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Keywords:

  • apoptosis;
  • development;
  • p53;
  • SirT1

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The SirT1 gene encodes a protein deacetylase that acts on a number of nuclear substrates. p53 was identified as a SirT1 substrate whose transcriptional activity was reported to be negatively regulated by SirT1-dependent deacetylation. We set out to determine whether developmental defects and perinatal lethality observed in SirT1-null mice were caused by p53 hyperactivity by creating mice deficient for both SirT1 and p53. Animals null for both proteins were smaller than normal at birth, had eyelid opening defects and died during the late prenatal and early postnatal periods, a phenotype indistinguishable from mice deficient for SirT1 alone. Upon re-examination of the role of SirT1 in modulating p53 activity, we found that while SirT1 interacts with p53, the SirT1 protein had little effect on p53-dependent transcription of transfected or endogenous genes and did not affect the sensitivity of thymocytes and splenocytes to radiation-induced apoptosis. These findings suggest that SirT1 does not affect many p53-mediated biological activities despite the fact that acetylated p53 has been shown to be a substrate for SirT1.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The silent information regulator (SIR) family of genes encodes a group of NAD+-dependent protein deacetylases. The founding member of this family is the sir2 gene of S. cerevisiae whose substrates are histones H4 and H3 acetylated on lysines 16 and 9, respectively (reviewed in Guarente, 1999). The mammalian SirT1 gene is the homologue of yeast sir2. In addition to deacetylating histones H3 and H4 (Imai et al., 2000), SirT1 has a remarkably varied spectrum of nuclear substrates that include TAF68 (Muth et al., 2001), p53 (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002; Cheng et al., 2003), FoxO (Brunet et al., 2004; Motta et al., 2004), p300/CBP (Motta et al., 2004), myoD (Fulco et al., 2003), NFB (Yeung et al., 2004), BCL6 (Bereshchenko et al., 2002), Ku70 (Cohen et al., 2004), and histone H1 (Vaquero et al., 2004).

The yeast sir2 gene regulates lifespan and response to stress, two biological processes regulated in mammalian cells by p53. The p53 transcription factor is thought to be acetylated in its active form and can be deacetylated by SirT1 (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002; Cheng et al., 2003). These observations suggest that an important role of the mammalian SirT1 may be to modulate p53 function and thus affect aging and response to stress. Experiments on cell lines in culture demonstrated that p53-dependent transcription and p53-dependent responses to ionizing radiation can be dependent on the SirT1 deacetylase. However, tests of p53-dependent cellular processes in SirT1-null mice and embryonic stem (ES) cells have yielded conflicting data. On the one hand, thymocytes from SirT1-null mice were shown to be hypersensitive to radiation-induced apoptosis (Cheng et al., 2003), consistent with the prediction that SirT1 normally serves to negatively regulate p53 function. However, cells of the testis of SirT1-null animals are not differentially sensitive to radiation (McBurney et al., 2003b), nor are SirT1-null ES cells (McBurney et al., 2003a).

Given the potential importance of SirT1 as a possible regulator of p53, we sought to examine the effect of SirT1 on some of the p53-dependent biological activities. If the main function of SirT1 were to regulate p53 activity, the prediction is that the phenotype of the SirT1-null mice should be a consequence of hyperactivity of p53. In fact, mice engineered to have high p53 activity through overexpression of the p44 short isoform of p53 have shortened lifespans, small size and bone morphological defects (Maier et al., 2004), characteristics that resemble those of the SirT1-null mice (Cheng et al., 2003; McBurney et al., 2003b). If p53 regulation were the main role of the SirT1 protein, one would predict that the phenotype of the SirT1-null mouse would be rescued in the absence of p53. We therefore set out to create animals that were null for both SirT1 and p53.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

SirT1-null phenotype is not rescued by the absence of p53

If the abnormalities in SirT1-null mice were the result of improper regulation of p53 activity, then animals carrying null alleles for both genes should resemble p53−/– mice, i.e. the absence of p53 should rescue the SirT1-null phenotype.

