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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:951-959 (2007)
© 2007 The Gerontological Society of America

Reduced Expression of the Caenorhabditis elegans p53 Ortholog cep-1 Results in Increased Longevity

Oge Arum and Thomas E. Johnson

1 Institute for Behavioral Genetics, 2 Molecular, Cellular, and Developmental Biology Department, and 3 Integrative Physiology Department, University of Colorado, Boulder.

Address correspondence to Oge Arum, PhD, Southern Illinois University, School of Medicine, 801 North Rutledge St., Rm. 4389, Springfield, IL 62702. E-mail: oarum{at}siumed.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Hyperactivation of mammalian p53 has been shown to result in segmental progeria and decreased survivorship. Repression of the p53 homolog in Drosophila melanogaster has also been shown to increase survival. We show that RNA interference (RNAi) or genetic knockout of the Caenorhabditis elegans p53 ortholog, cep-1, leads to increased life span, which is dependent upon functional daf-16. Furthermore, one other DNA damage–responsive C. elegans mutant, hus-1(op241), exhibits a life-span increase. The cep-1(gk138) knockout mutant does not show increased resistance to heat, oxidative, or ultraviolet stress; nor to bacterial pathogenicity. cep-1 RNAi does not extend the life span of a sir-2.1(geIn3) overexpressing strain. cep-1 RNAi does not alter dauer formation propensity or nuclear-localization of DAF-16::GFP, even under heat stress; nor does it change nuclear-persistence and/or retention of DAF-16::GFP. This study clarifies the inverse relationship between cep-1 expression and C. elegans life span, and, by extrapolation, that between p53 expression and mammalian life span.

AN inverse relationship between DNA damage-response and aging has long been hypothesized (1). That the actions of DNA damage-response proteins could be detrimental to an organism's health is a very recent concept. Donehower and colleagues (2) showed that a hyperactive isoform of the mammalian tumor-suppressor p53 led to mice that displayed segmental progeria (a premature display of some, yet not all, age-associated phenotypes) and a decrease in median (23%) and maximum (21%) life span. Another study in which p53 activity was elevated revealed similar results (3). Recently, dominant-negative suppression of the Drosophila melanogaster homolog of p53 was shown to increase life span (4). Other studies, including ones showing that hypoactive p53 isoforms are preferentially found in centenarians (5,6), and those that correlate the presence of age-associated molecules and DNA damage-response proteins (7), support the notion that DNA damage-response mechanisms may lead to more rapid senescence.

The Caenorhabditis elegans p53 ortholog, cep-1, has similar molecular function to its human tumor-suppressor counterpart. Although C. elegans cannot develop cancer, and thus the worm ortholog does not function as a tumor suppressor, experimental corroboration utilizing the C. elegans germline (the only part of the adult worm where apoptosis can occur) supports the notion that cep-1 and p53 are homologous in form and function (8,9). CEP-1 acts as a transcription factor to regulate the response to DNA damage within the germline, a response that is composed of cycle arrest, damage repair, and apoptosis. In worms treated with cep-1 RNA interference (RNAi) or in cep-1 mutant worms, fewer cells than expected respond to DNA damage as noted above; furthermore, worms with decreased levels of CEP-1 function have increased genomic instability, as evidenced by the number of chromosomal nondisjunction events observed (8). Thus, these worms clearly have a reduced ability to properly assess DNA damage, at least within germinal cells, and/or are less able to initiate the proper molecular brakes, which would allow for the repair of that damage or the suicide of the egregiously damaged cells.

In addition, CEP-1 functions as a transcription factor that can bind consensus human p53 binding sites, and its DNA binding domain contains residues that are those most frequently mutated in human cancers (9). Quite intriguingly, CEP-1 overexpression leads to widespread caspase-independent cell death, which is lethal to the organism at any stage of life (8), as p53 overexpression leads to embryonic lethality in Mus musculus (2). This latter phenotype further connects p53 and cep-1 as true orthologs, and implies that reduced p53 or CEP-1 levels may be advantageous in an organism that can evade the life-span-blunting threat of a neoplasm, such as D. melanogaster or C. elegans (10).

