Cerebral ischemia produces laddered DNA fragments distinct from cardiac ischemia and archetypal apoptosis.

The electrophoretic pattern of laddered DNA fragments which has been observed after cerebral ischemia is considered to indicate that neurons are dying by apoptosis. Herein the authors directly demonstrate using ligation-mediated polymerase chain reaction methods that 99% of the DNA fragments produced after either global or focal ischemia in adult rats, or produced after hypoxia-ischemia in neonatal rats, have staggered ends with a 3' recess of approximately 8 to 10 nucleotides. This is in contrast to archetypal apoptosis in which the DNA fragments are blunt ended as seen during developmental programmed cell death in dying cortical neurons, neuroblastoma, or thymic lymphocytes. It is not simply ischemia that results in staggered ends in DNA fragments because ischemic myocardium is similar to archetypal apoptosis with a vast majority of blunt-ended fragments. It is concluded that the endonucleases that produce this staggered fragmentation of the DNA backbone in ischemic brain must be different than those of classic or type I apoptosis.

Detection of higher-order 50- and 10-kbp DNA fragments before apoptotic internucleosomal cleavage after transient cerebral ischemia.

DNA fragments of 50 and 10 kbp were found in ischemic brain in adult rats following two-vessel occlusion or in neonates following hypoxia-ischemia. These higher-order fragments were detected before any laddered oligonucleosomal DNA fragmentation characteristic of apoptosis. Both the 50- and 10-kbp fragments were also detected during necrosis produced by decapitation, but these led to smeared smaller fragments, not laddered patterns. End-group analysis showed the presence of both 3'-OH and 5'-OH ends in both the 50- and 10-kbp fragments but the predominance of 3'-OH ends in the laddered fragments. A higher proportion of 5'-OH to 3'-OH ends was found in the 10-kbp fragment compared to the larger 50-kbp fragment, suggesting a selective degradation of the 50-kbp DNA fragment to the laddered oligonucleosomal patterns. Overall, the mode of DNA fragmentation appeared different from that described in classic apoptosis of thymocytes.

Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.

The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.

Hyperglycemia enhances DNA fragmentation after transient cerebral ischemia.

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.

A new intrinsic fluorescent probe for proteins. Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin.

The tryptophan analog, 5-hydroxytryptophan (5HW), has a significant absorbance between 310-320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca2+ binding protein, oncomodulin. Fluorescence anisotropy and time-resolved fluorescence decay measurements of the interaction between anti-oncomodulin antibodies and the 5HW-incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use of 5HW as an intrinsic probe in the study of structure and dynamics of protein-protein interactions.

Neuronal apoptosis inhibitory protein expression after traumatic brain injury in the mouse.

Apoptosis of brain cells is triggered by traumatic brain injury (TBI) and is blocked by caspase inhibitors. The neuronal apoptosis inhibitor protein (NAIP), which has been shown to inhibit apoptosis by both caspase-dependant and caspase-independent mechanisms, is neuroprotective in rat models of cerebral ischemia and axotomy. In order to gain a better appreciation of CNS apoptosis following head injury in general and the possible involvement of NAIP specifically, we have configured a mouse model of TBI. In addition to demonstrating apoptosis, the spatiotemporal expression or levels of a number of proteins with apoptosis modulating effects have been determined. Apoptosis of neurons and oligodendrocytes following TBI was observed in brain sections which were triple-stained with in situ end labeling, bisbenzimide and immunofluorescent stain for neuron specific nuclear protein and myelin-associated glycoprotein, respectively. Further evidence for apoptosis following TBI in this model was obtained in brain samples using ligation-mediated PCR amplification of DNA fragments and gel electrophoresis. The temporal profile of apoptosis was similar to the temporal profile of microglial activation determined by CD11b staining and TNFa expression induced by TBI. NAIP staining in sections of cerebral cortex and subcortical white matter increased at 6 h and decreased towards control levels at 24 h post-TBI. Temporal changes in the expression of NAIP were also observed using Western blot analysis of brain samples removed from injured cortex and sub-cortical white matter. At the time that NAIP expression decreased markedly (24 h post-TBI), procaspase-3 levels also decreased, PARP cleavage increased, and the highest levels of apoptosis were observed. These findings have implications in our understanding of traumatically induced programmed cell death and may be useful in the configuration of therapies for this common injury state.