We created animals heterozygous for both SirT1 and p53 and intercrossed these compound heterozygotes. Among the offspring from this cross were animals of the expected nine genotypes shown in Table 1. Consistent with previous studies (Cheng et al., 2003; McBurney et al., 2003b), the proportion of viable offspring from these crosses that were null for SirT1 was lower than expected (Table 1) indicating loss of some SirT1−/– fetuses in utero. Animals that were null for p53 were also born in proportions that were lower than expected. However, viable SirT1−/– p53−/– pups were recovered in this experiment. The genotypes of these animals were obtained by PCR and immunoblots verified that both SirT1 and p53 proteins were absent from knockout animals (Fig. 1). The SirT1−/– p53−/– animals had characteristics indistinguishable from those of the SirT1−/– p53+/+ animals (Fig. 2). On the 129/Sv genetic background, SirT1-null animals are smaller than their normal littermates, have a characteristic eyelid defect, and die before reaching 1 month of age. None of these characteristics was ameliorated by the absence of p53. Each of the 7 SirT1−/–p53−/– animals obtained in our experiment died before reaching 4 weeks (Fig. 3), all were smaller than their littermates that carried a wild-type SirT1 allele, and all had the eyelid defect.

Table 1.  SirT1+/− p53+/− crosses produce viable offspring
 Offspring from heterozygous crosses
p53+/+p53+/–p53−/–
  1. Offspring from crosses between SirT1+/–p53+/– animals were genotyped by PCR analysis of DNA from tail clips and classified into the nine categories above. A total of 212 animals were obtained. SirT1−/– and p53−/– animals were present in less than expected Mendelian ratios (P < 0.01, χ2-test).

SirT1+/+203510
SirT1+/–376021
SirT1−/– 913 7
image

Figure 1. Intercross of SirT1+/– p53+/– mice yields offspring lacking SirT1, p53 or both proteins. Protein lysates were obtained from livers of mice whose PCR-determined genotypes were those indicated above each lane. Proteins were separated on SDS 4–12% polyacrylamide gradient gels and blots probed with antibodies to SirT1, p53, and β-actin.

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image

Figure 2. Knockout of p53 does not rescue the SirT1−/– phenotype. A and B show SirT1−/–p53+/+ and SirT1+/+p53+/+ 3-week-old littermates while C and D show SirT1−/–p53−/– and SirT1+/+p53−/– littermates of the same age. In all panels, the arrow indicates the SirT1−/– mutant. The eyelid phenotype of the SirT1−/– animals is clear in A and C while the smaller size of the SirT1−/– animals is evident in B and D. The SirT1−/–p53+/+ and SirT1−/–p53−/– animals are indistinguishable in sharing the eyelid opening defect and small stature.

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image

Figure 3. SirT1−/– and SirT1−/– p53−/– animals die shortly after birth. Survival of SirT1−/–p53+/+ (solid line) and SirT1−/–p53−/– (dashed line) mice is shown. Neither SirT1−/–p53+/+ nor SirT1−/–p53−/– mice survive past 5 weeks of age on the 129/Sv background. There is no significant difference in lifespan between these genotypes.

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The failure of p53 deficiency to rescue the SirT1-null phenotype caused us to reexamine the role of SirT1 on p53 function.

Mammalian SirT1 interacts with p53

Previous reports that SirT1 physically interacts with the p53 tumor suppressor protein (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002; Cheng et al., 2003) were based primarily on experiments in cells transfected with DNA encoding one or both proteins. Both SirT1 and p53 proteins are abundantly expressed in ES cells (Aladjem et al., 1998; McBurney et al., 2003a). Although there have been reports that p53 is cytoplasmic in ES cells (Aladjem et al., 1998), we found that immunostaining of p53 is nuclear in the R1 lines of ES cells used for our experiments (data not shown). Similarly, the SirT1 protein is nuclear. We carried out immunoprecipitation experiments to determine whether these two proteins are associated when expressed from their endogenous genes. Extracts from mouse ES cells were immunoprecipitated with an anti-p53 antibody and immunoblot analysis showed that SirT1 was coimmunoprecipitated with p53 (Fig. 4A). In the reciprocal experiment, cell lysates were immunoprecipitated with anti-SirT1 antibody and the immunoblot showed that p53 was present in the precipitate (Fig. 4B). Thus, the endogenous p53 and SirT1 proteins do interact in mouse ES cells.

image

Figure 4. SirT1 interacts with p53. (A) Cell lysates from SirT1+/+ and SirT1−/– ES cells were immunoprecipitated with antibody to p53 and the precipitated proteins electrophoresed, blotted and probed with antibody to SirT1. The amount of material loaded in the control cleared lysate lanes was 10% that used for immunoprecipitation. (B) The same cell lysates were immunoprecipitated with antibody to SirT1 and the immunoblot of precipitated proteins probed with antibody to p53.