This study shows that RNAi or genetic knockout of the C. elegans p53 ortholog, cep-1, leads to an increase in life span, which is dependent upon functional daf-16. The life-span increase of the cep-1(gk138) knockout mutant is not due to increased resistance to heat, oxidative, or ultraviolet (UV) stress. Moreover, not only is the life span of the cep-1(gk138) knockout mutant not due to increased resistance to bacterial pathogenicity, but a greater life-span increase is revealed when bacterial pathogenicity is reduced. cep-1 RNAi does not extend the life span of an sir-2.1(geIn3) overexpressing strain. cep-1 RNAi does not alter dauer formation propensity or nuclear localization of DAF-16::GFP, even under heat stress; nor does it change nuclear persistence and/or retention of DAF-16::GFP. The cumulative results of this investigation further strengthen the theory that p53-like genes contribute to organismal senescence.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Maintenance of Strains
The C. elegans worm strains used in these experiments were N2(CGCb), N2 Bristol, CB1370 [daf-2(e1370)] (11), TJ1052 [age-1(hx546)] (12), TJ1 [cep-1(gk138)] (8) backcrossed 10 times to N2, WS2277 [hus-1(op241)] (13), TJ1060 [fer-15(b26ts); spe-9(hc88ts)] (14), TJ1081 [daf-16(m26); fer-15(b26ts)], TJ356 [daf-16::gfp(zIs356)] (15), JR2474 [cep-1::gfp] (8), MT1082 [egl-1(n487)] (16), MT2547 [ced-4(n1162)] (17), MT4770 [ced-9(n1950)] (18), MQ125 [clk-2(qm37)] (19), SP506 [rad-5(mn159)] (20), CB1480 [him-7(e1480)] (21), CB5348 [mrt-2(e2663)] (22), NW1613 [msh-2(ev679::Tc1)] (23), WS2265 [hus-1(op244)] (13), and LG100 [sir-2.1(geIn3)] (24).

Worms were kept frozen in liquid nitrogen or at –80°C or –72°C for long-term storage and then stored as dauers or at other larval stages prior to use, when they were cultured as described below (25,26).

Construction of RNAi Plate
Standard Nematode Growth Medium (NGM) plates were made as usual (25) but with the addition of ampillicin at 100 µg/mL (RPI, Mt. Prospect, IL) and 1 mM isopropyl-B-D-thiogalactoside (IPTG) (LabScientific, Inc., Livingston, NJ). The plasmid-containing Escherichia coli strains derived from HT115 were obtained from a Chromosome I RNAi Library (27) and were cultured in Luria Broth (LB) with ampicillin at 100 µg/mL.

Survivorship Assays
Eggs were laid at 25.5°C on either NGM agar plates spotted with E. coli strain OP50 or RNAi plates spotted with E. coli strain HT115, and then placed at 25.5°C. Beginning on day 3 of life, the adult worms were transferred to fresh plates similar to the ones that they were being transferred from, in order to separate the parents from their progeny and to prevent the starvation of the parents. At about 10 days of life, the adults became postreproductive and were transferred only weekly. For survivorship assays using fluorodeoxyuridine (FUDR), similar procedures were used except that new plates contained 25 µM FUDR; these worms were only transferred once more to a fresh FUDR-containing plate to cull them away from the few, L1-arrested progeny that they produced. For RNAi survivorship assays, a bacterial strain producing double-stranded RNA targeting green fluorescent protein (GFP) messenger RNA (mRNA) transcripts was used as a negative control to the experimental bacterial strains producing double-stranded RNA targeting mRNA transcripts present in the worms. Worms were henceforth scored for survival based on whether they responded to mechanical stimuli.

