Endonuclease Activation Research Articles

Among substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetraones, the lead antitumor triptycene bisquinone TT24 blocks nucleoside transport, induces apoptotic DNA fragmentation and decreases the viability of L1210 leukemic cells in the nanomolar range of daunorubicin in vitro.

In contrast to their inactive parent compound triptycene (code name TT0), several new synthetic analogs (TT code number) have antileukemic activities and remain effective in daunorubicin (DAU)-resistant tumor sublines in vitro. Among variously substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetraones, a total of six lead antitumor compounds have been identified, and their code names are TT2, TT13, TT16, TT19, TT21 and TT24. These active antitumor triptych structures have bisquinone functionality, and various bromo, methoxy, methylamino and/or dimethylamino substitutions with or without longer alkyl chains on the amino function. Like the anthracycline quinone antibiotic DAU, these triptycene (TT) bisquinones also inhibit DNA synthesis and induce DNA cleavage in relation with their cytotoxic activities, but have the additional advantage of blocking the cellular transport of purine and pyrimidine nucleosides, an effect which DAU cannot do. As demonstrated by intact chromatin precipitation and agarose gel electrophoresis, the ability of TT bisquinones and DAU to induce DNA fragmentation is biphasic with a peak that shifts to lower concentrations with increasing times of drug exposure. The most effective lead antitumor compound, TT24, induces DNA cleavage in the same concentration-dependent manner as DAU at 24 h (similar peak in response to 1.6 microM) and is nearly equipotent to DAU against L1210 tumor cell viability at day 4 (IC50 values of TT24 and DAU: 48 and 25 nM, respectively). The mechanism by which TT24 induces DNA fragmentation is inhibited by actinomycin D, cycloheximide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone and ZnSO4, suggesting that TT bisquinones trigger apoptosis by caspase and endonuclease activation. Since TT24 is cytotoxic in the nanomolar range of DAU, but might have a more versatile mechanism of action than DAU in wild-type and multidrug-resistant tumor cells, this new class of DNA-damaging quinone antitumor drugs inhibiting nucleoside transport might be valuable to develop new means of polychemotherapy.

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca 2+-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H 2O 2 (200 μM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.

Regulation of DNAS1L3 Endonuclease Activity by Poly(ADP-ribosyl)ation during Etoposide-induced Apoptosis: ROLE OF POLY(ADP-RIBOSE) POLYMERASE-1 CLEAVAGE IN ENDONUCLEASE ACTIVATION

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.

Oxidative stress and acclimation mechanisms in plants.

One of the most crucial functions of plant cells is their ability to respond to fluctuations in their environment. Understanding the connections between a plant's initial responses and the downstream events that constitute successful adjustment to its altered environment is one of the next grand challenges of plant biology. Oxidative stress from environmental sources and developmental transitions such as seed maturation involves the formation of reactive oxygen species (ROS) in plant cells. The redox-modulated changes that follow are central events in cellular responses. Thiol redox regulation (Figure 1) partially mediated through the redox state of the glutathione pool (GSH/GSSG), regulation of the glutathione biosynthetic pathway, and ROS themselves are each thought to have important roles as environmental sensors and/or modulators of global patterns of gene expression in development and defense. Exposure of green tissue to potentially damaging light intensities involves redox sensing molecular events throughout the plant, originating at the plastoquinone (PQ) pool in the thylakoid membrane. Major defense genes whose expression is affected by the redox state of the PQ pool include both cytosolic and chloroplast ascorbate peroxidases (APX) ( Karpinska et al. 2000 ). The superoxide dismutase (SOD) gene families appear to be specialized in function with respect to subcellular location and other as yet unknown factors (Alscher et al. 2002). In the case of peroxisomes, the imposition of oxidative stress gives rise to organelle proliferation, thus adding another layer of complexity to stress responses (Lopez-Huertas et al. 2000). Defense mechanisms involving molecular chaperones and methionine sulfoxide reductase are becoming recognized as important players in resistance to oxidative stress throughout the cell. Figure 1. Thiol redox control and stress defense Intracellular origins of ROS and their multiple damaging effects Any circumstance in which cellular redox homeostasis is disrupted can lead to oxidative stress or the generation of ROS (Asada 1994). Production of ROS during environmental stress is one of the main causes for decreases in productivity, injury, and death that accompany these stresses in plants. ROS are produced in both unstressed and stressed cells, and in various locations (Halliwell and Gutteridge 1989) (Figure 2). They are generated endogenously during certain developmental transitions such as seed maturation and as a result of normal, unstressed, photosynthetic and respiratory metabolism. An initial oxyradical product, the superoxide radical (O2-.), upon further reaction within the cell, can form more ROS such as hydroxyl radicals and singlet oxygen. Superoxide is a charged molecule and cannot cross biological membranes. Subcellular compartmentation of defense mechanisms is, therefore, crucial for efficient removal of superoxide anions at their sites of generation throughout the cell. Hydrogen peroxide, on the other hand, which is formed as a result of SOD action, is capable of diffusing across membranes and is thought to fulfil a signaling function in defense responses (Mullineaux et al. 2000). Figure 2. Reactive oxygen species (ROS) arise throughout the cell. ROS play an important role in endonuclease activation and consequent DNA damage (Hagar et al. 1996). In the presence of metal ions such as Fe or Cu(II), hydroxyl radicals are formed very rapidly. Hydroxyl radicals can cause damage to all classes of biologically important macromolecules, especially nucleic acids. Hydroxyl radicals can also modify proteins so as to make them more susceptible to proteolytic attack. There is evidently considerable specificity associated with this degradative process since proteins have widely differing susceptibilities to attack by ROS (Davies 1987). Once damaged, proteins can be broken down further by specific endopeptidases such as the one found bound to the thylakoid membrane (Casano et al. 1994). A multicatalytic proteinase complex has been demonstrated in plant systems, with the capacity to selectively break down oxidatively damaged proteins (Van Nocker et al. 1996)

