Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death

Dexamethasone and corticosterone kill mouse thymocytes, as measured by eosin uptake, after several hours of in vitro incubation. This killing requires RNA and protein synthesis, because it is inhibited by cycloheximide, emetine, or actinomycin D. An earlier event than cell death is the extensive fragmentation of nuclear DNA into oligonucleosomal subunits; this fragmentation also requires RNA and protein synthesis. The DNA cleavage results from the action of an endonuclease that preferentially cleaves DNA in the linker regions between nucleosomes. This endonuclease is found constitutively in the nuclei of thymocytes and some other cells, and requires calcium and magnesium ions for its activation; if isolated fresh thymocyte nuclei are incubated with these ions, as much as 77% of their DNA is cleaved within 90 min. Thus, the protein for which synthesis is necessary for glucocorticoid-induced thymocyte death is not the endonuclease itself, but is in some way involved in its activation; we suggest that it may be part of a system for transporting calcium into the nucleus. The endonuclease is inhibited by zinc, which also prevents thymocyte death. It appears that glucocorticoids cause thymocyte death by activating an enzyme that rapidly and extensively degrades DNA. This "death from within" is biochemically and morphologically different from toxic or accidental cell death, such as that induced by azide, heat, or antibody and complement treatment. Although mature T cells also contain the endogenous endonuclease, they lack the glucocorticoid-inducible mechanism for activating it, and are thus glucocorticoid-resistant.     

Endonucleases and apoptosis in animals

Endonucleases are the main instruments of obligatory DNA degradation in apoptosis. Many endonucleases have marked processive action; initially they split DNA in chromatin into very large domains, and then they perform in it internucleosomal fragmentation of DNA followed by its hydrolysis to small fragments (oligonucleotides). During apoptosis, DNA of chromatin is attacked by many nucleases that are different in activity, specificity, and order of action. The activity of every endonuclease is regulated in the cell through its own regulatory mechanism (metal ions and other effectors, possibly also S-adenosylmethionine). Apoptosis is impossible without endonucleases as far as it leads to accumulation of unnecessary (defective) DNA, disorders in cell differentiation, embryogenesis, the organism’s development, and is accompanied by various severe diseases. The interpretation of the structure and functions of endonucleases and of the nature and action of their modulating effectors is important not only for elucidation of mechanisms of apoptosis, but also for regulation and control of programmed cell death, cell differentiation, and development of organisms.     

Nonsteroidal antiestrogens are estrogen-receptor-targeted growth inhibitors that can act in the absence of estrogens

The mechanism of the antiproliferative effect of nonsteroidal antiestrogens (tamoxifen, hydroxytamoxifen) is discussed from studies performed in human breast cancer cell lines. At least two types of mechanism have been evidenced. In the presence of estrogens, antiestrogens behave as classical antihormones and their inhibition of cell proliferation is likely due to inhibition of the synthesis and release of several estrogen-induced mitogens (growth factors and proteases). In the absence of estrogens (cells cultured in phenol-red-free medium), antiestrogens can still inhibit the effect of growth factors (EGF, insulin). At concentrations less than or equal to 4 microM, antiestrogens are also cytotoxic and they require accessible estrogen receptors for their action. 'Estrogen-receptor-targeted drugs' is therefore a better general term than 'antiestrogens' to describe the mechanism of action of these drugs, which can also function without inhibiting estrogen action.     

Extracellular ATP as a trigger for apoptosis or programmed cell death

Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.     

Involvement of proton-sensing receptor TDAG8 in the anti-inflammatory actions of dexamethasone in peritoneal macrophages

Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.     

Glucocorticoid-induced lymphocytolysis: State of the genetic analysis

The glucocorticoid-induced lysis of lymphoid cell lines offers a genetic approach to steroid hormone action because unresponsive variants can easily be selected as resistant to this lytic effect. The present state of analysis of lymphocytolysis in two murine cell lines, the S49 T-lymphoma and the W7 thymoma, is reviewed. All glucocorticoid-resistant variants isolated so far result from various defects in the glucocorticoid receptor. The absence of variants blocked at another step of the lytic mechanism is discussed. The observed hemizygosity of the glucocorticoid receptor locus in the S49 line and the instability of cell hybrids illustrate some of the potential problems encountered in somatic cell genetics.     

Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s)

Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced higher order chromatin degradation: A novel mechanism of oxidative genotoxicity

The genotoxicity of reactive oxygen species (ROS) is well established. The underlying mechanism involves oxidation of DNA by ROS. However, we have recently shown that hydrogen peroxide (H2O2), the major mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers at matrix-attachment regions. The activation of endonuclease that catalyzes HOCD is a signalling event triggered specifically by H2O2. The activation is not mediated by an influx of calcium ions, but resting concentrations of intracellular calcium ions are required for the maintenance of the endonuclease in an active form. Although H2O2-induced HOCD can efficiently dismantle the genome leading to cell death, under sublethal oxidative stress conditions H2O2-induced HOCD may be the major source of somatic mutations.     

Metabolic correlations of glucocorticoids and polyamines in inflammation and apoptosis

Glucocorticoid hormones (GC) are essential in all aspects of human health and disease. Their anti-inflammatory and immunosuppressive properties are reasons for therapeutic application in several diseases. GC suppress immune activation and uncontrolled overproduction and release of cytokines. GC inhibit the release of pro-inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Investigation of GC’s mechanism of action, suggested that polyamines (PA) may act as mediators or messengers of their effects. Beside glucocorticoids, spermine (Spm) is one of endogenous inhibitors of cytokine production. There are many similarities in the metabolic actions of GC and PA. The major mechanism of GC effects involves the regulation of gene expression. PA are essential for maintaining higher order organization of chromatin in vivo. Spermidine and Spm stabilize chromatin and nuclear enzymes, due to their ability to form complexes with negatively charged groups on DNA, RNA and proteins. Also, there is an increasing body of evidence that GC and PA change the chromatin structure especially through acetylation and deacetylation of histones. GC display potent immunomodulatory activities, including the ability to induce T and B lymphocyte apoptosis, mediated via production of reactive oxygen species (ROS) in the mitochondrial pathway. The by-products of PA catabolic pathways (hydrogen peroxide, amino aldehydes, acrolein) produce ROS, well-known cytotoxic agents involved in programmed cell death (PCD) or apoptosis. This review is an attempt in the better understanding of relation between GC and PA, naturally occurring compounds of all eukaryotic cells, anti-inflammatory and apoptotic agents in physiological and pathological conditions connected to oxidative stress or PCD.     

Hydrocortisone and 'macrocortin' inhibit the zymosan-induced release of lyso-PAF from rat peritoneal leucocytes

Hydrocortisone and the glucocorticoid-induced anti-phospholipase protein macrocortin, were tested as inhibitors of PAF generation. The steroid produced a dose-dependent inhibition of the release of the PAF precursor 2-lyso-PAF, and this effect was mimicked by affinity-purified macrocortin. Neither agent had any effect on the acetylation of lyso-PAF to PAF. Of other drugs tested only phospholipase inhibitors blocked lyso-PAF release and sulphydryl reagents blocked the acetylation step. It is concluded that glucocorticoids inhibit the generation of PAF and this could be an important component of their anti-anaphylactic and anti-inflammatory action.     

Spermine prevents endonuclease activation and apoptosis in thymocytes.

