Cellular senescence in vitro and organismal ageing
Maier, A.B.
Citation
Maier, A. B. (2008, December 11). Cellular senescence in vitro and organismal ageing. Retrieved from https://hdl.handle.net/1887/13335
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Influence of the TP53 codon 72 polymorphism on the cellular responses to X-irradiation in fibroblasts from nonagenarians
P. Martijn den Reijer, Andrea B. Maier, Rudi G.J. Westendorp, Diana van Heemst
Mech. Ageing Dev. 2008; 129, 175-182
Summary
In mice, genetic modification of the gene encoding p53 affects both cancer incidence and longevity. In humans, we recently found that a TP53 codon 72 Arginine (Arg) to Proline (Pro) polymorphism affected both cancer incidence and longevity as well. The TP53 codon 72 polymorphism has previously been shown to influence the apoptotic potential of human cells in response to oxidative stress.
Here, we studied the influence of this polymorphism on the cellular responses to X- irradiation of fibroblasts obtained from nonagenarians. We found that the average clonogenic survival after X-irradiation was similar for the three TP53 codon 72 genotype groups. As described before, X-irradiation did not induce an appreciable degree of apoptosis in human fibroblasts. However, percentages of senescence- DVVRFLDWHG ȕ-galactosidase positive cells (p < 0.001), micronucleated cells (p <
0.001) and cells displaying abnormal nuclear morphologies (p < 0.001) significantly increased with the radiation dose. Compared to Arg/Arg fibroblasts, Pro/Pro fibroblasts exhibited higher irradiation dose-dependent increases in ȕ- galactosidsae positive cells (pinteraction = 0.018), micronucleated cells (pinteraction = 0.005) and cells displaying abnormal nuclear morphologies (pinteraction = 0.029) at three days after irradiation. Possibly, these differences in cellular responses to stress between the TP53 codon 72 genotypes contribute to the differences in cancer incidence and longevity observed earlier for these genotypes.
Introduction
In various mouse models, genetic modification of the gene encoding p53 affects both cancer incidence and longevity. Mice deficient for p53 die of cancer within the first year (Donehower et al., 1992). However, the rare mice only partially deficient for p53 (p53±) that by chance avoided tumors were reported to have increased life spans (Donehower, 2002). On the other hand, mutant mice having constitutively activated p53 not only show enhanced resistance to spontaneous tumors but also an early onset of diverse aging phenotypes, including general loss of cellularity and reduced longevity (Donehower, 2002; Tyner et al., 2002; Maier et al., 2004). In humans, a common codon 72 polymorphism in TP53 encodes either Arginine (Arg) or Proline (Pro) (Weston et al., 1994). Recently, in a prospective follow-up study of people aged 85 years and over, we demonstrated that for TP53 codon 72 Pro/Pro carriers, despite an increased mortality from cancer, old age survival was significantly increased compared to Arg/Arg carriers (van Heemst et al., 2005). Moreover, in a meta-analysis of the vast array of literature on the relationship of this polymorphism with cancer incidence, we found that Pro/Pro carriers were more susceptible to cancer than Arg/Arg carriers (van Heemst et al., 2005). Together, these data hint at a link between tumor suppression and longevity in organisms with renewable tissues.
Such a link can be explained, because on the one hand it is necessary to constantly replace damaged and lost cells to maintain tissue homeostasis, whereas at the same time cellular proliferation must be carefully controlled to suppress cancer (Campisi, 2003, 2005). One strategy to suppress cancer involves the prevention and repair of accumulated damage. Another strategy to suppress cancer is to eliminate cells with accumulated damage by apoptosis or to prevent such cells from proliferating by permanent cell cycle arrest, i.e., senescence. Despite the important anti-tumorigenic functions of apoptosis and senescence, these actions at the same time contribute to the loss of tissue cellularity and integrity thought to underlie aging (Campisi, 2003, 2005; Higami and Shimokawa, 2000; Pelicci,
2004). Yet, on a cellular level, it is unclear what the relative importance of p53- dependent apoptosis and senescence is in striking the balance between cancer suppression and longevity.