DNA damage consistent with apoptosis in transient focal ischaemic neocortex.

Transient focal ischaemia was produced in rat right neocortex by temporary middle cerebral artery occlusion. DNA damage was visualized in situ in cells of this right hemisphere but not in the contralateral hemisphere. The extracted damaged DNA exhibited laddered fragmentation which is indicative of apoptotic degradation. The amount of DNA damage was quantified by an end-labelling technique and shown to increase with the duration of the ischaemic insult. We conclude that the neurodegeneration resulting from focal ischaemia has an apoptotic component.

DNA fragmentation indicative of apoptosis following unilateral cerebral hypoxia-ischemia in the neonatal rat.

DNA extracted from regional brain samples of hypoxic/ischemic neonatal rats showed internucleosomal cleavage indicative of apoptosis. Cells containing cleaved DNA were identified by in situ labelling in the cortex, hippocampus, striatum and thalamus of the ipsilateral hemisphere. When the effects of increasing the length of the hypoxia were examined, increases were seen in the amount of internucleosomally cleaved DNA and in the number of labelled cells.

Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain.

Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.

Increased expression of the transcription factor E2F1 during dopamine-evoked, caspase-3-mediated apoptosis in rat cortical neurons.

The transcription factor E2F1 mRNA and protein levels increased in rat cortical neurons in response to dopamine (DA)- or 6-hydroxydopamine (OHDA)-evoked apoptosis. Increased E2F1 protein was detected in the nucleus of neurons by double fluorescent immunocytochemistry using antibodies to E2F1 and NeuN. DA and 6-OHDA induced caspase-3-mediated apoptosis of cortical neurons which was attenuated by the addition of antioxidants or caspase-3 inhibitors to the cultures. Antioxidants prevented DA-evoked neuronal apoptosis, and also attenuated the increase in E2F1 expression. These findings suggest that increased expression of the transcription factor E2F1 may serve as a death signal during DA-evoked neuronal apoptosis.

Studies on the use of plant extracts in assessing the effects of plant metabolism on the mutagenicity and toxicity of pesticides.

We have carried out studies on the effects of plant metabolism on the mutagenicity of agricultural chemicals. Our approach is to use a cell-free plant extract, as a source of metabolic enzymes, in a standard Ames test. Using a number of test compounds, we observe that plant metabolism can alter the mutagenicity of several pesticides, and can in some instances give rise to metabolites apparently unique from those which are formed in animal cells. A number of parameters of the assay have been examined, and we find that the assay temperature and preincubation of the pesticide with the extract can significantly alter the outcome of the test. We also have devised a method of controlling for the effects that natural extracts can have on the spontaneous reversion rate of the Salmonella tester strains, in an effort to distinguish slight mutagenic responses from the effects of nutrients (e.g. histidine for his- bacteria) in the assay.

Apoptotic human SH-SY5Y neuroblastoma cells have regularly spaced single strand DNA breaks and increased DNA-dependent protein kinase activity.

SH-SY5Y human neuroblastoma cells died by apoptosis when treated with staurosporine or ceramide. The treated cells had both the nuclear morphology and patterns of DNA fragmentation which are characteristic of apoptosis. Higher order DNA fragments separable by pulse field gel electrophoresis were shown to contain regularly spaced single-strand nicks by producing a laddered pattern upon alkali treatment. Further evidence of DNA damage in treated cells was shown by increased activity of DNA-dependent protein kinase. This human cell model may prove useful in delineating the role of a cellular repair response to DNA damage prior to the irreversible steps of the cell death program.

A "CAT" family of repetitive DNA sequences in Saccharomyces cerevisiae.