Stress Resistance Assays
For all stresses, eggs were laid at 25.5°C onto solid NGM plates spotted with OP50. On approximately day 3, the plates were transferred to the appropriate stress. For heat stress, plates were put at 35°C for 10 hours, allowed 3 hours for recovery, and scored for survival. For oxidative stress, the plates were put in a hyperbaric O2 chamber at 40 p.s.i. O2 for 48 hours at 20°C, allowed to recover for 22 hours, and then scored for survival. For UV radiation stress, worms were transferred to plates without OP50, irradiated with 254 nm light at 2000 J/m2 in a UV Stratalinker 2400 (Stratagene, La Jolla, CA), returned to the plates with OP50 to recover for 24 hours, and then scored for survival every 24 hours.

Dauer Formation at 27°C Assays
Eggs laid on RNAi plates at 20°C or 25.5°C were transferred to 27°C as embryos or early first-stage larvae. After about 3 days, the resulting worms were subjected to 1% sodium dodecylsulfate (Sigma-Aldrich, St. Louis, MO) to kill all nondauers. Then each plate was scored for live dauers versus all other worms (28).

Visualization of DAF-16::GFP and CEP-1::GFP
Worms from strain TJ356 (15) were placed on either E. coli strain HT115 producing double-stranded RNA transcripts targeting cep-1 or daf-2 mRNA for RNAi-mediated degradation, or OP50. Similarly, worms from JR2474 were grown on both OP50 or HT115 targeting daf-16 or daf-2. Some strains were subjected to 35°C or starvation on NGM plates, for some of the analysis. Visualization of DAF-16::GFP subcellular localization or CEP-1::GFP utilized an MZFL III Fluorescence Stereomicroscope (Leica Microsystems, Wetzler, Germany) and an Axioskop Fluorescence Stereomicroscope (Carl Zeiss International, Oberkochen, Germany), and pictures were captured with an Axioskop Fluorescence Stereomicroscope (Carl Zeiss) equipped with a digital imaging system (Intelligent Imaging Innovations, Inc., Denver, CO).

Data Analysis
Means and standard errors of the means (SEM) for all survival assays were obtained by using the survival analysis function of the Statistica ‘99 Edition software package (Statsoft, Inc., Tulsa, OK). Comparisons of survival curves between strains were made by using the log-rank test function of this package, as well as that of Russell Thompson at The Walter and Eliza Hall Institute of Medical Research (http://bioinf.wehi.edu.au/software/russell/logrank/index.html). The inter-strain comparisons of the means for the O2 stress, heat stress, and dauer formation experiments utilized independent, two-tailed t tests of the means on the SigmaPlot 2000 for Windows Version 6.00 software package (SPSS Inc., Chicago, IL).


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Longevity
In a screen of Chromosome I, we noted an effect of cep-1 RNAi on life span. cep-1 is an ortholog of mammalian p53, so we decided to study this effect in more detail, using both RNAi and mutant analysis. Figure 1A shows survival curves for a data set that pools results of four replicate experiments. The effect of cep-1 RNAi was modest—an increase of only 9% in mean longevity—but was statistically significant (p <.002). (Table 1 compiles the relevant statistics for all incidences of mild life-span increase observed in this study.) The percentage increases in mean life span for these four replicate experiments were, respectively, 10%, 18%, 8%, and 13%. We also measured the life span of the cep-1(gk138) knockout mutant in three replicate experiments, using the N2 Bristol stock as the control. cep-1 mutant worms were longer-lived in each experiment: Mean life span increased by 3%, 11%, and 17%, respectively. Data pooled across all three replicates show an increase of 12% in mean longevity (Figure 1B; p <.0001). The pooled data also showed that the cep-1(gk138) mutant had a maximal life-span increase of 33% relative to the N2 Bristol controls. Using the quantile test proposed by Wang and colleagues (29), we found that survival to the last decile, an estimate of maximal longevity, was significantly better in cep-1(gk138) mutants than in controls (p <.0001).


