Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 2115, USA
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Abstract |
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Abbreviations: B[a]P, benzo[a]pyrene; DSB, double-strand break.
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Introduction |
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The observation that DNA damage can induce elevated levels of p53 and cell cycle arrest has led to the concept that p53 might be involved predominantly in a DNA damage recognition and response pathway (3), independent of the glucocorticoid-mediated apoptotic pathway (6,7). Studies have also shown a gene dosage effect in irradiated cells: p53 null cells are more resistant to DNA damaging agents than p53 wild-type cells, with p53 heterozygous cells exhibiting an intermediate sensitivity (68). The effects of p53 on cell cycle and apoptosis are thought to be mediated through its transcriptional activity following binding to a consensus DNA sequence, the p53 response element. This binding activity is promoted by the presence of short single-stranded DNA sequences (9). Once bound to DNA, p53 may regulate the transcriptional activation of downstream effectors such as p21/WAF1 and GADD45 (1012). It has been proposed that p53 might also participate more directly in the DNA repair machinery. In vitro, p53 binds preferentially to short single-stranded DNA ends and catalyzes DNA renaturation and strand transfer (13). A 3'5' exonuclease activity has also been reported (14). Thus p53 has the potential to play a complex and integral role in the DNA damage control and processing pathways.
The murine pink eyed unstable (pun) mutation results from the tandem duplication of 70 kb internal to the p gene (Figure 1; 15,16). The wild-type p gene is expressed in melanocytes and codes for an integral membrane protein required for the assembly of a high molecular weight complex, giving rise to the black coat pigmentation of C57BL/6J wild-type mice (17). The pun mutation reverts to wild-type by deletion of one copy of the duplicated sequences. Clonal expansion of revertant embryonic premelanocytes results in black spots on the gray fur of the animals. Such reversion events are inducible by X-rays and benzo[a]pyrene (B[a]P), as well as other carcinogenic compounds (18,19).
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Materials and methods |
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Analysis of p53 status
The presence of a p53 deletion allele was diagnosed by Southern blot analysis of DNA from tail biopsies. Briefly, 58 mm of the tail were removed using sharp scissors. The tissue was placed in a sterile Eppendorf tube with 400 µl digestion buffer [50 mM Tris, pH 7.5, 100 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg/ml proteinase K (Sigma, St Louis, MO)] and incubated at 37°C overnight. The proteins were precipitated by addition of 200 µl saturated NaCl solution, the supernatant collected and the DNA precipitated with 600µl 95% ethanol, washed in 75% ethanol and resuspended in 150 µl sterile water. DNA samples were digested with BamHI and the fragments separated on a 0.7% agarose gel, transferred onto a nitrocellulose membrane and crosslinked with a Stratalinker (Stratagene, La Jolla, CA). Blots were hybridized with an [-32P]dCTP-labeled probe and the films exposed for 2448 h at 80°C.
Determining the frequency of pun reversions
The protocol used in this test was similar to the `mouse spot test' (for reviews see refs 21,22). A reversion event in a premelanocyte of the embryo results in a dark spot on the fur of the offspring. Matings were set up between pun/pun p53+/ mice and pregnancy was timed from the discovery of a vaginal plug. Sperm entry into the egg was assumed to have occurred in the early morning hours of the day on which the plug was found and noon of that day was defined as 0.5 day post-conception. Offspring were examined for spots at 10 days and again at 34 weeks of age. Only spots that were clearly visible at both examination points were recorded.
Carcinogen exposure
Animals were exposed to X-rays or an acute dose of B[a]P 10.5 days post-conception. Irradiation was performed in a Westinghouse 150 self-rectified industrial X-ray machine with an inherent filtration of 1.65 mm aluminum and 2 mm aluminum added filtration. The unit was operated at 130 kVp and 8 mA, yielding an exposure dose of 24 ± 2 R/min at 40 cm distance. Mice were exposed in individual sterile polypropylene containers resting on a 24 cm diameter steel turntable, which was rotated to ensure uniform irradiation. The quantity of radiation delivered was measured for each sample with a Victoreen C-r 570 meter. B[a]P (Sigma) was dissolved in corn oil at a concentration of 22.5 or 4.5 mg/ml for doses of 150 or 30 mg/kg, respectively, and 0.2 ml of solution/30 g mouse was injected i.p.
