Increased cell surface exposure of phosphatidylserine on propidium iodide negative thymocytes undergoing death by necrosis.

Phosphatidylserine (PS) exposure on propidium iodide negative cells using FITC labelled annexin-V has been used to quantify apoptosis in vitro and in vivo. Detection of PS within cells undergoing necrosis is also possible if labelled annexin-V specific for PS enters the cell following early membrane damage. Necrotic or late apoptotic cells can be excluded from flow cytometric analysis using propidium iodide which enters and stains cells with compromised membrane integrity. Here we show that thymocytes undergoing death exclusively by necrosis show early exposure of PS prior to loss of membrane integrity. This early exposure of PS occurs in cells treated with agents which both raise intracellular calcium levels and are also capable of interacting with protein thiol groups. We also demonstrate that PS exposure in thymocytes induced to undergo apoptosis by three different agents does not correlate with calcium rises but correlates with and precedes DNA fragmentation.

Antitumor activity of gold(i), silver(i) and copper(i) complexes containing chiral tertiary phosphines.

The in vitro cytotoxicities of a number of gold(I), silver(I) and copper(I) complexes containing chiral tertiary phosphine ligands have been examined against the mouse tumour cell lines P815 mastocytoma, B16 melanoma [gold(I) and silver(I) compounds] and P388 leukaemia [gold(I) complexes only] with many of the complexes having IC(50) values comparable to that of the reference compounds cis-diamminedichloroplatinum(ll), cisplatin, and bis[1,2-bis(diphenylphosphino) ethane]gold(I) iodide. The chiral tertiary phosphine ligands used in this study include (R)-(2-aminophenyl)methylphenylphosphine; (R,R)-, (S,S)- and (R(*),R(*))-1,2-phenylenebis(methylphenylphosphine); and (R,R)-, (S,S)- and (R(*),R(*))-bis{(2-diphenylphosphinoethyl)phenylphosphino}ethane. The in vitro cytotoxicities of gold(I) and silver(I) complexes containing the optically active forms of the tetra(tertiary phosphine) have also been examined against the human ovarian carcinoma cell lines 41M and CH1, and the cisplatin resistant 41McisR, CH1cisR and SKOV-3 tumour models. IC(50) values in the range 0.01 - 0.04 muM were determined for the most active compounds, silver(I) complexes of the tetra(tertiary phosphine). Furthermore, the chirality of the ligand appeared to have little effect on the overall activity of the complexes: similar IC(50) data were obtained for complexes of a particular metal ion with each of the stereoisomeric forms of a specific ligand.

Apoptosis induced by gliotoxin is preceded by phosphorylation of histone H3 and enhanced sensitivity of chromatin to nuclease digestion.

The fungal toxin gliotoxin induces apoptotic cell death in a variety of cells. Apoptosis induced in thymocytes by gliotoxin is rapid, and DNA fragmentation is observable within 4 h treatment. Apoptosis induced by gliotoxin is calcium-independent and unaffected by protein synthesis inhibitors. We have previously shown that gliotoxin results in phosphorylation of a 16.3-kDa protein within 10 min treatment of thymocytes. Here we show that this protein is histone H3 and phosphorylation occurs on Ser-10. Cyclic AMP levels and activity of protein kinase A (PKA) are raised in cells treated with gliotoxin. Apoptosis is inhibited by genistein which also inhibits PKA and histone H3 phosphorylation. Apoptosis is also inhibited by a number of specific inhibitors of PKA suggesting apoptosis induced by gliotoxin is modulated by this kinase. The agents forskolin and cholera toxin do not induce rapid phosphorylation of H3 although some increase in phosphorylation of H3 does occur after 8 h with these agents. Forskolin and cholera toxin also induce apoptosis but over a longer time course than gliotoxin. In all cases levels of apoptosis correlate with degree of H3 phosphorylation. Cells treated with gliotoxin show an early sensitivity to micrococcal nuclease and DNase I digestion indicating a functional relationship between DNA fragmentation and H3 phosphorylation.

DNA synthesis precedes gliotoxin-induced apoptosis.

