Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not influenced by p66Shc expression. In basal conditions, p66Shc−/− and p53−/− cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.
One popular theory of ageing proposes that reactive oxygen species (ROS) cause cumulative oxidative damage over a lifetime (Kirkwood and Austad, 2000; Harman, 1981). ROS (superoxide anions, hydroxyl radicals, and H2O2) are produced by different intracellular redox reactions, which involve molecular oxygen as the final electron acceptor, and are the most recognized mediators of cell damage (Harman, 1981; Lambeth et al., 2000). In support of the free radical theory of ageing, antioxidant treatment or over-expression of antioxidant genes increase life span in invertebrates (Guarente and Kenyon, 2000; Melov et al., 2000). In mammals, instead, supporting evidence is largely correlative, mainly based on findings of increased oxidative damaged DNA/proteins in aged individuals (Van Remmen and Richardson, 2001). In both invertebrates and mammals increased longevity correlates with increased resistance to oxidative stress, both at the organism and cellular levels (Finkel and Holbrook, 2000). Whether this reflects the existence of a common genetic/biochemical pathway that regulates life span and response to oxidative signals is, however, unknown.
We report here our investigations on the molecular mechanisms underlying oxidative stress resistance of the p66Shc−/− longevity mice. P66Shc is a splice variant of p52Shc/p46Shc, a cytoplasmic signal transducer involved in the transmission of mitogenic signals from tyrosine kinases to Ras (Pelicci et al., 1992). P66Shc has the same modular structure of p52Shc/p46Shc (SH2-CH1-PTB) and contains a unique N-terminal region (CH2); however, it is not involved in Ras regulation but rather functions in the intracellular pathway that converts oxidative signals into apoptosis (Migliaccio et al., 1997, 1999). P66Shc is serine-phosphorylated (within the CH2 region) in cells treated with UV or other inducers of oxidative stress, such as H2O2, and p66Shc−/− fibroblasts are resistant to UV- or H2O2-induced apoptosis, a finding mirrored by the increased UV- or H2O2-sensitivity conferred by overexpression of p66Shc (Migliaccio et al., 1999). Finally, p66Shc−/− mice survive significantly better to paraquat intoxication, another inducer of oxidative stress and live longer (Migliaccio et al., 1999).
Results and discussion
P66Shc is a downstream target of p53
The p53 tumour suppressor plays a central role in the regulation of oxidative stress-induced apoptosis (Vogelstein et al., 2000). H2O2 and UV activate p53, and p53−/− mouse embryo fibroblasts (MEFs), like p66Shc−/−, are resistant to H2O2- or UV-induced apoptosis (Migliaccio et al., 1999; Yin et al., 1998). To investigate whether p66Shc is a downstream target of p53, we analysed p66Shc expression in WT and p53−/− MEFs, following UV (Figure 1a) or H2O2 (Figure 1b). Both treatments induced marked and persistent up-regulation of p66Shc protein levels in WT, but not p53−/−, MEFs. Furthermore, over-expression of p53 in WT MEFs (Figure 1c) or in DLD-1 colorectal cancer cells (Figure 1d), which contain inactive p53 (Polyak et al., 1996), provoked p66Shc up-regulation. RNase protection experiments showed no significant variations of p66Shc transcripts following H2O2 or UV treatment of WT MEFs (Figure 1e). Pulse chase experiments using 35S-labelled cells, instead, showed increased p66Shc protein stability in WT, but not p53−/−, cells following UV treatment (Figure 1f). These results indicate that p53 induces p66Shc protein up-regulation by increasing its stability.
P66Shc regulates p53-dependent apoptosis
To evaluate the biological significance of p66Shc up-regulation following p53 activation, we analysed: (i) the effects of p66Shc over-expression on p53-induced apoptosis and (ii) the apoptogenic effect of over-expressed p53 in p66Shc−/− cells. Over-expression of p53 in DLD-1 cells induced apoptosis (from 2% to 30% at day 3), which was further enhanced by concomitant over-expression of p66Shc (from 30% to 80% at day 3) (Figure 2a). Notably, over-expression of p66Shc alone slightly increased apoptosis (Figure 2a). Over-expression of p53 in MEFs induced apoptosis of WT (approximately 15%, 2 days after infection), but not p66Shc−/− (3%) cells (Figure 2b). It appears, therefore, that p66Shc regulates p53-dependent apoptosis.
