breaks and their association with the development of radiation-induced peripheral lymphocyte apoptosis were studied in healthy donors exposed to in vitro gamma-irradiation in a dose of 1 Gy. It was shown that irradiation in 1-Gy dose caused a significant (p < 0.05) increase in the frequency of cells in late apoptosis 4 hours after irradiation and a rise in their frequency in early apoptosis 24 hours following this procedure. A significant correlation (r = 0.52, p < 0.05) was recorded between the primary level of radiation-induced DNA double-stranded breaks and the frequency of cells in late apoptosis following 4 hours, which suggests that DNA double-stranded breaks as a signal to trigger cell apoptotic death are of great importance.     

Radioprotective effect of combinations of WR-2721 and mercaptopropionylglycine on mouse bone marrow chromosomes

The radioprotective and toxic effects of low to moderate doses of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) and its combination with mercaptopropionylglycine (MPG, 20 mg/kg body wt) on the chromosomes of the bone marrow cells of Swiss albino mice were studied at 24 h and 14 days postirradiation. Significant protection against radiation-induced chromosome aberrations was observed with 50 mg/kg WR-2721. The protection increased with the dose of the drug administered, and the degree of protection per unit dose increment was more pronounced at lower than at higher doses. A combination of WR-2721 and MPG given before exposure resulted in a significantly greater number of normal metaphases at 24 h postirradiation compared to the respective single-drug treatment groups. On Day 14 postirradiation, when the presence of WR-2721 resulted in an increase in the frequency of aberrant cells, combination with MPG helped to reduce this value markedly, especially at WR-2721 doses below 200 mg/kg. On the basis of these results it is suggested that 150 mg/kg WR-2721 may be considered an optimum dose for combination with MPG for protection of chromosomes of bone marrow cells when repeated drug administrations are not needed. Changes in the level of glutathione (GSH) in the blood were studied at different times following the administration of 150 mg/kg WR-2721 and its combination with MPG (20 mg/kg) before sham irradiation or exposure to 4.5 Gy 60Co gamma rays. The results showed that WR-2721 elevated blood GSH levels significantly above normal values by the time radiation was delivered, while MPG did not. Glutathione appears to have an important role in the action of WR-2721, while protection by MPG may not be mediated through GSH. Injection of MPG after WR-2721 helps to maintain the higher GSH level for a longer duration compared to treatment with WR-2721 alone. It is possible that MPG delays the metabolism of GSH.     

Protection of bone marrow of Swiss albino mouse against whole body gamma irradiation by WR-2721.

Preinjected with a radioprotective drug, WR-2721, the Swiss albino mice were whole body irradiated with 5 Gy of 60Co gamma rays. The animals were sacrificed at different intervals and bone marrow films were prepared for differential counting of lymphocytes, pronormoblasts and normoblasts and granulocytes. The results indicated significant protection of the bone marrow cells by the drug against radiation induced damage. It is therefore concluded that WR-2721 protects all types of cells including as sensitive ones as lymphocytes, pronormoblasts and normoblasts.     

Intact Cell Evidence for the Early Synthesis, and Subsequent Late Apopain-Mediated Suppression, of Poly(ADP-ribose) during Apoptosis

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after ∼4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual,intact cells,demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.     

Modification of radiation induced damage in mouse intestine by WR-2721

Intestinal protection in mice against radiation injury by WR-2721 (300 mg/kg body wt, i.p., 30 min before irradiation) was studied after whole body gamma irradiation (0.5, 1.5, 3.0, 4.5, or 6.0 Gy). Crypt survival and induction of apoptosis, and abnormal mitoses in crypt cells in the jejunum were studied on day 1, 3 and 7 after irradiation. Irradiation produced a significant decrease in crypt survival, whereas apoptosis and abnormal mitoses showed a significant increase from sham-treated control animals. Maximum changes in all the parameters were observed on day 1 after irradiation and the effect increased linearly with radiation dose. There was recovery at later intervals, which was inversely related to radiation dose. WR-2721 pre-treatment resulted in a significant increase in the number of surviving crypts, whereas the number of apoptotic cells in the crypts showed a significant decrease from respective irradiated controls on day 1 after exposure. The recovery was also faster in WR-2721 pre- treated animals. It is concluded that WR-2721 protects against gastrointestinal death by reducing radiation induced cell death, thereby maintaining a higher number of stem cells in the proliferating compartment.     