Recent insights into the mechanism of glucocorticosteroid-induced apoptosis.

Glucocorticosteroid hormones induce apoptosis in lymphocytes. Therefore, glucocorticoids are commonly used as immunosuppressive and chemotherapeutic agents. This review examines many facets of the process by which glucocorticoids induce apoptosis. This process is divided into three stages, an initiation stage that involves glucocorticoid receptor-mediated gene regulation, a decision stage that involves the counterbalancing influence of prosurvival and proapoptotic factors, and the execution stage which involves caspase and endonuclease activation. Many aspects of glucocorticoid-induced apoptosis, such as mitochondrial dysfunction and caspase activation, are important steps in virtually all forms of apoptosis. But the process glucocorticoid-induced apoptosis differs from other forms of apoptosis in terms of initiation at the transcriptional level and involvement of the multicatalytic proteasome and calcium. Moreover, the abundant opportunity for crosstalk between the glucocorticoid receptor and other signaling pathways increases the complexity of glucocorticoid-induced apoptosis and its regulation.

Modulation of apoptosis during infection with Chlamydia

This chapter describes methods used to measure apoptosis or inhibition of apoptosis during infection, particularly techniques that reveal host cell morphological changes, caspase activation, mitochondrial membrane depolarization, cytochrome c release, and DNA fragmentation. Apoptosis is apparently blocked through inhibition of cytochrome c release from mitochondria and subsequent caspase-3 activation, In contrast, induction of host cell apoptosis has also been observed in macrophages and epithelial cells infected by C. psittaci during late stages of the infection, and the apoptosis requires intracellular bacterial replication. C. psittaci-induced apoptosis may require secretion of a bacterial apoptotic factor, potentially via the type III secretion apparatus and/or may be a stress response in the infected cell. Furthermore, the hallmarks of apoptosis are condensation of the nuclear chromatin and cytoplasm, activation of proteases (caspases) and endonucleases, loss of plasma membrane phosphatidylserine (PS) asymmetry, cleavage of the DNA into 200 base-pair oligonucleosomal fragments, and segmentation of the dying cell into membrane-bound apoptotic bodies. Intracellular microbes can also modulate apoptosis of the host cell, either inhibiting or promoting cell death, and it has been proposed that the persistence and pathogenesis of several pathogenic microbes may be related to their ability to dysregulate apoptosis.