The TP53 codon 72 polymorphism was found to influence the apoptotic potential of human cells. Both in p53-inducible cell lines (Dumont et al., 2003; Pim and Banks, 2004; Sullivan et al., 2004) and in normal diploid fibroblasts from centenarians (Bonafe et al., 2004) it was found that the Arg variant had a significantly higher apoptotic potential than the Pro variant. Here, the influence of the TP53 codon 72 polymorphism on cellular responses to X-irradiation was examined in normal human fibroblasts obtained from 90-year old participants of the prospective Leiden 85-plus Study. X-irradiation was reported to induce senescence in a p53-dependent manner in normal human fibroblasts (Di Leonardo et al., 1994; Linke et al., 1997), while no appreciable degree of apoptosis was found (Belyakov et al., 1999; Brammer et al., 2004; Lara et al., 1996; Enns et al., 1998; Goldstein et al., 2005). Therefore, we compared 6 Arg/Arg, 6 Arg/Pro and 6 Pro/Pro cell strains for differences in attachment frequency, cloning efficiency, clonogenic survival, senescence-associated ȕ-galactosidase activity, incidence of micronuclei (MN) formation and apoptosis after X-irradiation.
Materials and Methods
Fibroblast strains
The Leiden 85-plus Study is a prospective population-based study consisting of inhabitants of Leiden, the Netherlands (der Wiel et al., 2002). Between 1997 and 1999, 599 people aged 85 years were enrolled and followed for specific causes of death until April 2004. In April 2004, about 50% of the participants had died.
Between December 2003 and May 2004, a three millimeter full thickness punch biopsy was taken from the sun unexposed medial site of the mid-upper arm of 68 of the surviving 90 year old subjects, and fibroblasts were cultured from these
biopsies as described previously (Maier et al., 2007). The Leiden 85-plus Study was approved by the medical ethical committee of the Leiden University Medical Centre. Informed consent was obtained from all participants after the nature and possible consequences of the studies were explained. For the current study, highly proliferating cultures from 18 fibroblast strains (6 Arg/Arg, 6 Arg/Pro and 6 Pro/Pro) were used, at replicative age ranging from 8 to 18 passages, which corresponds to 21.8–39.8 cumulative population doublings (PDs). The growth speed during the early replicative phase and the onset of senescence did not differ significantly between the three TP53 codon 72 genotype groups (Table 1).
Attachment frequency
For analysis of attachment frequency, cells derived from cultures at a confluence of 70–80% were seeded at a number of 15,000 cells per well on plastic well plates (Nunc, VWR, Amsterdam, the Netherlands). After the cells had fully attached, but before any subsequent growth (4 h), cells were washed, attached cells were trypsinized and counted. The attachment frequency assays were repeated twice on separate days for each cell strain. Results from the two independent experiments were averaged.
X-irradiation
Cells were irradiated at room temperature with a linear X-ray generator (ELEKTA Precise Linac) at a distance of 1 m and at a rate of 4 Gy/min.
Clonogenic cell survival
Prior to irradiation, cells derived from cultures at a confluence of 70–80% were seeded at a number of 500 cells per plate in 94-mm Petri dishes and allowed to attach. Cells were seeded in triplicate at 0 Gy, and in duplo for the other radiation doses. After irradiation, cells were grown for two weeks at 37°C and 5% CO2 to allow formation of colonies. Medium was not changed during this period. After two weeks, cells were fixed with 0.9% NaCl (Merck, Amsterdam, the Netherlands)
Table 1. Characteristics of the fibroblast strains stratified for TP53 codon 72 genotype.
Arg/Arg n = 6
Arg/Pro n = 6
Pro/Pro n = 6
PD, when tests were performed, mean (SD) 35.6 (4.4) 32.0 (5.4) 32.3 (7.3) Growth speed during the early replicative
phase, mean (SD) PD per day 0.31 (0.02) 0.3 (0.04) 0.31 (0.04) Maximal number of PD, mean (SD) 78.3 (7.3) 74.8 (2.8) 79.4 (20.1)
and stained with methylene blue (2.5 g per L; Sigma, Zwijndrecht, the Netherlands). Colonies were scored as such if they contained 20 cells or more. The surviving fractions at 1, 2, 4 and 6 Gy were calculated taking into account the cloning efficiencies of the unirradiated controls. Only clonogenic survival assays with an average cloning efficiency of at least 10% were included. All survival assays were repeated twice on separate days for each cell strain. Results from the two independent experiments were averaged.