Figure 01
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  		Figure 1. Life span of cep-1 mutants. A, Longevity using cep-1 feeding-RNA interference (RNAi) confers a 9% life-span increase, relative to gfp feeding-RNAi. TJ1060 had a life expectancy of 12.6 ± 0.3 days on cep-1 RNAi (n = 183), whereas the control TJ1060 had a life expectancy of 13.7 ± 0.2 days on gfp RNAi (n = 138) (p =.0016). B, The cep-1(gk138) mutant allele grants a 12% life-span increase relative to its wild-type counterpart. N2 Bristol had a life expectancy of 15.5 ± 0.2 days (n = 91), whereas cep-1(gk138) had a life expectancy of 17.3 ± 0.4 days (n = 87) (p =.00008)

 

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  		Table 1. Data and Statistical Analyses for Incidences of Mild Longevity.

 
We then asked if any other strains with mutations in genes involved in DNA repair were also long-lived by surveying the seven other genetic loci known to modulate DNA repair in C. elegans (Table 2). We found only one, hus-1, with a positive effect on longevity (Figure 2). hus-1 is required for DNA damage-induced cell-cycle arrest, apoptosis, and genomic stability. Following DNA damage, HUS-1 relocalizes and forms distinct foci that overlap with chromatin (13). Longevity of hus-1 mutant worms was measured in three experiments, which showed increases in longevity, compared to control, of 6%, 17%, and 10%, respectively. In the pooled data set (Figure 2A), mean longevity of the hus-1 mutant was 11% greater than that of the control (p <.00001). To see if hus-1 and cep-1 affect longevity through distinct pathways, we evaluated the effect of cep-1 RNAi on the life span of worms carrying the hus-1 point mutation allele. Figure 2B shows data pooled from three independent experiments. cep-1 RNAi does not increase longevity in hus-1 point mutants, consistent with the notion that the two proteins influence longevity by the same pathway or process.


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  		Table 2. Life Span of C. elegans DNA Damage-Response Mutants.

 

Figure 02
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  		Figure 2. Life spans of hus-1. A, The hus-1(op241) allele bestows an 11% life-span increase relative to its wild-type counterpart in the N2 Bristol negative control. N2 Bristol had a life expectancy of 17.3 ± 0.3 days (n = 95), and hus-1(op241) had a life expectancy of 19.3 ± 0.4 days (n = 87) (p =.00001). B, cep-1 feeding-RNA interference (RNAi) does not increase or decrease the life span of the hus-1(op241) mutant relative to gfp feeding-RNAi. The life expectancy of hus-1(op241) on cep-1 RNAi was 17.2 ± 0.2 days (n = 171); that of hus-1(op241) on gfp RNAi was 16.9 ± 0.2 days (n = 176) (p =.27)

 
Overexpression of the C. elegans homolog of the Saccharomyces cerevisiae gene SIR2, sir-2.1, leads to an increase in life span (24). We found extended longevity of sir-2.1(geIn3) worms in three replicate experiments (increased mean survival of 6%, 14%, and 16% over controls). Pooled data (Figure 3) gave an increase in mean life span of 11%, less dramatic than in the original publication (24), but statistically significant (p <.0001). cep-1 RNAi did not alter the survival of sir-2.1(geIn3) worms in three independent experiments, with relative life spans of –2%, 0%, and –2% (pooled data set [p =.81] is shown in Figure 3B). SIR-2.1 and CEP-1 may modulate life span through the same pathway or process.


Figure 03
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  		Figure 3. A, The sir-2.1(geIn3) allele confers an 11% increase in life expectancy relative to the wild-type counterpart, N2 Bristol. sir-2.1(geIn3) = 19.3 ± 0.4 days (n = 109); N2 Bristol = 17.3 ± 0.3 days (n = 95) (p =.00001); daf-2(e1370) = 29.9 ± 0.8 days (n = 69) (p =.00001). B, cep-1 feeding-RNA interference (RNAi) does not alter the life span of the sir-2.1(geIn3) mutant relative to gfp feeding-RNAi. sir-2.1(geIn3) on cep-1 RNAi = 15.0 ± 0.2 days (n = 94); sir-2.1(geIn3) on gfp RNAi = 15.1 ± 0.2 days (n = 102) (p =.81); sir-2.1(geIn3) on daf-2 RNAi = 17.3 ± 0.4 days (n = 50) (p =.00001)