Depending on the activating stimulus, p53 mediates apoptosis or cell cycle arrest (Vogelstein et al., 2000). To investigate whether p66Shc is involved in p53-dependent cell cycle arrest, MEFs were treated with low concentration (200 μM) H2O2, which is known to induce growth arrest of human diploid fibroblasts (Bladier et al., 1997). Low dose H2O2 provoked a dramatic reduction of cycling cells in both WT and p66Shc−/− cells, as measured by bromodeoxyuridine (BrdU) incorporation (from 25–30% to less than 5% 4 h after treatment) (Figure 2c). Similarly, γ-irradiation or oncogenic Ras expression induced cell cycle arrest of both WT and p66Shc−/− MEFs (unpublished results). It appears, therefore, that p66Shc regulates p53-dependent apoptosis, while leaving unaffected other p53 functions. These findings might explain why p66Shc−/− mice, carrying a selective functional alteration of p53, do not develop tumours at increased frequency. Complete inactivation of p53 functions in p53−/− mice, in fact, leads to high tumour susceptibility (75% of mice die of cancer by 26 weeks of age) (Purdie et al., 1994). P66Shc−/− mice, instead, showed a frequency of spontaneous tumour formation comparable to their control littermate (in the first year of life, 7/211 WT (3.3%); 1/35 p66Shc+/− (2.8%) and 4/136 p66Shc−/− (2.9%) died of tumours). In addition, we observed no differences between WT and p66Shc−/− mice in two TPA/DMBA-induced carcinogenesis experiments (unpublished results).
P66Shc controls p53-induced cytochrome c release and ROS up-regulation
P53 activation involves protein stabilisation and post-translational modifications, transcriptional activation of a number of pro-apoptotic genes, release of apoptogenic factors from mitochondria, including cytochrome c, and cytosolic assembly of the apoptosome (Green and Reed, 1998). H2O2 treatment induced a similar extent of p53 up-regulation, phosphorylation and acetylation in WT and p66Shc−/− MEFs (Figure 3a). Accordingly, the ability of over-expressed p53, H2O2 or UV, to regulate p53-target promoters was not affected by p66Shc expression (Figure 3b and not shown). However, following H2O2 treatment, a 2–3-fold increase of cytoplasmic cytochrome c was observed in WT, but not in p66Shc−/− MEFs (Figure 3c). Re-expression of p66Shc restored the ability of H2O2 to induce cytochrome c release in p66Shc−/− MEFs (Figure 3c), thus demonstrating that, in p66Shc−/− cells, H2O2 activates p53 but is unable to significantly induce cytochrome c release from mitochondria.
The mechanisms involved in the release of mitochondrial proteins during apoptosis are not entirely clear. P53 induces transcriptional activation of redox-related genes and a sustained rise in ROS levels, possibly involved in opening of the mitochondrial pore and activation of the mitochondrial permeability transition (PT) (Polyak et al., 1997; Li et al., 1999; Schuler et al., 2000). Indeed, antioxidant treatment inhibits apoptosis following p53 over-expression (Polyak et al., 1997; Johnson et al., 1996). To measure levels of intracellular ROS we used 2′,7′-dichlorofluorescin diacetate (DCFDA), which is oxidised, mainly by H2O2, and generates fluorescent dichlorofluorescein. Over-expression of p53 in WT MEFs lead to a twofold increase in DCFDA fluorescence (Figure 3d). In contrast, no significant variations of DCFDA fluorescence after p53 expression were seen in p66Shc−/− MEFs (Figure 3d). To investigate whether p66Shc interferes with mitochondrial PT, we analysed the effects of Cyclosporin A (CsA) on p66Shc-mediated apoptosis. CsA binds to cyclophilin D, a component of the mitochondrial PT pore, thus inhibiting PT and apoptosis (Nicolli et al., 1996). CsA treatment almost completely prevented H2O2-induced cell death of WT fibroblasts (Figure 3e), showing that oxidative stress-induced apoptosis involves activation of the mitochondrial pathway. Notably, CsA also prevented the ability of re-expressed p66Shc to restore H2O2-induced apoptosis in p66Shc−/− cells (Figure 3e), suggesting that p66Shc acts upstream of the mitochondrial PT and its release of apoptogenic factors.