Elucidating Neuronal and Vascular Injury Through the Cytoprotective Agent Nicotinamide

Both neuronal and cerebrovascular endothelial cell (EC) injury are intimately involved in the development of acute and chronic neurodegenerative disorders. Recently, this cellular injury has been shown to consist of two distinct pathways of programmed cell death (PCD) that involve the degradation of genomic DNA and the exposure of membrane phosphatidylserine (PS) residues. In addition, it is the downstream cellular and molecular cascades that are considered to be the vital for the prevention and reversal of neuronal and vascular injury. These include free radical injury, the independent mechanisms of programmed cell death, loss of mitochondrial membrane potential, release of cytochrome c, DNA repair enzymes, changes in intracellular pH, endonuclease activation, cysteine protease generation, and MAP kinase activation. As a novel cytoprotectant, the agent nicotinamide can maintain DNA integrity and prevent the progression of membrane PS exposure. Immediate and delayed injury paradigms with nicotinamide also illustrate the ability of this agent to maintain mitochondrial membrane potential, prevent specific cysteine protease activation, and reverse a previously sustained insult. As an investigational tool, nicotinamide has assisted in identifying several of the cellular pathways that modulate ischemic neuronal and vascular injury and has offered new therapeutic strategies for central nervous system degenerative disorders.

The Heterocyclic Amine, 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole Induces Apoptosis in Cocultures of Rat Parenchymal and Nonparenchymal Liver Cells

In this study, we investigated the mechanism of apoptosis by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in cocultures of parenchymal and nonparenchymal liver cells, since the liver consists of various cell types and they cooperatively respond to chemicals. It was found that cocultures were more susceptible to cell death by Trp-P-1 than culture of each cell type alone. In cocultures, Trp-P-1 induced DNA fragmentation accompanied by the activation of 18-kDa endonuclease. Trp-P-1 (30 μM) caused a rapid increase in Bid protein level in mitochondria and the leakage of cytochrome c from mitochondria into the cytosol 15 min after treatment. On the other hand, an increase in Bax protein and a decrease in Bcl-2 protein were detected in the mitochondrial fraction 2 h after treatment following the increases in p53 protein level and DNA binding activity of NF-κB. Caspase-8 was activated within 30 min followed by the activation of downstream caspases as measured using the corresponding peptide substrates. The activation of caspases was also confirmed by cleavage of caspase-3, poly(ADP-ribose)polymerase, and protein kinase C-δ as analyzed by Western blotting. A peptide inhibitor of caspase-8 diminished DNA ladder formation and the activation of downstream caspases, but a caspase-9 inhibitor and pyrrolidinedithiocarbamate as an inhibitor of NF-κB showed only partial inhibition, suggesting that caspase-8 is the apical caspase in the cascade. These results led to the conclusion that Trp-P-1 mainly drives the caspase-8-mediated pathway that involves Bid, accompanied by a delay in the p53/NF-κB-mediated side pathway that involves Bax, Bcl-2, and caspase-9.

Thapsigargin-Induced Calcium Entry and Apoptotic Death of Neutrophils Are Blocked by Activation of Protein Kinase C

Intracellular free calcium ([Ca²⁺]_i) concentration, free oxygen radical (FOR) production, DNA breakdown, and plasma membrane phosphorylation were studied in human neutrophils activated with thapsigargin and phorbol myrisate acetate (PMA). Thapsigargin produced a rapid and sustained rise of [Ca²⁺]_i, activated the endonuclease, and caused the breakdown of the neutrophil’s DNA with a half-time close to 6 h. The protein kinase C activator PMA failed to inhibit the initial rise of [Ca²⁺]_i, but inhibited the second phase of thapsigargin-induced calcium transient and completely blocked the activation of the endonuclease induced by thapsigargin. Thapsigargin induced a minor and delayed production of FOR, whereas PMA caused an abrupt and sustained FOR production that was enhanced by thapsigargin. Two plasma membrane proteins close to 50 and 64 kD were phosphorylated in PMA-activated neutrophils. These results suggest that the nonphosphorylated form of the membrane protein permits basal and thapsigargin-induced calcium entry. Phosphorylation by PMA of plasma membrane protein inhibits calcium uptake in both resting and thapsigargin-activated neutrophils and contributes to the block of the activation of the apoptotic endonuclease.

Galpha11 induces caspase-mediated proteolytic activation of Rho-associated kinase, ROCK-I, in HeLa cells.