An oligonucleotide probe was used to isolate yeast genomic clones containing DNA sequences with repetitive elements consisting primarily of a tandemly arranged trinucleotide, CAT. Hybridization analyses estimate that the yeast genome contains 40-50 CAT clusters, representing the first repetitive DNA sequence family found in yeast. Sequence analyses show short spacers between the CAT repeats consisting of closely related trinucleotides, primarily CGT. Some of the CAT clusters are located in longer repeating elements with lengths of 7 nucleotides or more. In one case a three-times-repeated 27-nucleotide sequence bears striking homology to the 21-base pair repeat region of the mammalian simian virus 40 promoter element. Hybridization studies further suggest that the "CAT" sequences may be widely dispersed in many diverse organisms including Escherichia coli, Drosophila, and man.

Despite the internucleosomal cleavage of DNA, reactive oxygen species do not produce other markers of apoptosis in cultured neurons.

The cell death induced by hydroxyl radicals generated by Cu-phenanthroline and peroxynitrite generated by 3-morpholinosydnonimine hydrochloride (SIN-1) in rat primary cortical neuronal cultures was compared with the apoptotic death induced by staurosporine and the necrotic death induced by glutamate. Both SIN-1 and Cu-phenanthroline were capable of generating internucleosomal cleavage of DNA-a hallmark of apoptosis. Other characteristics of this cell death, such as nuclear morphology by light microscopy; DNA breaks by single-cell gel electrophoresis; the effects of the apoptotic inhibitors cycloheximide, aurintricarboxylic acid, and tosyl-l-lysine chloromethyl ketone; the measurement of caspase activity; and the effects of antioxidants, were then analyzed. The conclusion from these hallmarks of apoptosis is that the cell death induced by these reactive oxygen species is not apoptosis.

Activation of DNA-dependent protein kinase may play a role in apoptosis of human neuroblastoma cells.

Treating SH-SY5Y human neuroblastoma cells with 1 microM staurosporine resulted in a three- to fourfold higher DNA-dependent protein kinase (DNA-PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA-PK activity: an initial increase that peaked by 4 h and a rapid decline that reached approximately 5-10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA-PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase-1 activity but stimulated caspase-3 activity by 10-15-fold in a time-dependent manner, similar to morphological changes. Similar time-dependent changes in DNA-PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 microM ceramide or UV radiation. In all these cases the increase in DNA-PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell-permeable inhibitor of DNA-PK, OK-1035, significantly reduced staurosporine-induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA-PK may be involved with the induction of apoptotic cell death.

Glutamate-treated rat cortical neuronal cultures die in a way different from the classical apoptosis induced by staurosporine.

The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized by in situ end-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.

The transcription factor E2F1 modulates apoptosis of neurons.

The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 -/- mice) were resistant to staurosporine-induced apoptosis as evidenced by the significantly lower caspase 3-like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild-type mice. Furthermore, overexpressing E2F1 alone using replication-deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3-like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis.

Increases in DNA lesions and the DNA damage indicator Gadd45 following transient cerebral ischemia.

Transient global or focal ischemia leads to the production of several types of lesions in the DNA backbone including alkali-labile sites, and both single-stranded (ss) and double-stranded (ds) breaks. The ds breaks result in high molecular weight fragments of 10-50 kbp that contain both 3'- and 5'-OH end-groups, suggesting that more than one endonuclease is involved. This lesioning of DNA is followed by the appearance of the damage-response indicator Gadd45 in the ischemic hemisphere following middle cerebral artery occlusion. By 6 h, gadd45 mRNA was shown to increase by semi-quantitative reverse transcriptase - polymerase chain reaction. In situ hybridization histochemistry indicated that these increases in gadd45 mRNA occurred in pyramidal neurons located on the edge of the infarcted cortex. Gadd45 immunostaining yielded similar findings with maximal protein staining detected at 18 h after occlusion. In neurons, in the infarct core with frank DNA fragmentation shown by in situ TdT-mediated dUTP-biotin nick end labeling (TUNEL) at 24 h, the Gadd45 immunostaining was not visible. Taken together, these findings suggest that Gadd45 responds to DNA damage following ischemia as part of a repair response mounted by brain cells attempting to survive the insult.