RTPCR characterization of pun reversion events
For molecular confirmation of the deletion events, 34-day-old pun/pun mice of each p53 genotype were killed and black and gray patches of skin were excised; skin sections from wild-type C57BL/6J mice were used as a positive control. Total RNA was isolated using guanidinium thiocyanate/phenol extraction as described (23). First strand cDNA synthesis was performed using the SuperScript II reverse transcriptase preamplification system and oligo(dT)1218 (Gibco BRL, Gaithersburg, MD) with 5 µg total RNA and 1x PCR buffer (2.5 mM MgCl2, 0.5 mM dNTP mix and 10 nM DTT; Gibco BRL, Grand Island, NY). PCR amplification of the p and pun cDNA was performed using primers homologous to sequences outside duplicated regions: 3' primer, CAA CCA GAT GGC ACC CAG AAT AGC; 5' primer, CTG TGT CAC CGC TGG AAA ACT ACT. The PCR reaction contained one tenth of the cDNA reaction mixture, 1x PCR buffer, 1.5 mM MgCl2, 200 µM dNTP mix, 100 nM of each primer and 2 U of Taq DNA polymerase (Gibco BRL). After initial denaturation for 3min, 35 PCR cycles were performed by annealing at 55°C for 1 min, synthesis at 72°C for 1.5 min and denaturation at 95°C for 1 min. PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining.
Statistical analysis
Statistical significance of the frequencies of induced pun reversions was examined using the G-test. Litter size and gender distribution were analyzed by Student's t-test (24).
Animal treatment for western blotting
Pregnant p+/ dams were treated with X-rays or B[a]P 10.5 days post-conception as described above. Animals were killed at given time points following treatment, the embryos immediately snap frozen in liquid N2 and stored at 20°C for further analysis.
Tissue preparation for blotting
Frozen embryos were thawed on ice, minced on ice with a scalpel blade in 150 µl ice-cold RIPA buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 100 µg/ml PMSF and 1 mM EDTA, homogenized through 18G, 21G and 27G syringes successively and incubated for 3 h at 4°C on a rotating platform. The lysates were spun in a microfuge at 14 000 r.p.m. for 15 min and the supernatants transferred into chilled 1.5 ml microfuge tubes. Before addition of protease inhibitors (10 µg/ml leupeptin, aprotinin and pepstatin in 10 µl RIPA buffer) small aliquots were taken for protein determination by the Bradford assay (Bio-Rad, Hercules, CA).
SDSPAGE and western blotting
Aliquots of 100 µg of protein extract in Laemmli buffer (25) were separated by electrophoresis on a 10% SDSpolyacrylamide gel, transferred onto a Hybond ECL nitrocellulose membrane (Amersham, Arlington Heights, IL) and blocked in PBS-T (1x PBS, 0.1% Tween 20) with 3% non-fat dry milk. The membrane was incubated with the sheep polyclonal anti-p53 antibody PAb-7 (Oncogene, Cambridge, MA) at a dilution of 1:2500 for a minimum of 3 h, washed three times for 15 min in PBS-T, incubated with the biotinylated secondary antibody (Oncogene) at a dilution of 1:100000, washed as previously, incubated with streptavidin-conjugated peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:2000 and washed as previously. The blot was incubated in ECL reagents (Amersham) for 1 min and exposed to an autoradiographic film (BioMax MR; Kodak, Rochester, NY) for the appropriate length of time. The p53 Western Blotting Standard from Oncogene was used as a positive control. Western blots were stained with Ponceau stain before immunoblotting to verify equal loading of proteins.
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Results |
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It has been previously reported that male p53/ mice are fertile (26), yet p53-deficient C57BL/6J mice suffer from a testicular giant cell degenerative syndrome (27). This might explain the reduced fertility of these mice. In addition, p53/ offspring had a shortened lifespan and suffered from development of neoplasms, particularly lymphomas, as seen by others (26,28,29).
B[a]P at 150 mg/kg significantly lowered the average litter size, probably as a result of toxicity. The lower dose of 30 mg/kg and X-rays had no effect on litter size (Table I).