Glucocorticoid hormones, Ca2+ ionophores, and some toxic chemicals activate a suicide process in thymocytes, known as apoptosis or programmed cell death. A crucial event in apoptosis is the activation of a Ca(2+)- and Mg(2+)-dependent endonuclease that promotes extensive DNA fragmentation. In this study, we investigated the effect of various polyamines on endonuclease activation leading to thymocyte apoptosis. We found that both glucocorticoid- and Ca2+ ionophore-induced DNA fragmentation and apoptosis were prevented by spermine. Other polyamines such as putrescine or spermidine had moderate or no effect. Moreover, spermine, and to a lesser extent spermidine, but not putrescine, prevented endonuclease activation in permeabilized liver nuclei incubated in the presence of Ca2+ and Mg2+, indicating that spermine efficiency in blocking DNA fragmentation was related to the interaction of this polyamine with the endonuclease or its substrate, DNA. Experiments with the fluorescent dye, ethidium bromide, and a purified preparation of liver endonuclease revealed that the protective effect of spermine on DNA fragmentation was related to its ability to modify the chromatin arrangement. Thymocytes incubated with methyl glyoxal bis(guanylhydrazone) to deplete intracellular spermine exhibited spontaneous DNA fragmentation, which suggests that modulation of the intracellular polyamine content and regulation of chromatin structure may play a critical role in the early phases of apoptosis. Finally, these results demonstrate that inhibition of DNA fragmentation also prevents the onset of apoptosis, directly linking endonuclease activation and cell death.     

Knockdown of zinc finger protein, X-linked (ZFX) inhibits cell proliferation and induces apoptosis in human laryngeal squamous cell carcinoma

Apoptosis is a natural form of cell death involved in many physiological changes in the cell. Defects in the process of apoptosis can lead to serious diseases. During some apoptotic pathways, proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin degradation together with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interacting partners like flap endonuclease 1, DNase I, and exonuclease III were already suggested, but also other interacting partners were proposed. We conducted a living-cell confocal fluorescence microscopy followed by an image analysis of fluorescence resonance energy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins during apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin degradation. The possible existence of a degradation complex consisting of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investigated.     

Endonucleases and their involvement in plant apoptosis

This review considers modern data about the set, nature, specificity of action, and other properties of plant endonucleases involved in various forms of programmed cell death (PCD) in various plant tissues (organs). Apoptosis is an obligatory component of plant development; plant development is impossible without apoptosis. In dependence on the conditions of plant growth, this process can be induced by various biotic and abiotic factors, including stressors. Endonucleases accomplishing apoptotic degradation of nuclear material in the plant cell play one of the main roles in PCD. Plant endonucleases belong to at least two classes: (1) Ca²⁺- and Mg²⁺-dependent and (2) Zn²⁺-dependent nucleases. The set and activities of endonucleases change with plant age and during apoptosis in a tissue-specific manner. Apoptosis is accompanied by the induction of specific endonucleases hydrolyzing DNA in chromatin with the formation firstly of large domains and then internucleosomal DNA fragments; the products produced are of about 140 nucleotides in length with their subsequent degradation to low-molecular-weight oligonucleotides and mononucleotides. About 30 enzymes are involved in apoptotic DNA degradation. Histone H1 modulates endonuclease activity; separate (sub)fractions of this nuclear protein can stimulate or inhibit corresponding plant endonucleases. In the nucleus and cytoplasm of the plant cells, Ca²⁺/Mg²⁺-dependent endonucleases recognizing substrate DNA methylation status were revealed and described for the first time; their action resembles that of bacterial restrictases, which activity is modulated by the donor of methyl groups, S-adenosylmethionine. This indicates that higher eukaryotes (higher plants) might possess the system of restriction-modification to some degree analogous to that of prokaryotes.     

Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity

Past studies have shown that serum-free cultures of PC12 cells are a useful model system for studying the neuronal cell death which occurs after neurotrophic factor deprivation. In this experimental paradigm, nerve growth factor (NGF) rescues the cells from death. It is reported here that serum-deprived PC12 cells manifest an endonuclease activity that leads to internucleosomal cleavage of their cellular DNA. This activity is detected within 3 h of serum withdrawal and several hours before any morphological sign of cell degeneration or death. NGF and serum, which promote survival of the cells, inhibit the DNA fragmentation. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases in vitro, suppresses the endonuclease activity and promotes long-term survival of PC12 cells in serum-free cultures. This effect appears to be independent of macromolecular synthesis. In addition, ATA promotes long-term survival of cultured sympathetic neurons after NGF withdrawal. ATA neither promotes nor maintains neurite outgrowth. It is hypothesized that the activation of an endogenous endonuclease could be responsible for neuronal cell death after neurotrophic factor deprivation and that growth factors could promote survival by leading to inhibition of constitutively present endonucleases.