Singapore grouper iridovirus,a large DNA virus,induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling

Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (ΔΨm) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.     

Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1

The effect of synthetic isothiocyanate ethyl-4-isothiocyanatobutanoate (E-4IB) on survival of mismatch repair-proficient TK6 and -deficient MT1 cell lines as well as the influence of proteasomal inhibitor MG132, caspase inhibitor Z-VAD-fmk, and ATM inhibitor caffeine on E-4IB modulation of cell cycle and apoptosis was evaluated. Flow cytometric analyses of DNA double strand breaks (γ-H2AX), mitotic fraction (phospho-histone H3), cell cycle modulation, apoptosis induction (sub-G₀ fraction and fluorescein diacetate staining), and dissipation of transmembrane mitochondrial potential (JC-1 staining) were performed. Western blotting was used for the evaluation of ERK activation, expression of p53, p21^cip1/waf1 and GADD45α proteins, as well as PARP fragmentation. Analysis of mitotic nuclei was performed for chromosomal aberrations assessment. MT1 cells were more resistant to E-4IB treatment then TK6 cells (IC₅₀ 8 μM vs. 4 μM). In both cell lines E-4IB treatment induced phosphorylation of H2AX, increase of p53 protein level, phospho-histone H3 staining, and G₂/M arrest. The sub-G₀ fragmentation was accompanied by PARP degradation, decreased mitochondrial transmembrane potential, and diminished p21^cip1/waf1 protein expression in TK6 cells. Caspase inhibitor Z-VAD-fmk decreased E-4IB induced sub-G₀ fragmentation and extent of apoptosis in TK6 cells, while proteasome inhibitor MG132 increased number of apoptotic cells in both cell lines tested. A number of aberrant metaphases and clastogenic effect of high E-4IB concentration was observed. The synthetic isothiocyanate E-4IB induced DNA strand breaks, increased mitotic fraction and apoptosis potentiated by MG132 inhibitor in both mismatch repair-proficient and -deficient cell lines. This work was supported in part by Slovak Governmental Research and Development sub-program Food-quality and safety No. 2003SP270280E010280E01, National Program “Use of Cancer Genomics to Improve the Human Population Health”, project 2003 SP 510280800/0280801, European Commission project (QLG1-CT-2000-01230), and VEGA projects 2/4069 and 2/3161/23.     

Modification of cyclophosphamide-induced clastogenesis and apoptosis in rats by alpha-lipoic acid

The aim of the present study was to investigate the protective efficacy of alpha-lipoic acid (LA) on the cyclophosphamide (CP)-induced chromosomal aberrations (CA) and apoptosis in the bone marrow of rats. Male Wistar rats of 140+/-20 g were categorized into eight groups. Five groups were administered CP (40 mg/kg body weight, intraperitoneally) to induce toxicity; four of these groups received a single intraperitoneal injection of LA at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to CP administration. A vehicle-treated control group and LA control groups were also included. Twenty-four hours after CP treatment, the frequency of CA in bone marrow cells were significantly increased in comparison with the controls. The CP-induced CA were associated with significant increase in DNA damage in the bone marrow as evidenced by increased single strand breaks, whereas in rats treated with LA and CP, the frequency of CA and single strand breaks were significantly decreased in comparison to those given CP alone. CP administration distinctly triggered the apoptotic and necrotic cell death, and LA pretreatment affected cell death by decreasing the number of apoptotic and necrotic cells. The protective effect of LA was found to be stronger at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage, indicating the dose dependent protective effect of LA. However, the protection by LA was not dependent on the time intervals between LA and CP administration. The results of this study illustrate the protective effect of LA on the CA and apoptosis induced by CP in the erythropoietic system of rats.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> glycosylphosphatidylinositols are not involved in <Emphasis Type="Italic">T. gondii</Emphasis>-induced host cell survival

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.     

Analysis of apoptosis by cytometry using TUNEL assay

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.