 
Bacterial Pathogenicity and Other Stressors
Bacterial pathogenicity has been shown to be a major contributor to death in C. elegans. When worms are fed bacteria killed by heat, UV irradiation, or antibiotics, they live longer than they do on live bacterial lawns (30). In four independent trials, we found that incubation of cep-1(gk138) knockout worms on heat-killed bacteria led to increased life span (replicate increases of 37%, 38%, 48%, and 22%) (pooled data in Figure 4A, mean longevity increase 29%, p <.0001). The greater longevity of the cep-1(gk138) worms on heat-killed bacteria suggests that the gk138 mutant strain is susceptible to bacterial pathogenicity, and in its absence can extend life span even more. Furthermore, we found that, in two independent trials, the incubation of hus-1(op241) mutant worms on heat-killed bacteria led to increased life span (replicate increases of 19% and 14%) (combined data in Figure 4B, mean longevity increase 16%, p <.03). The mildly increased longevity of the hus-1(op241) worm on heat-killed bacteria suggests that the op241 mutant strain is also susceptible to bacterial pathogenicity, and in its absence can extend life span further.


Figure 04
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  		Figure 4. On heat-killed Escherichia coli, the cep-1(gk138) allele grants a 29% life-span increase relative to its wild-type counterpart in the N2 Bristol negative control. A, cep-1(gk138) = 24.7 ± 0.6 days (n = 57); N2 Bristol = 19.1 ± 0.5 days (n = 36) (p <.00001). On heat-killed E. coli, the hus-1(op241) allele grants a 16% life-span increase, relative to its wild-type counterpart in the N2 Bristol negative control. B, hus-1(op241) = 25.5 ± 1.7 days (n = 26); N2 Bristol = 21.9 ± 1.1 days (n = 36) (p <.03)

 
Resistance to a variety of other stressors correlates significantly with longevity, and many long-lived mutants are resistant to one or more stressors, including heat, hyperbaric oxygen and other oxidants, and UV radiation (31). Worms bearing the cep-1(gk138) mutation do not differ from controls in their resistance to heat stress (Figure 5A), high oxygen levels (Figure 5B), or UV stress (Figure 5C).


Figure 05
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  		Figure 5. The cep-1(gk138) allele does not affect stress resistance relative to its wild-type counterpart in the N2 Bristol negative control. A, Heat resistance. cep-1(gk138) = 27.1 ± 9.6% (n = 154); N2 Bristol = 22.7 ± 8.2%; (n = 138); daf-2(e1370) = 96.5 ± 1.8% (n = 70) (p =.74). B, Oxidative stress resistance. cep-1(gk138) = 15.2 ± 7.5% (n = 153); N2 Bristol = 21.4 ± 7.0% (n = 115) (p =.58); daf-2(e1370) = 95.1 ± 3.6% (n = 86). C, ultraviolet stress resistance. cep-1(gk138) = 44.9 ± 1.0% (n = 85); N2 Bristol = 43.0 ± 1.3% (n = 67) (p =.25); daf-2(e1370) = 78.2 ± 2.6% (n = 50)

 
daf-16, a gene required for dauer larva formation, is also a major modulator of life span in C. elegans. To determine whether daf-16 is epistatic to cep-1 for life span, we evaluated the effect of cep-1 RNAi on daf-16(m26) mutant worms. The relative life spans of the daf-16 hypomorph on cep-1 RNAi were essentially unchanged (p =.24; pooled data in Figure 6), consistent with a role for daf-16 in regulating cep-1–mediated increases in longevity.