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Since 90% of our p53/ offspring were male, any gender bias in the spotting frequency may correlate with the lack of X-ray-induced pun reversions among the p53/ offspring. Therefore, we analyzed the gender distribution of spots in p53 wild-type and p53 heterozygous offspring (Table III). The percentage of spotted offspring was not significantly different between genders with either genotype. In addition, there was no significant difference between the spontaneous spotting frequencies of untreated male and female p53/ animals (data not shown); furthermore, we have already shown that X-ray exposure of p53/ mice had no effect on the gender distribution (Figure 2
). Thus the reduced spotting frequency observed in X-ray-treated p53/ offspring appears to be independent of any gender distribution.
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To determine whether there was any variation in the number of reversion events in each mouse depending on genotype or treatment, we calculated the average number of spots per mouse as well as the average number of spots on spotted mice. In controls and X-ray-treated animals, irrespective of genotype, the majority of mice (>98%) had only one spot, with the mean number of spots per spotted mouse between 1 and 1.1 (data not shown). Treatment with 30 or 150 mg/kg B[a]P increased the number of spots per spotted mouse to mean values between 1.3 and 1.6 in all genotypes, none of the values being statistically different from each other (data not shown). Therefore, we concluded that p53 genotype has no effect on the number of independent reversion events in any treatment group examined.
We also analyzed the litter distribution of the spotting frequency by plotting the number of spotted mice in a litter against litter size, regardless of p53 status. The proportion of spotted mice per litter was uniformly increased after all treatments (data not shown); the strongest correlation was observed with 150 mg/kg B[a]P (correlation coefficient 0.90). This analysis demonstrates that there was no bias in the litter distribution of spotted mice, thus excluding the possibility of a founder effect within the colony.
Molecular analysis of reversion events
Spontaneous reversion of the pun mutation to wild-type results from homologous intrachromosomal recombination leading to a deletion (15). It is possible, however, that the black spots observed in the p53/ background might have arisen through an alternative pathway, such as by mutation or activation of other genes. Moore (30) reported a bypass suppression of the dsu gene (dilute suppressor) which suppresses the dilute coat color phenotype of mice homozygous for the dilute leaden and ashen mutations. Hence, we investigated at the molecular level whether spots on p53/ mice occurred as a result of pun reversion.
Black fur spots and gray fur were excised from the same p53/ offspring and the RNA was isolated and analyzed using the RTPCR protocol described previously (19). The primers used amplified a 1.3 kb fragment from p wild-type cDNA (Figure 3, lane 2) while the pun transcript resulted in a 2.6 kb fragment. In gray pun skin samples, however, we found both amplification products at a ratio of ~1:1 (lane 3). The presence of the p+-specific fragment in gray skin samples could be explained by the fact that there is a background frequency of ~1 in 104 cells exhibiting the revertant phenotype and that the shorter 1.3 kb fragment is amplified preferentially. Alternatively, or in addition, it could be the result of a lower expression or lower stability of the pun transcript (19). RTPCR analysis of black spots from pun animals showed an increased amount of the 1.3 kb product (p+) relative to the 2.6 kb product (pun): the ratio of p+- to pun-specific bands varied between 4:1 and 12:1 (lanes 46). The presence of some 2.6 kb pun transcript in the spots is expected for at least two reasons. Firstly, most likely only one of the two pun alleles has reverted to the wild-type p gene, leaving the other allele as a pun duplication. Secondly, there may be contaminating surrounding tissue excised together with the spot. These results show that all spots, regardless of the p53 genotype, are derived from p+ expression.
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Discussion |
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Segregation of p53
In contrast to published data (26) we found fewer than expected p53/ offspring, mainly as a result of the loss of female offspring during intra-uterine development. Donehower's group (20,26) reported 23% of p53/ offspring from the mating of p53 heterozygous mice, with no mention of any gender bias amongst them. Others (31,32) reported a loss of p53/ offspring similar to that observed here, predominantly linked to female-associated defects in neural tube closure. This gender distribution appears to be dependent on the genetic background of the parents. In an outbred background about twice as many p53/ male as female offspring survived to weaning, whereas a ratio of six male to one female was found in an inbred 129/Ola background (31). Here, in the C57BL/6J background, this imbalance was even more pronounced, with 10 times as many p53/ male as female offspring. A background specificity might explain why the gender bias was not detected in experiments performed in an outbred background (20).
