Injury‐induced spinal motor neuron apoptosis is preceded by DNA single‐strand breaks and is p53‐ and Bax‐dependent

The mechanisms of injury‐induced apoptosis of neurons within the spinal cord are not understood. We used a model of peripheral nerve‐spinal cord injury in the rat and mouse to induce motor neuron degeneration. In this animal model, unilateral avulsion of the sciatic nerve causes apoptosis of motor neurons. We tested the hypothesis that p53 and Bax regulate this neuronal apoptosis, and that DNA damage is an early upstream signal. Adult mice and rats received unilateral avulsions causing lumbar motor neurons to achieve endstage apoptosis at 7–14 days postlesion. This motor neuron apoptosis is blocked in bax^?/? and p53^?/? mice. Single‐cell gel electrophoresis (comet assay), immunocytochemistry, and quantitative immunogold electron microscopy were used to measure molecular changes in motor neurons during the progression of apoptosis. Injured motor neurons accumulate single‐strand breaks in DNA by 5 days. p53 accumulates in nuclei of motor neurons destined to undergo apoptosis. p53 is functionally activated by 4–5 days postlesion, as revealed by immunodetection of phosphorylated p53. Preapoptotically, Bax translocates to mitochondria, cytochrome c accumulates in the cytoplasm, and caspase‐3 is activated. These results demonstrate that motor neuron apoptosis in the adult spinal cord is controlled by upstream mechanisms involving DNA damage and activation of p53 and downstream mechanisms involving upregulated Bax and cytochrome c and their translocation, accumulation of mitochondria, and activation of caspase‐3. We conclude that adult motor neuron death after nerve avulsion is DNA damage‐induced, p53‐ and Bax‐dependent apoptosis. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 181–197, 2002; DOI 10.1002/neu.10026     

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.     

DNA fragmentation during apoptosis is caused by frequent single-strand cuts.

One of the hallmarks of apoptosis is the digestion of genomic DNA by an endonuclease, generating a ladder of small fragments of double-stranded DNA. We have examined the nature of the DNA breaks produced in mouse thymocytes triggered to undergo apoptosis by steroids or by stimulation of the T cell receptor. Whereas the typical ladder pattern of oligonucleosomal fragments was observed after agarose gel electrophoresis, numerous single-strand cuts were detected after electrophoresis under denaturing conditions. Single-strand nicks were found to be very frequent in the internucleosomal regions, but also to occur in the core particle-associated DNA. An identical pattern of single-strand nicks was obtained when chromatin DNA was exposed to the single-strand cleaving deoxyribonuclease I. The nicked DNA fragments, extracted from apoptotic thymocytes, were sensitive to the action of S1-nuclease. We propose that DNA fragmentation induced during apoptosis is not due to a double-strand cutting enzyme as previously postulated, but rather is the result of single-strand breaks. This ensures the dissociation of the DNA molecule at sites where cuts are found within close proximity.     

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine-NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO-induced apoptosis-like cell death because Bax- or p53-deficient astrocytes are much more resistant than wild-type cells to the same NO treatment. We further demonstrate that LY294002-sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro-apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002-sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO-induced death in astrocytes is mediated by p53- and Bax-dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.     

A direct interaction between the survival motor neuron protein and p53 and its relationship to spinal muscular atrophy.

Mutations in the SMN1 (survival motor neuron 1) gene cause spinal muscular atrophy (SMA). We now show that SMN protein, the SMN1 gene product, interacts directly with the tumor suppressor protein, p53. Pathogenic missense mutations in SMN reduce both self-association and p53 binding by SMN, and the extent of the reductions correlate with disease severity. The inactive, truncated form of SMN produced by the SMN2 gene in SMA patients fails to bind p53 efficiently. SMN and p53 co-localize in nuclear Cajal bodies, but p53 redistributes to the nucleolus in fibroblasts from SMA patients. These results suggest a functional interaction between SMN and p53, and the potential for apoptosis when this interaction is impaired may explain motor neuron death in SMA.     

Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification

The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.     

A role for endogenous and radiation-induced DNA double-strand breaks in p53-dependent apoptosis during cortical neurogenesis

Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.     

DNA repair is activated in early stages of p53-induced apoptosis

p53 is a complex molecule involved in apoptosis, cell cycle arrest, and DNA repair. Since apoptosis may play an important role in deletion of neoplastic cells, an understanding of the mechanism of p53-induced apoptosis may be critical for possible future therapeutic interventions. Recent evidence suggests that p53-induced apoptosis may involve members of the nucleotide excision repair (NER) family, linking these two cellular events. Our work using a temperature-sensitive p53 construct further analyzes p53-induced apoptosis in cultured murine mammary epithelial cells and also suggests that DNA repair plays a role in that process. Although p21 is induced in our system, apoptosis occurs without a detectable preceding G1 cell cycle arrest and independent of cellular alterations brought on by the temperature shift. In addition, clonogenic assays suggest that early stages of p53-induced apoptosis may be reversible upon removal of the apoptosis stimulus. As a possible explanation for this reversibility, our results show that general DNA repair activity increases early in p53-induced apoptosis. We also show that caspase-3 is activated at a timepoint when colony formation begins to drop, suggesting a possible mechanism for the point of no return in p53-induced apoptosis.     

In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction

Phenanthroline and bipyridine, strong chelators of iron, protect DNA from single-strand break formation by H2O2 in human fibroblasts. This fact strongly supports the concept that these DNA single-strand breaks are produced by hydroxyl radicals generated by a Fenton-like reaction between intracellular Fe2+ and H2O2: H2O2 + Fe2+----Fe3+ + OH- + OH: Corroborating this idea is the fact that thiourea, an effective OH radical scavenger, prevents the formation of DNA single-strand breaks by H2O2 in nuclei from human fibroblasts. The copper chelator diethyldithiocarbamate, a strong inhibitor of superoxide dismutase, greatly enhances the in vivo production of DNA single-strand breaks by H2O in fibroblasts. This supports the idea that Fe3+ is reduced to Fe2+ by superoxide ion: O divided by 2 + Fe3+----O2 + Fe2+; and therefore that the sum of this reaction and the Fenton reaction, namely the so-called Haber-Weiss reaction, H2O2 + O divided by 2----O2 + OH- + OH; represents the mode whereby OH radical is produced from H2O2 in the cell. EDTA completely protects DNA from single-strand break formation in nuclei. The chelator therefore removes iron from the chromatin, and although the Fe-EDTA complex formed is capable of reacting with H2O2, the OH radical generated under these conditions is not close enough to hit DNA. Therefore iron complexed to chromatin functions as catalyst for the Haber-Weiss reaction in vivo, similarly to the role played by Fe-chelates in vitro.     

Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks

Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.     

Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage.     

Induction of DNA single-strand breaks in barley by sodium azide applied at pH 3.

Sodium azide (1 to 50 mM), adjusted to pH 3 and applied for 2 h to presoaked barley seeds, induced a dose-dependent frequency of single-strand breaks in DNA of non-germinating embryos. This was demonstrated by sedimentation analyses of isolated DNA samples in alkaline sucrose gradients and in neutral sucrose gradients with 80% formamide. The doses applied also inhibited dose dependently the root length, seed germination and partially the seedling height. Only the sub-lethal doses (10 and 12.5 mM) induced a low frequency of chromatid breaks and translocations in the root tip metaphases. The sedimentation rate (in alkaline sucrose gradients) of calf thymus DNA treated with sodium azide at pH 3, was similar to that of the control DNA treated with buffer (pH 3) alone.