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Effect of SirT1 on p53 transcriptional activity

Several lysine residues on p53 are known to be acetylated and the acetylated form is thought to be a more active transcription factor, perhaps because it binds its cognate DNA sequence more avidly (Luo et al., 2004). SirT1 has been shown repeatedly to deacetylate lysine 382 of human p53 both in vitro and in vivo (Luo et al., 2001; Vaziri et al., 2001). In addition, the transcription of p53-dependent reporter genes has been reported to be suppressed by SirT1 and elevated by inhibition of SirT1 (Luo et al., 2001;Vaziri et al., 2001).

We used the CAT reporter gene driven by either the p53-dependent p21 promoter or the p53-dependent maspin promoter to assess p53 transcription-inducing activity in 293T cells. Cotransfection with an expression vector encoding p53 resulted in significant reporter gene expression (Fig. 5A). This level of expression was elevated further, albeit by less than twofold, by cotransfection with an expression vector encoding SirT1. An expression vector encoding a mutant version of SirT1 lacking catalytic activity resulted in no change of expression, suggesting that the increased expression from the reporter was dependent on the deacetylase activity of SirT1.

image

Figure 5. SirT1 enhances p53-dependent transcription. (A) 293T cells were transfected with plasmid carrying the CAT (chloramphenicol acetyl-transferase) reporter gene under the control of the maspin promoter along with plasmids carrying expression vectors encoding p53 or SirT1 as indicated. The SirT1H355Y mutation renders the SirT1 protein catalytically inactive. The mutant p53 (p53L22Q/W235) is transactivation deficient (Lin et al., 1994). CAT activity was measured 24 h after transfection. The results shown represent the averages and standard errors of three experiments. (B) SirT1+/+ and SirT1−/– ES cells were transfected with CAT under control of the p21 promoter. The HCT116 human colorectal carcinoma cell line and its p53-null derivative (Brattain et al., 1981; Bunz et al., 1999) were used as controls to verify that the expression from the p21 promoter was dependent on endogenous p53. CAT activity was determined as in (A). Transfection efficiency was normalized by cotransfection with a plasmid carrying GFP driven by the mouse Pgk-1 promoter. GFP-derived fluorescence was assessed in a fluorometer.

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The endogenous level of p53 is low in 293T cells so expression of the p53-dependent reporter genes depends on the cotransfected expression vector encoding p53. To investigate the effect of endogenous p53 and SirT1 on reporter gene expression, we transfected the reporter genes into ES cells that were normal or null for SirT1. p53 is abundant in ES cells and reporter CAT activity was easily detected. In cells lacking SirT1, CAT reporter activity was diminished to about 50% of that found in wild-type ES cells (Fig. 5B). Thus, contrary to previously published reports (Luo et al., 2001; Langley et al., 2002; Cheng et al., 2003), our data indicate that SirT1 does not negatively regulate p53 transcriptional activity. Rather, our data suggest that SirT1 has a small activating effect on p53-dependent transcription of transfected reporter genes.

To assess the induction of endogenous p53-dependent genes in vivo, SirT1+/+ and SirT1−/– mice were irradiated and RNA harvested 6 h later from thymus and spleen. Quantitative PCR analysis of this RNA indicated elevated levels of the cell-cycle inhibitor protein p21 and pro-apoptotic bbc3 following irradiation (Fig. 6). These transcripts were induced to similar extents in SirT1+/+ and SirT1−/– tissues although bbc3 appeared to be induced to a higher level in the SirT1−/– thymus. Western blots from irradiated testes indicated induction of p21, Bax, and mdm2 to similar levels in SirT1+/+ and SirT1−/– mice (data not shown).