The toxin gliotoxin induces apoptosis or programmed cell death in a variety of immune cells including thymocytes. Apoptosis induced by gliotoxin in thymocytes is unaffected by protein synthesis inhibitors nor is it associated with early changes in intracellular calcium levels (Beaver and Waring, 1994). This work shows that the cell lines P815 and WEHI7 and murine thymocytes when treated with gliotoxin show an early incorporation of tritiated thymidine over the concentration range which causes apoptosis. Proliferating cell nuclear antigen (PCNA), a marker for S phase, is elevated in cells following gliotoxin treatment and S phase DNA content is increased. Thymidine incorporation is inhibited by hydroxyurea, an inhibitor of replicative DNA synthesis not repair. Free radical scavangers have no effect on apoptosis induced by gliotoxin in thymocytes. Hydrogen peroxide-treated cells showed no enhanced thymidine incorporation and no apoptosis. Thus oxidative stress does not appear to be a factor in gliotoxin-induced apoptosis. Thymocytes treated with gliotoxin show increased phosphorylation of a 16.3 kDa protein, and apoptosis is inhibited by the tyrosine kinase inhibitor genistein, which also inhibited the increased thymidine incorporation in P815 cells. We conclude that one mechanism by which gliotoxin can cause apoptosis may be the induction of inappropriate entry of cells into the cell cycle followed by death.

Extracellular calcium is not required for gliotoxin or dexamethasone-induced DNA fragmentation: a reappraisal of the use of EGTA.

The immunomodulating agent gliotoxin and related toxins cause apoptotic cell death in a variety of cell types including macrophages and thymocytes [Waring et al. (1988) J. biol. Chem., 263, 18,493-18,499; Waring et al. (1990) Int. J. Immunopharmac., 12, 445-457]. The mechanism of induction of apoptosis by gliotoxin is not yet known, although it does not require protein synthesis [Waring (1990) J. biol. Chem., 14,476-14,480], unlike dexamethasone-induced apoptosis in thymocytes. Because of the reported requirement for extracellular calcium in apoptosis induced by dexamethasone, we studied the effects of extracellular calcium on gliotoxin-induced apoptosis in macrophages. Initial experiments using calcium-depleted media showed no inhibition of apoptotic DNA fragmentation by gliotoxin. By measuring residual 45Ca2+ remaining in cells pulsed with labelled calcium over the time period required for DNA fragmentation, we could demonstrate some uptake of extracellular calcium into treated cells as assessed by residual, slowly exchanging calcium. When cells were treated with the calcium chelator EGTA at 0.5-2 mM, calcium uptake was abolished but DNA fragmentation was unaffected. EGTA at higher concentrations, up to 8 mM, did inhibit DNA fragmentation without any additional inhibition of calcium uptake. Similar results were found for dexamethasone-treated thymocytes. Thymocytes treated with 8 mM EGTA, however, were not rescued from apoptosis but died by necrosis. These results indicate that extracellular calcium is not essential for apoptosis induced by these agents and that the use of high concentrations of EGTA to establish a requirement for extracellular calcium in apoptosis should be treated with caution.

Gliotoxin inactivates alcohol dehydrogenase by either covalent modification or free radical damage mediated by redox cycling.

The fungal metabolite gliotoxin shows selective toxicity to cells of the immune system and has been implicated in the aetiology of invasive aspergillosis. The related toxin sporidesmin is the causative agent of facial eczema in sheep. The toxicity of these compounds has been related to their ability to redox cycle intracellularly and thus produce damaging free radicals. These toxins are also potentially capable of forming mixed disulphides with thiol groups on proteins by virtue of their bridged disulphide structure. We show here that gliotoxin can inactivate horse liver alcohol dehydrogenase by either oxidative damage or covalent modification of thiol groups on the enzyme. Either Cys-281 or Cys-282 is selectively modified. Neither of these residues are at the active site. Covalent modification occurs in the absence of reducing agents such as dithiothreitol. In the presence of dithiothreitol no protection is observed and the rate of inactivation is enhanced although as expected no covalent modification occurs. Gliotoxin can therefore inhibit alcohol dehydrogenase by either pathway and this will depend on the availability of reducing agents such as glutathione and/or how readily the reactive oxygen species generated are removed.

Cellular uptake and release of the immunomodulating fungal toxin gliotoxin.