ȕ-Galactosidase staining
Prior to irradiation, cells derived from cultures at a confluence of 70–80% were seeded at a number of 15,000 cells per chamber on plastic chamber slides (Lab-Tek chamber slide system, Nunc) and allowed to attach and stained for ȕ-galactosidsae activity at three days after irradiation (Salvioli et al., 2005). Before staining, cells were washed twice, fixed with 9% paraformaldehyde for 3 min at room temperature, washed again and stained overnight (16 h) with ȕ-galactosidsae (40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2) as described earlier (Dimri et al., 1995). Cells were then double stained with Mayer’s hematoxylin for 5 min. All chemicals were obtained from Sigma (Zwijndrecht, the Netherlands). Cells were viewed with a Leica microscope and photographed at a 100x magnification using a Leica DC 300F digital color camera. At least 500 cells were counted per chamber, at a confluence of maximally 70% to avoid counting cells stained as a result of
confluence. For every cell strain, 2 chambers were counted, and results were averaged.
dRib treatment
As a positive control in the apoptosis assays, fibroblast cell strains were treated with 2-deoxy-D-ribose (dRib, Sigma), a well known inducer of apoptosis in human fibroblasts (Bonafe et al., 2004; Kletsas et al., 1998). Briefly, cells were seeded on plastic chamber slides, grown until confluence was reached and then incubated overnight in medium containing 0.5% fetal calf serum to synchronize cells. The next day, medium was changed to medium containing 20 mM dRib, to which the cells were exposed for 72 h.
Hoechst staining
Prior to irradiation, cells derived from cultures at a confluence of 70–80% were seeded at a number of 15,000 cells per chamber on plastic chamber slides and allowed to attach. Cells were incubated immediately after irradiation for three days at 37°C and 5% CO2. Subsequently, cells were washed twice with PBS and fixed for 10 min at room temperature with 9% paraformaldehyde. Cells were then washed twice, permeabilized for 5 min with 0.2% Triton X-100 and after three additional washes stained with Hoechst 33258 (5 mg per mL Bisbenzimide H 33258, Sigma) for a minimum of 30 min at 37°C in the dark. Stained cells were viewed and photographed immediately at a 250x magnification using a Leica fluorescence microscope (Leica Microsystems, Rijswijk, the Netherlands).
Apoptotic, micronucleated and abnormal cells were all viewed at the same magnification and 400–500 cells were counted per chamber. For each cell strain two chambers were counted and results were averaged.
Apoptotic cells were readily observed by nuclear condensation. MN were scored as described before (e.g., Abend et al., 1995; Keshava et al., 1995). Briefly, cells were only scored as micronucleated if MN within the cell were visible as clearly delineated round compartments separated from the main nucleus. The MN
had to be smaller than one-third of the size of the main nucleus while their fluorescence was of the same color and intensity as that of the main nucleus. Cells that did not fall in any of the three categories normal, apoptotic or micronucleated were considered as abnormal cells. These were mainly cells with main nuclei displaying dim fluorescent protrusions and cells displaying multiple large nuclei.
Detection of nuclear matrix protein
Apoptosis was also quantified by assaying the supernatant of cell cultures on the presence of nuclear matrix protein (NMP), which is only released by apoptotic cells. Culture supernatants from cell strains irradiated in chamber slides were collected five days after irradiation, centrifuged at 2000 rpm for 10 min and stored at -20°C. Culture supernatants from control apoptotic cells were collected three days after treatment with dRib. NMP concentration was measured using the Cell Death Detection ELISA kit (Calbiochem, Temecula, CA), according to the manufacturer’s instructions. Culture supernatant samples from two chambers were assayed for each dose and cell strain and results were averaged.