Expression of the constitutively active mutant of Galpha(11) (Galpha(11)QL) induces the formation of vinculin-containing focal adhesion-like structures in HeLa cells. This was found to be inhibited by Y-27632, a specific inhibitor of Rho-associated kinases (ROCK), but not by co-expression with a dominant negative mutant of RhoA, suggesting Rho-independent activation of ROCK by Galpha(11)QL. Investigation of trypan blue exclusion and immunocytochemistry with an antibody against cleaved caspase revealed the cellular phenotype of Galpha(11)QL-expressing cells to be identical to that displayed by cells undergoing apoptosis, and the caspase inhibitor zVAD-fmk blocked all morphological changes induced by Galpha(11)QL. Transfection of Galpha(11)QL induced cleavage of ROCK-I, and this proteolysis was also prevented by zVAD-fmk. ROCK-I C-terminally truncated at its authentic caspase sites also induced the formation of vinculin-containing focal adhesion-like structures. In addition, cleavage of ROCK-I was observed when cells overexpressing m1 muscarinic acetylcholine receptors were stimulated with carbachol. These results suggest that Galpha(11) induces proteolytic activation of ROCK-I by caspase and thereby regulates the actin cytoskeleton during apoptosis.

Induction of apoptosis by a ribosome-inactivating protein from Agrostemma githago is associated with down-regulation of anti-apoptotic bcl-2 protein expression.

Agrostin is a type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Agrostemma githago. 3[H]-thymidine incorporation in human leukemic HL-60 cells was significantly reduced after incubation with agrostin showing that the RIP was inhibitory on the cell proliferation. Results from flow cytometric studies illustrated that apoptosis was induced by agrostin, which was associated with activation of endonucleases and DNA fragmentation of the leukemic cells. Anti-apoptotic bcl-2 protein level was also reduced by agrostin showing that the associated apoptotic pathway might involve down-regulation of the intracellular bcl-2 protein level.

Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25–45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca 2+-Mg 2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.

The dynamics of cellular injury: transformation into neuronal and vascular protection.

Despite the immediate event, such as cerebral trauma, cardiac arrest, or stroke that may result in neuronal or vascular injury, specific cellular signal transduction pathways in the central nervous system ultimately influence the extent of cellular injury. Yet, it is a cascade of mechanisms, rather than a single cellular pathway, which determine cellular survival during toxic insults. Although neuronal injury associated with several disease entities, such as Alzheimer's disease, Parkinson's disease, and cerebrovascular disease was initially believed to be irreversible, it has become increasingly evident that either acute or chronic modulation of the cellular and molecular environment within the brain can prevent or even reverse cellular injury. In order to develop rational, efficacious, and safe therapy against neurodegenerative disorders, it becomes vital to elucidate the cellular and molecular mechanisms that control neuronal and vascular injury. These include the pathways of free radical injury, the independent mechanisms of programmed cell death, and the downstream signal transduction pathways of endonuclease activation, intracellular pH, cysteine proteases, the cell cycle, and tyrosine phosphatase activity. Employing the knowledge gained from investigations into these pathways will hopefully further efforts to successfully develop effective treatments against central nervous system disorders.

Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species

Cadmium poses a serious environmental threat in aquatic ecosystems but the mechanisms of its toxicity remain unclear. The purpose of this work was first to determine whether cadmium induced apoptosis in trout hepatocytes, second to determine whether or not reactive oxygen species (ROS) were involved in cadmium-induced apoptosis and genotoxicity. Hepatocytes exposed to increasing cadmium concentrations (in the range of 1–10 μM) showed a molecular hallmark of apoptosis which is the fragmentation of the nuclear DNA into oligonucleosomal-length fragments, resulting from an activation of endogenous endonucleases and recognized as a ‘DNA ladder’ on conventional agarose gel electrophoresis. Exposure of hepatocytes to cadmium led clearly to the DEVD-dependent protease activation, acting upstream from the endonucleases and considered as central mediators of apoptosis. DNA strand breaks in cadmium-treated trout hepatocytes was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect DNA primary damage in individual cells. Simultaneous treatment of trout hepatocytes with cadmium and the nitroxide radical TEMPO used as a ROS scavenger, reduced significantly DNA fragmentation, DEVD-related protease activity and DNA strand breaks formation. These results lead to a working hypothesis that cadmium-induced apoptosis and DNA strand breaks in trout hepatocytes are partially triggered by the generation of ROS. Additional studies are required for proposing a mechanistic model of cadmium-induced apoptosis and genotoxicity in trout liver cells, in underlying the balance between DNA damage and cellular defence systems in fish.

Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay.