Uptake of the immunomodulating agent gliotoxin into a panel of cells using biosynthetically radiolabelled 35S toxin showed rapid association of the toxin with all cell types studied with 70-85% of the total counts in the media becoming cell associated. A difference in kinetics was observed for cell lines when compared to the primary cells thymocytes, activated T-cells and macrophages. In the latter uptake was maximal after 10-15 min and radiolabel was lost from the cells as early as 100 min. In the cell lines studied, uptake was complete in less than 1 min with no loss of label after 100 min. The exception to this was a Wilms tumour line. Analysis of the fate of gliotoxin taken up into sensitive (activated T-cells) and resistant (human fibroblast) cells by HPLC showed: (a) up to 30% of the original gliotoxin taken up by sensitive cells was released as free gliotoxin over a 22 hr period. The remainder was metabolized to inorganic sulphate; (b) in T-cells gliotoxin is reduced to the dithiol form in significant amounts and this reduction may be modulated by glutathione; and (c) no reduced gliotoxin could be detected in the resistant fibroblast cell line 27Sk even though up to 50% of the original gliotoxin was still present in the free form in these cells at 22 hr. Gliotoxin became covalently associated with macromolecules in both cell types studied. Very little free gliotoxin is released into extracellular medium by the fibroblast cell line. Gliotoxin at 500 nM was found to induce apoptosis or programmed cell death in the Wilms tumour cell line but not in any other cell line studied, and this may account for the different kinetics of release of the toxin from the Wilms tumour cell line.

Apoptosis induced in macrophages and T blasts by the mycotoxin sporidesmin and protection by Zn2+ salts.

Incubation of 48 h concanavalin A stimulated spleen cells (T blasts) and murine peritoneal macrophages with the mycotoxin sporidesmin results in DNA fragmentation characteristic of apoptosis. Morphological changes, particularly condensed chromatin, observed following incubation of these cells with sporidesmin and the immunotoxin gliotoxin and related epipolythiodioxopiperazines (ETP) also show changes characteristic of apoptosis. The presence of Zn2+ salts in the culture medium at concentrations non toxic to the cells over the time period studied protects against DNA damage and morphological change. Interaction between Zn2+ and the reduced form of a simple ETP compound assessed by spectral changes demonstrated the formation of a weak complex between the two molecules. Complex formation between zinc and thiol however was insufficient to prevent oxidative damage to plasmid DNA in vitro by inhibiting auto-oxidation of the reduced ETP compound because of the looseness of the interaction. Cd2+, which appears to form a tighter complex with the dithiol does inhibit cleavage of plasmid DNA. These results establish that the toxicity of sporidesmin may be due in part to its ability to induce apoptosis or programmed cell death in sensitive cells. In addition the immunotoxin gliotoxin and related compounds have now been shown to induce the same characteristic morphological changes in cells of haemopoietic origin. The inhibition of apoptosis induced by ETP compounds by Zn2+ appears to be due to direct inhibition of apoptosis rather than Zn2+ acting as an antioxidant. These results demonstrate the inhibition of apoptosis induced by ETP compounds by Zn2+ and suggest an alternate explanation for the known prophylactic effect of Zn2+ on sporidesmin induced tissue damage.

Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties.

We have previously shown that the fungal metabolite and immunomodulating agent gliotoxin induces apparently random double-stranded fragmentation of genomic DNA in a variety of cell types and double- and single-stranded scission in isolated plasmid DNA. The in vitro damage to plasmid DNA appears to be mediated by reactive oxygen species, but the mechanism of damage to genomic DNA is not yet known. In this paper we show that treatment of macrophages with gliotoxin and some analogues gives rise to discrete DNA fragments with molecular weight 170 +/- 30 base pairs. This pattern of DNA fragmentation has the characteristics of apoptosis, a programmed form of cell death. Three structural analogues of gliotoxin and two S-acetylated precursors capable of intracellular hydrolysis to the thiol form induce identical DNA degradation patterns. Only those compounds with the epipolythiodioxopiperazine (ETP) bridged disulfide structure or those capable of extracellular conversion to ETP compounds are equipotent with gliotoxin in their effects on macrophage phagocytosis, although all are capable of generating reactive oxygen species intracellularly. These results suggest that the effect of gliotoxin on macrophage function as assessed by adherence to plastic surfaces is unrelated to DNA damage and in addition suggests a new mechanism by which the toxin and other ETP compounds may damage cells.

Prevention of graft-versus-host disease by treatment of bone marrow with gliotoxin in fully allogeneic chimeras and their cytotoxic T cell repertoire.

Gliotoxin, a secondary fungal metabolite, at nanomolar concentrations, irreversibly inhibits murine T cell proliferation to mitogen. Treatment of allogeneic spleen cells with gliotoxin allows their transfer into sublethally irradiated recipients without inducing a GVH reaction. Gliotoxin treatment of bone marrow allows the establishment of fully allogenic bone marrow chimeras free of GVH disease. The cytotoxic T cell repertoire against influenza virus in these animals is restricted to both host- and donor-type MHC. However, their immune competence is severely compromised by their lack of host MHC-type stimulator cells.