Statistical analysis
Cell survival data of the irradiation experiments were analyzed by nonlinear regression analysis, giving equal weight to each dose point, using the linear- quadratic equitation -ln SF = Į' + ȕ'2. SF is the surviving fraction and D is the radiation dose. Besides the linear-quadratic parameters Į and ȕ, the survival parameters SF2 (surviving fraction at 2 Gy) and D0.01(dose required for a surviving fraction of 1%) were calculated for each cell strain. The Arg/Pro and Pro/Pro groups were compared with the wild-type Arg/Arg group on these four parameters using linear regression, allowing adjustment for any differences in replicative age between cell strains at the time of irradiation.
5HVXOWV RI ȕ-galactosidase and Hoechst staining were analyzed using a linear mixed model. The p value describes the significance of the increase in a particular cellular response dependent on irradiation dose or TP53 codon 72 genotype. The
pinteraction value describes the significance of the difference in increase in a particular cellular response upon radiation dependent on the interaction between genotype and irradiation dose. Equal weight was given to each dose point and additional analyses data were adjusted for any differences in replicative age between cell strains. The correlations between frequency of micronucleated cells and cell survival were obtained as the Pearson correlation coefficient. For statistical analysis SPSS version 11.0 (SPSS Inc., Chicago, IL) was used.
Results
Outline of the study
In total 18 fibroblast stains (6 Arg/Arg, 6 Arg/Pro and 6 Pro/ Pro) were used for the experiments described in this article (Table 1). All 18 fibroblast strains were in the exponential phase of the growth curve and the in vitro replicative age was comparable between the three TP53 codon 72 genotype groups. We first used (subsets of) these strains to determine whether the attachment frequency and cloning efficiency at 0 Gy were dependent on the TP53 codon 72 genotype. Next we used (subsets of) these strains to determine whether clonogenic survival after X-irradiation and the different cellular responses to X-irradiation were dependent on TP53 codon 72 genotype.
Attachment frequency dependent on TP53 codon 72 genotype
We used 3 Arg/Arg and 3 Pro/Pro strains to determine the attachment frequency of cells dependent on TP53 codon 72 genotype without X-irradiation. The average attachment frequency of these strains was 70% and did not differ significantly between Arg/Arg carriers compared to Pro/Pro carriers (Table 2).
Cloning efficiency dependent on TP53 codon 72 genotype
We used all 18 fibroblast strains to determine the cloning efficiency of cells dependent on TP53 codon 72 genotype without X-irradiation. The average cloning
efficiency was 24% and was similar for the three TP53 codon 72 genotype groups (Table 2).
Post-irradiation survival dependent on TP53 codon 72 genotype
For the 6 Arg/Arg, 6 Arg/Pro and 6 Pro/Pro cell strains dose-response survival curves were determined after X-irradiation. For these 18 strains, the average values (S.E.) for the survival parameters SF2 and D0.01were, respectively, 0.28 (0.02) and 5.66 (0.17). Coefficients of variation between duplicate experiments were 26.7%
and 12.1% for, respectively, SF2 and D0.01. When cell strains were grouped according to their TP53 codon 72 genotype, three almost identical survival curves were obtained (Figure 1). The average survival parameters of the three TP53 codon 72 groups were similar (Table 3), and did not materially change after adjustment for replicative age.
Induction of apoptosis in fibroblasts after X-irradiation
To determine whether X-irradiation induces apoptosis in the fibroblasts obtained from nonagenarians, 6 Arg/Arg and 6 Pro/Pro cell strains were assayed for nuclear condensation at three days after irradiation. Staining of fibroblasts with Hoechst revealed that nuclear condensation at 2 and 4 Gy for all cell strains was less than 2%, while nuclear condensation was observed in almost all control cells treated with the apoptosis-inducing agent 2-deoxy-D-ribose (dRib) for 72 h. Nuclear con-
Table 2. Attachment frequency and cloning efficiency at 0 Gy dependent on TP53 codon 72 genotype.
Arg/Arg Arg/Pro Pro/Pro
n Mean
(SD) n Mean
(SD) n Mean
(SD)
Attachment frequency (%) 3 65 (19) nd nd 3 74 (9)
Cloning efficiency (%) 6 23 (12.1) 6 24 (9.3) 6 24 (10.6) nd: not determined.
densation remained absent at five and nine days after irradiation (data not shown).