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Figure 6. SirT1 deficiency does not affect expression of p53-induced genes in vivo. SirT1+/+ and SirT1−/– sex-matched littermates were irradiated with 10 Gy of 250 keV X-rays. RNA was collected from spleen and thymus 6 h postirradiation and analyzed by real-time quantitative RT-PCR using primers for p21(Cip1) and bbc3. The ratio of mRNA levels to 18S rRNA is shown.

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SirT1 does not affect p53-dependent apoptosis

Thymocytes are extremely sensitive to ionizing irradiation and undergo apoptosis in a p53-dependent fashion following radiation (Lowe et al., 1993). We isolated thymocytes from SirT1-null mice, from their sex-matched SirT1 wild-type and heterozygous littermates, and from p53-null mice. Cultures of these thymocytes were irradiated and the proportion of live cells assessed 24 h later. While the p53−/– thymocytes were very resistant to radiation-induced apoptosis, we found no difference in the survival of SirT1+/+, SirT1+/–, and SirT1−/– thymocytes (Fig. 7). This is in contrast to a previous study that demonstrated an increased sensitivity to ionizing radiation in SirT1-null thymocytes (Cheng et al., 2003).

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Figure 7. SirT1 deficiency does not affect p53-dependent survival of irradiated thymocytes. Thymocytes from age and sex matched SirT1+/+ (n = 3, •), SirT1+/– (n = 4, ▪), SirT1−/– (n = 4, ○) and p53−/–(n = 4, □) mice were collected and irradiated at the doses indicated with 250 keV X-rays. Cells were cultured for 24 h and viable cell number determined microscopically.

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Apoptosis in SirT1-deficient tissue was also assessed in vivo. Tissues from irradiated mice were collected, sectioned and apoptotic cells identified using a fluorescein TUNEL apoptosis detection kit (Fig. 8). Spleen and thymus from nonirradiated mice showed very few TUNEL-positive nuclei, while those from irradiated animals showed widespread cell death (Fig. 8A). No difference was observed between SirT1+/+ and SirT1−/– animals (Fig. 8B).

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Figure 8. SirT1 deficiency does not affect p53-dependent apoptosis in vivo. (A) Fluorescein TUNEL apoptosis staining on frozen sections of spleens from irradiated and nonirradiated SirT1+/+ and SirT1−/– mice. TUNEL staining is green and Hoescht nuclear counter stain is blue. Scale bar: 200 µm. (B) The proportion of TUNEL positive cells in thymus and spleen were quantified by counting three representative fields from samples from each of the mice.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Both p53 and SirT1 are key proteins involved in the regulation of cellular behavior. p53 is a transcription factor whose activity is thought to be modulated by acetylation (Sakaguchi et al., 1998). SirT1 is a protein deacetylase able to deacetylate p53 both in vitro and in vivo. An attractive model for how SirT1 might affect cellular behavior is by modulating the acetylated status of p53. A number of reports have appeared in the literature that support this model (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002; Cheng et al., 2003). However, the results reported above provide evidence that p53 is not the only cellular substrate for SirT1 and, in fact, a number of p53-dependent functions such as apoptosis and transcription appear to be independent of SirT1.

If p53 were a critical substrate for SirT1, one would predict that the phenotype of SirT1-null animals should be rescued in animals that are also p53 null. We found that the developmental abnormalities characteristic of the SirT1-null animals (in utero death, small size, perinatal lethality, eyelid defect) were all present in animals that were null for both SirT1 and p53. This evidence indicates that p53 hyperacetylation is not responsible for these developmental defects. Some other substrate(s) for SirT1 must be responsible for the biological defects that result in these characteristics.

One of the critical roles of p53 is the regulation of cell death. Thymocytes are known to undergo apoptosis very rapidly following irradiation and this effect is dependent on p53 (Lowe et al., 1993). We found that SirT1-null thymocytes and splenocytes underwent apoptosis with similar sensitivity to normal thymocytes. This result is in contrast to what has been reported (Cheng et al., 2003) but is consistent with the other results from our study that failed to confirm that p53-induced transcription is down-regulated by SirT1. It is now evident that p53-dependent apoptosis may be independent of the transcriptional activity of p53 per se (Erster & Moll, 2005) although a recent report (Chipuk et al., 2005) indicates a role for the bbc3 protein, a p53-dependent transcript, in triggering p53-dependent apoptosis.