P53 and p66Shc regulate steady-state levels of intracellular ROS
To investigate whether the effect of p66Shc on p53-induced ROS up-regulation reflects a more general function of p66Shc in the regulation of ROS metabolism, we performed DCFDA staining of WT and p66Shc−/− cells, grown under standard conditions. Fluorescence microscopy evaluation (Figure 4a) and FACS quantification (not shown) revealed 30–40% lower fluorescence in the p66Shc−/− MEFs, mouse adult fibroblasts (MAFs) and primary endothelial cells (not shown). Likewise, p53−/− MEFs and MAFs showed decreased DCFDA staining (Figure 4a). Expression of p66Shc in p66Shc−/− MEFs or p53-null DLD1 cells markedly increased DCFDA fluorescence (Figure 4b). Notably, the observed differences do not reflect biological variabilities since antimycin A treatment, which blocks electron flow at cytochrome b thus maximizing ubiquinone reduction and O2−. production by mitochondria (Gniadecki et al., 2000), induced comparable ROS rises in WT and p66Shc−/− MEFs (Figure 4c). It appears, therefore, that p53 and p66Shc regulate intracellular ROS levels under steady-state culture conditions (e.g. in the absence of apoptogenic signals) and that p66Shc functions downstream to p53.
P53 and p66Shc regulate levels of intracellular oxidative damage
We next investigated whether the variations of intracellular ROS observed in WT and p66Shc−/− or p53−/− MEFs are biologically significant, e.g. whether they correspond to different levels of oxidation-damaged macromolecules. Since mitochondrial DNA (mtDNA) is a critical cellular target for ROS (Yakes and Van, 1997), we analysed its integrity using Extra Long PCR (XLPCR). This assay is based on the fact that, in the presence of ROS-induced DNA lesions, the yield of PCR amplification of long (8616 bp) mtDNA fragments is significantly reduced (Yakes and Van, 1997). Higher XLPCR yields were found in p66Shc−/− MEFs and MAFs (Figure 5a), and in p53−/− MEFs (Figure 5b), compared to WT controls. Notably, a marked reduction of the XLPCR yield was detected in p66Shc−/− MEFs after p66Shc re-expression (Figure 5c). Together, these results suggest that p53- and p66Shc-dependent variations in ROS levels result into increased mitochondrial oxidative damage, thereby providing a putative mechanism for the proposed effect of p66Shc on mitochondrial integrity.
Accumulation of ROS-damaged DNA, both nuclear and mitochondrial, is considered a critical event during the ageing process (Ames et al., 1993). Oxidation of the C8 of guanine (8-oxo-dG) is among the most abundant type of oxidation-damaged nuclear DNA (Helbock et al., 1999). Analysis of nuclear (by 8-oxo-dG determination) or mitochondrial (by XLPCR) DNA from WT and p66Shc−/− tissues revealed decreased 8-oxo-dG and higher XLPCR yields in the lung, spleen, liver and skin from p66Shc−/− mice (Figure 5d). Slightly increased XLPCR yields and/or decreased 8-oxo-dG amounts were also observed in p66Shc−/− muscles (quadriceps femoris) and kidney, while no significant difference were found in brain and heart from WT and p66Shc−/− mice. Notably, heart, muscles and kidney express little amounts of p66Shc (unpublished), while brain expresses no p66Shc (Cattaneo and Pelicci, 1998). Finally, to obtain a further, independent confirmation of the presence of decreased mtDNA alterations in p66Shc−/− tissues, we searched specific mtDNA rearrangements. MtDNA rearrangements accumulate with age in tissues of a variety of animals, including humans. One of the most common deletions involves loss of a 3867 bp DNA segment, identified as a diagnostic PCR fragment of 748 bp (Tanhauser and Laipis, 1995). Strikingly, this deletion was detected in the mtDNA from liver samples of young and old WT mice, while it was barely detectable in matched tissues from p66Shc−/− mice. Furthermore, it was equally present in the brain of WT and p66Shc−/− mice (Figure 5e). In conclusion, p66Shc expression correlates with the extent of oxidation-damaged DNA, suggesting that decreased intracellular ROS are responsible for the increased life span of p66Shc−/− mice.
We reported here that p66Shc regulates intracellular levels of ROS and mediates ROS-upregulation during p53-induced apoptosis. Our findings are consistent with a model whereby the p53-p66Shc pathway is a sensor of the levels of intracellular oxidative signals and regulates intracellular levels of oxidants and of oxidative damage. High intensity oxidative signals, as when cells are exposed to acute oxidative stress, would result in high-level activation of the p53-p66Shc pathway and apoptosis. Low intensity oxidative signals, as may occur in metabolically active cells, would result in chronic, low-level activation of the p53-p66Shc signalling pathway, thus allowing moderate ROS rises and accumulation of oxidative damage.