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.

DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.

Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca(2+) / Mg(2+)-dependent endonuclease ubiquitously expressed in mammalian tissues, is believed to play a role in this process. To analyze the in vivo function of this enzyme in human cells, we have generated a cell line with targeted disruption of the DNase I gene, as well as several stable cell lines which overexpress the DNase I gene. Inactivation of the human DNase I gene was obtained in the Jurkat T cell clone JA3, characterized by high susceptibility to apoptotic cell death induced by pharmacological stimuli. JA3 cells, after disruption of the DNase I gene, became resistant to apoptotic stimuli. DNase I was overexpressed in the human cell lines JA3, K562 (erythroleukemia), M 14 (melanoma) and CEM (T cell lymphoma). Remarkably, stable overexpression of DNase I gene resulted in accelerated apoptosis in JA3 cells and induced apoptosis in K562, CEM and M14 cell lines, which are otherwise resistant to internucleosomal DNA degradation following pharmacological stimuli. Our study provides the first in vivo evidence that DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.

Mutagenic analysis of the 5' arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity.

Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5' arm of the influenza A virus viral RNA (vRNA) was constructed to determine the cis-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5' hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5' terminus, and (iii) the presence of base pair interactions between the 5' and 3' segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.

Calpains and apoptosis

Calpains are a family of cysteine proteases existing primarily in two forms designated by the Ca2+ concentration needed for activation in vitro, μ‐calpain (calpain‐I) and m‐calpain (calpain‐II). The physiological roles of calpains remain unclear. Many groups have proposed a role for calpains in apoptosis, but their patterns of activation are not well characterized. Calpains have been implicated in neutrophil apoptosis, glucocorticoid‐induced thymocyte apoptosis, as well as many other apoptotic pathways. Calpain activation in apoptosis is usually linked upstream or downstream to caspase activation, or in a parallel pathway alongside caspase activation. Calpains have been suggested to be involved in DNA fragmentation (via endonuclease activation), but also as effector proteases that cleave cellular proteins involved in DNA repair, membrane associated proteins and other homeostatic regulatory proteins. Recently, our laboratory demonstrated μ‐calpain activation in NAD(P)H: quinone oxidoreducatse 1 (NQOl)‐expr...

Expression of a Cytokeratin 18 Neo-epitope is a Specific Marker for Trophoblast Apoptosis in Human Placenta

In epithelial cells the caspase-mediated cleavage of cytokeratin 18 during apoptosis leads to the formation of a specific neo-epitope, recognized by the antibody M30. To test whether this antibody can be used as a specific marker for apoptotic trophoblast, we have stained serial sections of villi and junctional zone of first and third trimester human placenta with antibodies against cytokeratins 7 and 18, and against active caspase 3, with M30 and with the TUNEL reaction. Comparison of M30 immunoreactivities with TUNEL positivity and immunoreactivities for cytokeratins 7 and 18 clearly demonstrates that M30 specifically labels late apoptotic trophoblast cells. This finding is supported by the fact that in trophoblast, M30 immunoreactivities largely overlap with those for active caspase 3. As compared to the TUNEL test, the M30 immune reaction appears to be a highly reproducible marker for apoptotic trophoblast. This antibody stains a larger number of cells within the apoptosis cascade as compared to the TUNEL reaction, since cytokeratin 18 cleavage starts earlier than cleavage of DNA and since endonuclease activation can be bypassed in some trophoblast cells. The data suggest that M30 is superior to the TUNEL reaction as a marker for the detection of trophoblast apoptosis since it is easier to handle, more specific for apoptosis and less prone to artifacts.

Granulocyte-colony stimulating factor induced intranuclear endonuclease in murine leukemia cell line

We have reported that murine leukemia cell line (C2M-A5) induced apoptosis by G-CSF. To clarify the mechanism, mRNA expression of apoptosis-related genes was studied. It revealed transient over-expression of c-myc, H-ras and p53 and down-expression of bcl-2. These changes were known as triggers of endonuclease induction. After 96 h culture with G-CSF, apoptosis was occurred simultaneously with endonuclease (37 kd) activation. This endonuclease induced the digestion of double-strand DNA and might be associated with caspase3. Although G-CSF accelerates cell growth and prevents apoptosis in general, it is a contradictory effect. We concluded that G-CSF induced endogenous endonuclease activity in C2M-A5.