Acetylated p53 is thought to be a more active transcription factor because it binds its cognate DNA binding sequence more avidly (Luo et al., 2004). Given that SirT1 has been shown to deacetylate p53 (Luo et al., 2001; Vaziri et al., 2001; Cheng et al., 2003) we were surprised to find that p53-responsive gene expression was not reduced in the presence of SirT1. These results are difficult to reconcile with previous reports that indicate down-regulation of p53 transcriptional activity by SirT1 (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002; Cheng et al., 2003). In fact, this confusion regarding the effect of SirT1 on p53 is not unique as another transcription factor, FoxO, has been reported to be both activated and repressed by the SirT1 deacetylase (Brunet et al., 2004; Motta et al., 2004). Indeed, the relationship between SirT1, p53, and FoxO is complex. A recent report (Nemoto et al., 2004) indicates that transcription of SirT1 itself can be modulated by the activity of FoxO in a p53-dependent fashion.

One possible explanation for some of these discrepancies is that many previous studies were based on the use of the antioxidant resveratrol as a SirT1 activator. It has since been shown that resveratrol activates SirT1 in a highly substrate-specific manner (Borra et al., 2005; Kaeberlein et al., 2005), indicating that any effects observed using this compound cannot be attributed to the activation of SirT1.

Another possible source of complication regarding the relationship between SirT1 and p53 is that the activities of both proteins are modulated by NAD+. The p53 tetramer binds NAD+ and its activity is enhanced (McLure et al., 2004) while SirT1 requires NAD+ as a cofactor and uses it up during the deacetylation reaction (Imai et al., 2000). Thus, local concentrations of NAD+ may play a role in mediating the effects of SirT1 on p53 activity.

The activity of p53 has been reported to be regulated through lysine methylation (Chuikov et al., 2004) in addition to acetylation and other post-translation modifications. Thus, p53 activity may be regulated by complex mechanisms which depend not on individual amino acid modifications but rather combinatorial effects similar to the ‘histone code’ (Jenuwein & Allis, 2001).

However, we favor the idea that the regulation of p53 activity is exquisitely sensitive to the concentrations of endogenous regulatory proteins and that transfection assays yield such unphysiological intracellular conditions that meaningful conclusions cannot be inferred. Transfection assays indicated that the C-terminal lysine residues of p53 are important for modulating stability and activity but recent studies report that mutating the 6 or 7 C-terminal lysine codons of the endogenous p53 gene does not affect p53 transcriptional activity or p53-dependent processes such as cell cycle arrest and apoptosis (Feng et al., 2005; Krummel et al., 2005). Thus, the SirT1 catalyzed deacetylation of p53 may have little effect because modification of these lysine residues may play no role in the regulation of those p53 functions we assessed. Whether SirT1 affects other p53 activities, such as tumor suppression, warrants further exploration.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Cell culture

R1 cells (Nagy et al., 1993) and SirT1−/– ES cells (McBurney et al., 2003b) were grown in the presence of air and 10% CO2 at 37 °C in humidified chambers. These cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and leukemia inhibitory factor (LIF). 293T and HCT116 cells were cultured in α-MEM supplemented with 10% serum [7.5% donor bovine serum (DBS) and 2.5% FCS] and grown in 20% O2, 5% CO2 at 37 °C.

Immunoprecipitation and immunoblots

R1 and SirT1−/– ES cells were grown to confluence and harvested in IP buffer (50 mm NaH2PO4, 300 mm NaCl, pH 8.0). Cells were lysed by passage through a 26-gauge needle. Lysates were precleared with protein-A agarose beads (Upstate, Lake Placid, NY, USA) for 1 h and subsequently incubated with anti-SirT1 (Upstate) or anti-p53 (Ab-1, Oncogene, Mississauga, ON, Canada) antibody overnight at 4 °C. Protein-A agarose was added to the lysates and rotated at 4 °C for 2 h. Immune complexes were collected, washed, resolved on 8% polyacrylamide tris-glycine gels and transferred to nitrocellulose. Nitrocellulose membranes were probed with either anti-p53 (Oncogene) or anti-SirT1 (Upstate) followed by detection with HRP-conjugated secondary antibodies and KPL LumiGLO chemiluminescent substrate (Mandel, Guelph, ON, Canada).