Accumulation of ROS-inflicted, oxidative-damaged macromolecules with age has been hypothesised as the proximal causative agent of ageing (Finkel and Holbrook, 2000). Basal levels of ROS, and the extent of its oxidative damage to macromolecules, are generally considered to be the random consequences of physiological functions, such as cellular respiration (Finkel and Holbrook, 2000). An important implication of our findings is that, in mammals, these phenomena are genetically determined and controlled by a stress-induced signal transduction pathway, involving p53 and p66Shc.
Genetic evidence for the putative function of p53 as ageing gene cannot be obtained from the p53−/− mice, which die of cancer within the first year of life. However, two p53 transgenic mutations have recently been described in mice, which result into p53 activation and cause reduced life span, progeria and tumor resistance, suggesting that p53 has a role in ageing (Tyner et al., 2002). Our findings that p66Shc−/− mice have no increased tumor formation and that the p53-p66Shc pathway is selectively involved in the propagation of pro-apoptotic oxidative signals, suggest that p66Shc separates, genetically and biochemically, the ageing and tumor suppressor activities of p53.
Materials and methods
Cells and infections
MEFs or MAFs were prepared according to standard procedures. Retroviral vector DNAs were transfected into the Phoenix packaging cell lines and, after 48 h, supernatants were used to infect target cells. The pBABE retroviral vector expresses the puromycin selectable marker. Recombinant p53 adenovirus was a gift of Dr M Capogrossi.
H2O2 was added to complete culture media. Four μM CsA (Sigma) was added to cells 45 h before the addition of H2O2. Cell viability was measured by the Trypan-blue dye exclusion test. Apoptosis was determined by propidium iodine (PI) staining and flow cytometry (CellQuest 3.1, Beckton Dikinson) or by the TUNEL assay (Roche Tunel Kit).
P53 dependent transactivation assays
Transactivation assays were performed in MEFs by transient transfection (lipofectamin) of various p53-target promoters/Luc constructs. Light units were normalised to expression of a co-transfected β-galactosidase vector.
Cells were incubated for 30 min with 2–10 μM DCFDA (Molecular probes) in complete media and analysed by fluorescence microscopy (JVC 3-CCD, Image Grabber PCI 1.0) or flow cytometry.
High molecular weight DNA was isolated with the QIAamp kit (Qiagen). Normalization of mtDNA copy number was performed by quantitative PCR (Taqman method) of a D-loop mtDNA fragment, normalised to the nuclear telomerase gene. XLPCR was performed on normalised DNAs by PCR of 8616 bp and 322 bp mtDNA fragments. XLPCR was performed using the GeneAmp PCR System 9700 in a 50 μl volume with 20 pmol of primers, 50–200 ng DNA, 0.8 mM dNTPs, 1.75 mM Mg acetate and 1.5 U rTth polymerase XL. The termocycler profile was: 95°C for 2′; 27 cycles at 95°C for 30 h min′, 68°C for 8 h and 72°C for 10 h. XLPCR amplification primers: bp 4963–4988 of mouse mtDNA (forward); bp 13604–13578 (backward); for the co-amplified 322 bp fragment: bp 467–492 (forward) and bp 762–788 (backward).
Determination of 8-oxo-dG
Plates were coated with salmon sperm DNA and treated with CuSO4 1 mM, H2O2 5 mM for 30 h at 37°C, washed and saturated with 3% BSA. DNA samples (quantified as above) or the 8-oxo-dG modified base (OXIS) were incubated in test tubes with anti 8-oxo-dG antibody 1F7 (Trevigen), left overnight at 4°C and added to coated plates. After 2 h at 37°C, plates were washed in PBST and bound antibodies revealed by HRP-secondary antibody, followed by TMB reaction, and read at 450 nm.
Detection of mtDNA deletions
MtDNA deletions were measured by PCR using specific primers (bp 8484–8505 and 13077–13098) in a short PCR cycle (94°C, 40′ min; 50°C, 20′ min; 72°C, 40′ min; 30 cycles).
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We thank L Luzi, L Pozzi, K Helin, R Carbone, M Capogrossi, A Cocito and R Cortese for many helpful discussions; G Giardina, G Pelliccia, M Bono, MT Sciurpi, S Ronzoni and M Faretta for technical help; A Ariesi for secretarial work. M Trinei, A Ventura and E Milia are recipient of fellowships from FIRC, VAR from M Curie. Supported by grants from EC, AIRC and CNR.
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Trinei, M., Giorgio, M., Cicalese, A. et al. A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis. Oncogene 21, 3872–3878 (2002). https://doi.org/10.1038/sj.onc.1205513
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