Assaying 2 Arg/Arg and 2 Pro/Pro cell strains on NMP release five days after irradiation confirmed these results, showing no increase in NMP concentrations in culture supernatants at 2 and 4 Gy compared to 0 Gy, whereas NMP release significantly increased after dRib treatment. Thus, apoptosis is not a major cellular response to X-irradiation in fibroblasts.
Post-LUUDGLDWLRQ ȕ-galactosidase activity dependent on TP53 codon 72 genotype
The same 18 cell strains were also examined on the expression of senescence DVVRFLDWHG ȕ-galactosidase activity, a well-known marker for cellular senescence (Dimri et al., 1995), at three days after irradiation (Salvioli et al., 2005). For all cell VWUDLQVWKHQXPEHURIȕ-galactosidase positive cells increased significantly between
Figure 1. Average clonogenic survival of the Arg/Arg, Arg/Pro and Pro/Pro genotype groups at 14 days after X-irradiation. The average survival curve of each group is obtained from 6 cell strains, the dotted average line from all 18 cell strains. Data are mean values ± standard deviations.
Table 3. Clonogenic survival parameters 14 days after X-irradiation dependent on TP53 codon 72 genotypes.
Arg/Arg Arg/Pro versus Arg/Arg Pro/Pro versus Arg/Arg
Mean ȕ 6( p value ȕ 6( p value
Į *\-1)a -0.50 -0.12 (0.15) 0.43 -0.08 (0.14) 0.55
ȕ *\-2)a -0.07 0.05 (0.03) 0.18 0.02 (0.03) 0.48
SF2b 0.29 -0.02 (0.05) 0.73 -0.03 (0.05) 0.57
D0.01 (Gy)c 5.38 0.85 (0.41) 0.07 -0.02 (0.29) 0.95
aDerived from fitting survival points with –ln SF = ĮD + ȕD2.bSF2 = proportion of surviving clonogenic cells after single dose of 2 Gy as estimated from the fit through all dose points.
cD0.01(Gy) = single dose in Gy that reduces proportion of surviving clonogenic cells to 1%.
Survival parameters of all cell strains were grouped according to genotype and the difference with the reference category (Arg/Arg) was determined using linear regression. For each genotype group 6 cell strains were tested.
0, 2 and 4 Gy (p < 0.001), indicating that X-irradiation indeed induces senescence (Figure 2A and B). However, there was considerable variation between cell strains LQ WKH PDJQLWXGH RI LQFUHDVH LQ ȕ-galactosidase positivity after irradiation. When WKH DYHUDJH SHUFHQWDJHV RI ȕ-galactosidase positive cells after irradiation are compared between the three genotype groups, a consistently higher staining of the Pro/Pro group compared to the Arg/Arg and Arg/Pro groups is observed (Figure 2B). Notably, the average radiation-LQGXFHG LQFUHDVH LQ ȕ-galactosidase positive cells proved to be significantly higher in the Pro/Pro group compared to the Arg/Arg group (pinteraction = 0.018). Adjustment for replicative age did not materially change these results.
Post-irradiation incidence of MN and nuclear abnormalities dependent on TP53 codon 72 genotype
The 6 Arg/Arg and 6 Pro/Pro cell strains were also stained with Hoechst to detect
nuclear condensation and the formation of micronuclei (MN). Although Hoechst
Figure 2. Dose-dependent increase in ȕ-galactosidase positive cells after X-irradiation.
(A) Representative example of a cell strain before and after X-LUUDGLDWLRQ$UURZVLQGLFDWHȕ- galactosidase activity.
(B) 7KH DYHUDJH SHUFHQWDJHV RI ȕ-galactosidase positive cells at 0, 2 and 4 Gy for the Arg/Arg, Arg/Pro and Pro/Pro genotype groups.
Each group consists of six cell strains. TP53 codon 72 genotypes are marked as indicated.
Data are mean values ± standard deviation 7KH QXPEHU RI ȕ-galactosidase positive cells increases with the radiation dose (p < 0.001), with a higher average radiation-induced LQFUHDVH LQ ȕ-galactosidase positive cells in the Pro/Pro group compared to the Arg/Arg group (pinteraction = 0.018).