Transfections and CAT assays

293T or mouse ES cells were transfected using Fugene6 (Roche, Laval, QC, Canada) or Genejuice (Novagen, Mississauga, ON, Canada) according to the manufacturer's instructions. Cells were transfected with the chloramphenicol acetyltransferase (CAT) gene driven by the p21 (el Deiry et al., 1995) or by the maspin gene promoters (Zou et al., 2000) (gifts of Bruce McKay). p53 and SirT1 expression constructs were based on cDNAs driven by the mouse Pgk-1 promoter (Adra et al., 1987) or the human CMV promoter (Boshart et al., 1985). A pgk-1-driven GFP construct was used as an internal transfection control. Cells were harvested 48 h post-transfection by scraping and lysed by three freeze-thaw cycles. GFP was assessed in a fluorometer and CAT assays were performed according to methods described by the manufacturer (Molecular Probes, Burlington, ON, Canada). TLC plates were visualized on a STORM phosphoimager using the blue fluorescence filter.

RNA preparation and quantitative PCR

SirT1+/+ and SirT1−/– mice were irradiated with 10 Gy of 250 keV X-rays and total RNA was prepared from tissues 6 h later using Trizol (Invitrogen, Burlington, ON, Canada). RNA preparations were treated with DNase and further purified with an RNeasy kit (QIAGEN, Mississauga, ON, Canada). One microgram of total RNA from individual samples was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Fermentas, Burlington, ON, Canada). Real-time PCR was performed on an ABI 7500 Real Time PCR System using TaqMan gene expression assays and TaqMan mastermix (Applied Biosystems, Streetsville, ON, Canada). The average threshold cycle (Ct) for each gene was determined from triplicate reactions and the levels of the gene expression were determined relative to the average Ct value of endogenous 18S rRNA.

Thymocyte apoptosis assays

Thymocytes (1–1.5 × 105) were isolated from age- and sex-matched SirT1+/+, SirT1+/–, SirT1−/– and p53−/– animals, irradiated at different doses and cultured for 24 h. Surviving cell numbers were counted using a haemocytometer.

TUNEL assays

Ten-week-old SirT1+/+ and SirT1−/– littermates were irradiated with 10 Gy from a 250 keV X-ray source. Tissues were harvested 6 h postirradiation, cryosectioned and apoptotic cells were detected by fluorescence microscopy using an in situ cell death detection kit (Roche) according to the manufacturer's instructions.

Animal experimentation

SirT1 knockout animals were previously described (McBurney et al., 2003b). Heterozygote animals (SirT1+/–) maintained on the 129/Sv background were mated with 129-Trp53tm1Tyj mice also on the 129/Sv background (Jackson Laboratories, Bar Harbor, ME, USA), and the offspring from these matings were genotyped from DNA obtained by a tail clip. Offspring that were SirT1+/– p53+/– were subsequently mated and all of their offspring were genotyped by PCR.

Genotyping of the p53 allele was done according to methods provided by Jackson Laboratories. Briefly, primers spanning exons 6 and 7 of p53 (reverse primer: 5′-ATA GGT CGG CGG TTC AT-3′; forward primer: 5′-CCC GAG TAT CTG GAA GAC AG-3′) or the neomycin selection marker (reverse primer: 5′-CTT GGG TGG AGA GGC TAT TC-3′; forward primer: 5′-AGG TGA GAT GAC AGG AGA TC-3′) were used to identify the wild-type (∼600-bp PCR product) or targeted (∼280-bp PCR product) p53 alleles. Similarly, primers spanning a region between exons 4 and 5 (forward primer: 5′-TTCACATTGCATGTGTGTGG-3′; reverse primer: 5′-TAGCCTGCGTAGTGTGGTG-3′) or the Pgk-1 promotor of the hygromycin resistance gene (reverse primer: 5′-ATTTGGTAGGGACCCAAAGG-3′) were used to identify the wild-type (∼420-bp product) and targeted (∼520-bp product) SirT1 alleles.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

We thank Dr Bruce McKay for providing p53-dependent CAT reporter constructs and Jeff Ishibashi for providing some of the p53−/– mice used. This work was supported by a grant from the National Cancer Institute of Canada and the Canadian Institutes of Health Research.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
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