Irene E. Hill

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.

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.

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.

The Transcription Factor E2F1 Modulates Apoptosis of Neurons

Abstract : 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.

A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis

The objective of this study was to examine the possible role of the cysteine protease cathepsin B (E.C. 3.4.22.1) in the delayed neuronal death in rats subjected to the two-vessel occlusion model of global ischemia. Immunohistochemistry of the hippocampus showed an alteration in the distribution of cathepsin B in CA1 neurons from a lysosomal pattern to a more intense label redistributed into the cytoplasm. This change was not detected until the neurons had become morphologically altered with obvious shrinkage of the cytoplasmic region. Western blotting and enzyme activity measurements of subcellular fractions, including lysosomes and a cell soluble fraction, demonstrated that there was an overall decrease in cathepsin B activity at this time but an increase in the proenzyme form, particularly in the soluble fraction. This was found to be completely different from the marked loss of all forms of cathepsin B in necrotic neurons following decapitation.

Activation of DNA‐Dependent Protein Kinase May Play a Role in Apoptosis of Human Neuroblastoma Cells

Abstract : Treating SH‐SY5Y human neuroblastoma cells with 1 μM 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 ~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 μM 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.

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.

A novel peptide designed for sensitization of terbium (III) luminescence

Several synthetic peptides, modelled from a Ca2+‐binding loop of the EF‐hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal‐binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb3+‐peptide complex was more than 10‐fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide‐based time‐resolved luminescence immunoassays.

Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein

The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca(2+)-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M-1 and 10(7) M-1, respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca(2+)-binding could be used to specifically visualize the scFv fragment.