Figure 3. Dose-dependent increase in micronucleated cells after irradiation.
(A) Representative example of a cell strain before and after X-irradiation. Arrows indicate micronuclei.
(B) The average percentages of micronucleated cells at 0, 2 and 4 Gy for the Arg/Arg and Pro/Pro genotype groups. Each group consists of six cell strains. TP53 codon 72 genotypes are marked as indicated. Data are mean values ± standard deviations. The incidence of MN increases with the radiation dose (p < 0.001), with a higher average radiation-induced increase in micronucleated cells in the Pro/Pro group compared to the Arg/Arg group (pinteraction = 0.005).
Figure 4. Dose-dependent increase in nuclear abnormal cells after irradiation.
(A) Some representative examples of abnormal cells found after irradiation.
(B) The average percentages of abnormal cells at 0, 2 and 4 Gy for the Arg/Arg and Pro/Pro groups. Each group consists of six cell strains. TP53 codon 72 genotypes are marked as indicated. Data are mean values ± standard deviations. The number of abnormal cells increases with the radiation dose (p < 0.001), with a higher average radiation-induced increase in abnormal cells in the Pro/Pro group compared to the Arg/Arg group (pinteraction= 0.029).
staining did not reveal any nuclear condensation, the incidence of MN was found to significantly increase in all 6 Arg/Arg and 6 Pro/Pro cell strains at three days after irradiation (p < 0.001) (Figure 3B). Furthermore, the average correlation between the frequency of micronucleated cells and survival fractions was strong and significant (r = -0.842, p < 0.001).,QWHUHVWLQJO\DVIRUWKHȕ-galactosidase activity, the average radiation-induced increase in micronucleated cells proved to be significantly higher in the Pro/Pro group compared to the Arg/Arg group (pinteraction
= 0.005) (Figure 3B). Adjustment for replicative age did not change these results.
Furthermore, the increase in abnormal cells did not fulfilling the criteria of either normal, apoptotic or micronucleated cells (Figure 4A) also significantly increased with the radiation dose (p < 0.001), and again this increase was significantly higher in the Pro/Pro group compared to the Arg/Arg group (pinteraction = 0.029) (Figure 4B). Adjustment for replicative age did not change these results.
Discussion
In this study, we examined the response of normal fibroblasts from nonagenarians to X-irradiation dependent on the TP53 codon 72 polymorphism. The results are two-fold. In comparison to the Arg/Arg strains, the Pro/Pro strains exhibited a significantly higher dose-dependent increase in the percentages of ȕ-galactosidase positive, micronucleated and nuclear abnormal cells at three days after irradiation.
In contrast, attachment frequency and cloning efficiencies were similar and no differences were found in the average clonogenic survival of both genotype groups after irradiation.
Our results on the influence of the TP53 codon 72 polymorphism on induction of cellular senescence are in line with other published reports. Recently, it was demonstrated that the TP53 codon 72 polymorphism influenced the response of fibroblasts obtained from centenarians to the chemotherapeutic agents camptothecin and doxorubicin, which are competent inducers of senescence at low
doses (Salvioli et al., 2005). At three days after exposure, stressed fibroblasts containing the Pro/Pro genotype showed significantly higher expression of the senescence-associated cell cycle regulator p21WAF1 DQG ȕ-galactosidase compared to fibroblasts with the Arg/Arg genotype. Earlier reports showed that different types of stably transfected lines expressing the TP53 codon 72 Pro variant induced a higher level of G1 arrest than those expressing the Arg variant (Pim and Banks, 2004; Sullivan et al., 2004). In both reports the Arg variant was confirmed to be more efficient in inducing apoptosis than the Pro variant (Pim and Banks, 2004;
Sullivan et al., 2004). These and our current findings, therefore, suggest that the Arg/Arg genotype of the TP53 codon 72 polymorphism may associate with a more apoptotic prone cellular response, while the Pro/Pro genotype may associate with an increased senescence response.