Mechanism of pun reversion in p53-deficient mice
Similar to results obtained with the wild-type mouse (19), all spots from p53-deficient mice examined appear to be the result of pun reversion events. In principle, induced reversion events can occur either by interchromosomal recombination between homologs, by unequal crossing-over or conversion, or intrachromosomally, by exchange or by single-strand annealing (Figure 1). In yeast, a similar construct, the DEL recombination system, has been studied in detail (33,34). The most likely mechanism for induced DEL recombination events is single-strand annealing, proceeding via a double-strand break (DSB) intermediate (34). Interestingly, mice containing a pun allele on one homolog and a p deletion allele on the other homolog also have spontaneous and B[a]P-induced spots (19), indicating that intrachromosomal recombination is a major pathway in this system as well. Whichever mechanism is involved, pun reversion can only be achieved by homologous recombination with perfect alignment of the duplicated sequences.
Both X-rays and B[a]P cause DNA lesions. One gray of ionizing radiation causes 1640 DSBs, 6001000 single-strand breaks and 250 damaged thymine residues per cell (35), DSBs being the most significant biological lesion (36). Unprotected DNA ends are highly reactive and have the potential to disrupt genomic integrity. In addition, they are vulnerable to exonucleolytic attack, which can result in loss of genetic information. Their removal is therefore essential for cell survival. The fact that DSBs in mitotic mammalian cells are removed predominantly by illegitimate recombination, or non-homologous end joining (37,38), could explain the lack of induction of pun reversion by X-rays. In contrast, B[a]P damages DNA by binding of its metabolites to form DNA adducts (39). Unlike unprotected double-stranded DNA ends, DNA adducts probably only become recombinogenic after repair proteins have recognized and processed them into other lesions. Such lesions may be better candidates for homologous recombination. As a result, B[a]P is a potent inducer of pun reversions.
In conclusion, the fact that X-ray- but not B[a]P-induced reversions are dependent on the presence of p53 may indicate that the protein could somehow be involved in the processing of unprotected DNA ends.
Effect of p53 status on spontaneous recombination
The frequencies of spontaneous pun reversions in wild-type, heterozygous and mutant p53 mice observed in this study were not significantly different from each other. This is in contrast to several other studies (40,41) where p53-deficient or abrogated cells exhibited up to a 100-fold increase in recombination frequency compared with wild-type cells. Although this discrepancy may originate from an in vitroin vivo difference, it is also likely that fundamental differences exist in the recombination mechanisms that produced the end-points observed. The above-mentioned studies involved intrachromosomal recombination between close sequences (<3 kb), which most likely resulted from `short patch' gene conversion or small deletions. In contrast, a pun reversion event can only be achieved by `proper' homologous recombination, with perfect alignment of the duplicated sequence and deletion of the intervening sequence (15,16). Deletion by single-stranded annealing would require exonucleolytic degradation of at least 70 kb before revealing a region of homology, a constraint that by itself may favor non-homologous end joining. In addition, the majority of events analyzed in Mekeel et al. (40) are the result of intrachromosomal gene conversion. This reaction cannot produce a reversion in the pun system. Therefore, even though both systems measure homologous recombination, pun reversions may be mediated by a different recombination reaction, such as unequal crossing-over or single-stranded invasion. Nevertheless, none of the studies mentioned above investigated the effects of p53 deficiency on induced recombination.
Possible reasons for the involvement of p53 in X-ray induced pun reversions
X-rays did not induce pun reversions in the absence of p53. In fact, the frequency of pun reversions in p53/ offspring was significantly reduced following X-ray exposure compared with controls. This lack of induction may be linked directly or indirectly to one of the numerous activities of p53, such as initiation of G1 arrest following DNA damage, interaction with recombination-specific proteins or direct participation in the recombination process. Alternatively, non-homologous recombination could be up-regulated in these cells. Some possibilities are discussed below.