Figure 06
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  		Figure 6. cep-1 feeding-RNA interference (RNAi) does not change the life span of the daf-16(m26) mutant relative to gfp feeding-RNAi. daf-16(m26); fer-15(b26) on cep-1 RNAi is 12.6 ± 0.2 days (n = 164); daf-16(m26); fer-15(b26) on gfp RNAi is 13.1 ± 0.2 days (n =106). The log-rank test p value between these two values is.24

 
cep-1 RNAi did not induce the formation of dauer larvae in the spe-9; fer-15 strain or in the N2 background, nor did cep-1 RNAi alter the proportion of dauer larvae produced by a daf-2(e1370) hypomorph in which 6%–8% of larvae spontaneously enter the dauer pathway. Furthermore, cep-1 RNAi was found not to induce nuclear localization upon treatment, or nuclear retention following treatment, of a DAF-16::GFP fusion protein; although nuclear localization of this molecule was induced by daf-2 RNAi, and by heat stress.


   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
Excess p53 activity leads to a 19% decrease in median life span and a 17% decrease in maximal life span in mice (2,3). Recent work in D. melanogaster has shown that decreased expression of its p53 homolog leads to increased median life span of 11%–26% and increased maximum life span of 10%–16% (4). We show here that decreased expression of the p53 ortholog, cep-1, leads to a similar increase in mean life span in C. elegans of 9%–12%, using either an RNAi bacterial feeding system or the cep-1(gk138) mutant allele. These data suggest that these homologous transcription factors regulate the production of proteins or RNAs that are detrimental to long-term organismic survival in these three evolutionarily divergent species, as predicted by evolutionary theory (32,33).

To see if other genes involved in DNA repair might also specify longevity, we assessed the life span of other C. elegans mutants defective for the response to DNA damage-induced cell-cycle arrest or apoptosis within the germline. Only one of these mutants, the hus-1(op241) point mutation mutant, exhibited an increase in life span. This finding suggests that the effects on life span seen for that mutant and for the cep-1(gk138) knockout mutant are not connected to their roles within general genomic maintenance or general DNA damage response.

Evaluating the genetic interaction of cep-1 reduction with hus-1(op241) and another longevity allele, sir-2.1(geIn3), we found that cep-1 RNAi did not significantly increase the life span of either mutant. This finding suggests that cep-1 specifies life span through a mechanism similar to that of hus-1 and sir2.1. Both cep-1 and hus-1 encode DNA damage-response genes, and both have been shown to be necessary for egl-1 mediated apoptosis (13). The mammalian SIR-2.1 homolog, SIRT1, has been shown to deacetylate the mammalian CEP-1 homolog, p53. If the effect of sir-2.1 on life span is mediated largely by cep-1 modification, the absence of an effect on the life span of sir-2.1–overexpressing mutants on cep-1 RNAi would be expected. Moreover, life-span extension via sir-2.1 overexpression is dependent upon functional daf-16 (24). Considering our observation that life-span extension upon repression of cep-1 is also daf-16-dependent, this further supports the concept that a physical interaction between CEP-1 and SIR-2.1, both of which interact with DAF-16, might modulate life span.

Different experimental conditions have been shown to result in different relative increases in life span for some long-lived mutants, to the point where the relative longevity may be completely abrogated in one milieu. An example of this phenomenon is seen here for cep-1(gk138) and hus-1(op241), which, independently, have greater relative life-span increases on heat-killed E. coli than on living bacteria. Thus, both mutants are more susceptible to bacterial toxicity, suggesting that CEP-1 and HUS-1 may be involved with mediating bacterial resistance. This concurs with the life-span analyses showing that cep-1 and hus-1 both regulate survivorship by similar means, and thus may function in the same cellular process that has biochemical outputs related to regulating both bacterial resistance and longevity. This hypothesis is obviously greatly intriguing, as genes promoting resistance to bacterial infection are known to be up-regulated in long-lived worms (34,35).

We have shown that the cep-1(gk138) allele does not affect susceptibility to heat, oxidative, or UV stress, although repression is known to cause sensitivity to hypoxia and starvation, and poor response to DNA damage (8). There are now a few mutations that extend life span without increasing stress resistance (31,36), and cep-1 provides another such example. There are other single-gene mutants that are stress-resistant but not long-lived (37–41). This lack of positive correlation between decreased levels of a stress and increased life span has also been found in D. melanogaster (42), as well as in M. musculus and other species (36,43). Finally, Drosophila made long-lived by neuronal expression of a dominant-negative version of the fly p53 ortholog is not resistant to starvation or heat stresses, although it is resistant to the reactive oxygen species generator paraquat (4).