Interestingly, in this study not only the dose-dependent increase in ȕ- galactosidase activity but also in micronucleated and nuclear abnormal cells were significantly higher in the Pro/Pro group compared to the Arg/Arg group at three days after irradiation. Formation of MN results from damaged chromosomes, which become excluded from the main nucleus during mitosis. Although still controversial, MN are considered hallmarks of genomic instability resulting in mitotic catastrophe, which some think precedes cell death (Chu et al., 2004; Nitta et al., 2004) and others consider a form of cell death on its own (Eom et al., 2005;
Roninson et al., 2001). Interestingly, after treatment with low-dose doxorubicin, human cancer cells were found to display both ȕ-galactosidase activity and MN formation (Eom et al., 2005). It was suggested that micronucleated cells remain in a viable, senescence-like state for some time, but eventually die through loss of membrane integrity. As both low-dose doxorubicin treatment and X-irradiation are thought to induce senescence through similar pathways, it can be questioned whether upon X-irradiation normal fibroblasts are undergoing senescence or instead mitotic catastrophe accompanied by a senescence-like phenotype. Indeed, theSHUFHQWDJHVRIȕ-galactosidase positive, micronucleated and nuclear abnormal
cells that we observed were similar under all circumstances. However, aforementioned cancer cells lacked a functional p53, making it uncertain whether this cell fate plays a role in normal fibroblasts.
In contrast to the observed differences in cellular responses to X-irradiation at three days after irradiation, the clonogenic survival assays revealed no difference in the mean postirradiation survival of the Arg/Arg, Arg/Pro and Pro/Pro cell strains at 14 days after irradiation. The average survival parameters of all 18 cell strains were very similar to those reported by others for normal human fibroblasts (Peacock et al., 2000), as was the variation in post-irradiation survival that we observed between the tested cell strains (Lu-Hesselmann et al., 2004; Rave-Frank et al., 2001; Wurm et al., 1994). Based on differences in cellular responses to X- irradiation, we would have expected the more senescence prone Pro/Pro strains to differ in their ability to form colonies after X-irradiation compared to the Arg/Arg strains. The higher apoptotic potential of the Arg/Arg cell strains seems unlikely to be a compensatory factor, because, in line with an extensive array of previous data (Belyakov et al., 1999; Brammer et al., 2004; Lara et al., 1996; Enns et al., 1998;
Goldstein et al., 2005), we did not observe any appreciable degree of apoptosis in the X-irradiated fibroblasts.
We cannot exclude the possibility that the presence of additional variant alleles may have influenced certain of the endpoints measured. The codon 72 allele is in strong LD with an intron 3 duplication, and the presence of the variant allele at both positions has been shown to result in lower levels of p53 mRNA. In line with these findings, we previously observed the strongest effect on longevity in the Leiden 85-plus cohort if both variant alleles (codon 72 Pro and the intron 3 duplication) were present (data not shown). In the small subset used here, codon 72 Pro-intron 3 duplication carriers may have been slightly under-represented as compared to the total cohort in which the effects on survival were observed. We, therefore, can not exclude the possibility that the effects on some of the endpoints analyzed here may have been stronger if a higher percentage of codon 72 Pro and
intron 3 duplication carriers would have been present among the Pro/Pro group.
However, the number of cell strains used here (n = 18) is too small to allow for such detailed genetic analysis.
The differences in predisposition of cells to senescence and genetic instability at three days post-irradiation dependent on TP53 codon 72 genotype may contribute to the differences in survival and cancer incidence found earlier to associate with these genotypes. Pro/Pro subjects were shown to have a significantly increased survival compared to the Arg/Arg subjects, despite an increased incidence of cancer (van Heemst et al., 2005). It has been argued that cellular senescence is a double-edged sword in that it evolved to suppress cancer early in life, but may actually contribute to tumor formation later in life as senescent cells accumulate to sufficiently large numbers (Campisi, 2003, 2005). It has been shown that senescent fibroblasts can stimulate growth of premalignant cells in culture, probably via the adaptation of a growth and inflammatory promoting secretory phenotype (Krtolica et al., 2001; Dilley et al., 2003; Martens et al., 2003). Senescent fibroblasts also become resistant to apoptosis (Hampel et al., 2004; Marcotte et al., 2004; Seluanov et al., 2001). The increased incidence of cancer and the increased survival in the Pro/Pro subjects might be explained by an increased senescence and/or reduced apoptotic potential.
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