The absence of X-ray-induced pun reversions in p53/ animals may be due to the absence of G1 arrest following irradiation (3,4,42). Without cell cycle arrest there may not be time to search for homology by single-stranded invasion or to expose homologous sequences through exonucleolytic degradation. This may result in the replication of broken chromosomes, missegregation and subsequent loss of genetic information. Alternatively, the broken chromosome may recombine illegitimately, creating other rearrangements that do not lead to a pun reversion event. Thus, induced pun reversion events may be largely impaired in the absence of G1 arrest. However, this does not explain why the frequency of induced pun reversion events is below the spontaneous frequency observed in p53/ mice (Table II).
Another possibility is that p53 could be involved more directly in the recombination repair process. p53 can interact with hRAD51, a homolog of the RecA protein that catalyzes strand exchange during recombination (43). Moreover, p53 binds Holliday junctions and facilitates their cleavage (44). Holliday junctions are recombination intermediates in the recombination mechanisms shown in Figure 1A, C and D. However, such integral roles for p53 would be expected to result in a reduced frequency of spontaneous pun reversion events in its absence. No such reduction was observed (Table II
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We favor the hypothesis that under specific conditions the non-homologous end joining reaction may be more proficient in p53-deficient cells. The observation that p53 null cells are more resistant to DNA-damaging agents and are less likely to undergo apoptosis (6,7) suggest a fundamental change in the cellular response to DNA-damaging agents in these cells. In addition, p53 deficiency partially rescues the SCID mutation phenotype (4547), which otherwise leads to abortive V(D)J recombination. It was suggested that the unprocessed DSB intermediates act as a signal for apoptosis, via a p53- dependent pathway (4547). If the cells survive better in the absence of p53 an alternative mechanism must take place to remove the DSBs. Thus we suggest that p53/ cells may up-regulate the non-homologous end joining reaction in response to certain stimuli such as X-ray- or Rag1/Rag2-induced DSBs. In the absence of such stimuli, there would be no change in the spontaneous frequency of pun reversions in p53-deficient mice in our system.
But why are spontaneous reversions suppressed in X-ray-treated p53/ mice? According to our hypothesis illegitimate recombination is up-regulated after irradiation in p53- deficient mice. This up-regulation is likely to remove both spontaneous and induced recombination substrates occurring after X-ray exposure. In the pun reversion system most spontaneous reversions are thought to occur after day 10 post-coitum (A.J.R.Bishop and R.H.Schiestl, unpublished observations), i.e. after the time of X-ray exposure used here. Thus, this would explain the absence of spontaneously occurring pun reversion events in X-ray-treated p53-deficient mice.
Up-regulation of p53 following X-ray but not B[a]P treatment
Post-translational stabilization and nuclear accumulation of p53 following treatment with various types of DNA-damaging agents can occur by different pathways (48). DNA-breaking agents such as ionizing radiation, which are most effective at rapidly inducing p53, do so by activation of ATM and NBS/p95 (48). Agents creating other types of lesions, such as DNA adducts or thymidine dimers, induce p53 by blockage of RNA polymerase II without requirement for a DSB (48). A similar effect is observed with RNA synthesis inhibitors like actinomycin D (48). X-rays readily increase p53 levels in vivo, in vitro and in utero (3,7,49,50) and the results reported here are consistent with the literature. For B[a]P the picture is less clear. B[a]P leads to p53 accumulation in lung carcinoma and breast adenocarcinoma cell lines (51), in Swiss 3T3 cells (52) and in mouse skin in vivo (53) but not in the rat liver (54). This latter report suggests that the single dose administered (80 mg/kg) did not induce sufficient DNA damage for p53 accumulation. Surprisingly though, a dose of 50 mg/kg leads to extensive DNA adducts in utero (55). Most importantly, in 3T3 fibroblasts B[a]P induced G1 arrest by a p53-independent mechanism (52). Thus, there is some indication that B[a]P may act independently of p53. Here we have demonstrated that B[a]P-induced pun reversion events are also p53 independent.
In conclusion, the results presented here demonstrate that p53 is required for ionizing radiation- but not B[a]P-induced pun reversions. These results further define the role of p53 in DSB repair by homologous recombination.
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Acknowledgments |
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Notes |
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2 To whom correspondence should be addressed Email: schiestl{at}hsph.harvard.edu
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References |
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