Our data suggest that wild-type daf-16 is required for cep-1–mediated effects on life span. However, when we looked for nuclear localization using a DAF-16::GFP translational construct, we found no evidence that cep-1 RNAi affects DAF-16 nuclear localization or retention, even under heat stress. This finding suggests that CEP-1 is not biochemically upstream of DAF-16 or responsible for regulating its nuclear localization, as is the case with AGE-1 and DAF-2. This genetic dependence of cep-1 upon daf-16 for life-span increase could therefore be interpreted to suggest that cep-1 is one of the genes regulated by daf-16. In this model, increased DAF-16 within the nucleus would increase transcriptional repression of cep-1, leading to increased longevity.

It is also possible that CEP-1 counteracts DAF-16's transactivation without altering its location. In this model, DAF-16 localization may not be affected by CEP-1, but both transcription factors may have binding sites near the same genes. If DAF-16 is bound to the promoter of a gene the expression of which would be detrimental for longevity, and is repressing its expression via attenuation of transcription, another means by which it could be causing for that repression would be through physical occlusion of CEP-1 from that promoter region; leading to a failure of CEP-1 to up-regulate that pernicious gene. Thus, functional CEP-1 alone would lead to the expression of a gene detrimental for longevity, and the organismic result would be decreased survivorship; functional DAF-16 alone would lead to the repression of that gene, and thus longevity; functional copies of both transcription factors would lead to competition for the site, with mixed, intermediate results upon life span; and functional copies of neither protein would lead to up-regulation of the gene by another transcription factor, leading to short life.

After DNA damage, phosphorylation of p53 leads to its accumulation and activation. The accumulation results because phosphorylation reduces interaction of p53 with Murine Double Minute 2 (MDM2), a ubiquitin ligase that targets proteins for proteasomal degradation. In mammalian cells, p53 can be phosphorylated by DNA damage–responsive kinases ataxia telangectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR), DNA-dependent protein kinase (DNA-PK), checkpoint kinase 1 (Chk1), and checkpoint kinase 2 (Chk2), among others (44,45). Further studies in C. elegans of these regulators of p53 might provide insight to the pathways that connect p53 to longevity. For example, it is possible that loss of p53 frees MDM2 to target effete and potentially detrimental proteins for degradation and turnover, preventing cellular malfunction. Expression analysis of the transcriptional targets of p53 (46,47) in the cep-1(gk138) mutant may suggest new pathways for analysis of cep-1 effects. For example, human p53 alters transcription of targets that modulate cell-cycle inhibition, DNA repair, and apoptosis (44), and learning more about expression of these targets in cep-1(gk138) worms may help to clarify the pathways that are responsible for the effects of p53 on life span in multiple species.


   Acknowledgments
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 Abstract
 Methods
 Results
 Discussion
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Support for this work was provided by National Institutes of Health grants RO1 AGO16219 and KO2 AA00195 (TEJ), AG012423 (Christopher D. Link), and by National Institute of Child Health and Human Development (NICHD) Research Training Grant T32 HD007289 (OA).

We thank W. Brent Derry and Joel Rothman for generously providing the JR2474 [cep-1::gfp] strain, Thomas Petes for graciously providing the NW1613 [msh-2(ev679::Tc1)] strain, Heidi Tissenbaum for providing the LG100 [sir-2.1(geIn3)] strain, and the C. elegans Reverse Genetics Core Facility at UBC (which is part of the International C. elegans Gene Knockout Consortium) and the Caenorhabditis Genetics Center for all other strains. We also thank Richard A. Miller, W. Brent Derry, and Christopher D. Link for helpful discussions.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received September 26, 2006

Accepted April 26, 2007


   References
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 References
 

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