- Email: [email protected]
p53 and a human premature ageing disorder
Mechanisms of Ageing and Development 124 (2003) 599 /603 www.elsevier.com/locate/mechagedev
p53 and a human premature ageing disorder Mary O’Neill a,*, Fatima Nu´n˜ez b, David W. Melton a a
Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK b Centro de Investigac¸ion del Cancer, Campus Unamuno, 37007 Salamanca, Spain Received 17 April 2002; received in revised form 19 December 2002; accepted 30 January 2003
Abstract It has been shown that enhanced levels of p53 activity contribute to reduced cancer susceptibility in mice, however longevity is compromised due to the onset of an early-ageing phenotype. The effects of enhanced levels of p53 in these in mice could therefore have implications for human premature ageing disorders. We examined the DNA damage response of p53 and its target p21WAF1 to UV and ionising radiation in fibroblasts from patients with the premature ageing disorder Hutchinson-Gilford Progeria (HGP). We report a normal p53 response to these DNA damaging agents suggesting that, in this particular human disorder, the premature ageing phenotype does not arise from an enhanced p53 response. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Progeria; p53; DNA damage; p21WAF1; Senescence
1. Introduction The p53 tumour suppressor protein plays a vital role in maintaining the control of the cell cycle. A defective p53 protein leads to increased cancer susceptibility while higher than normal levels of the protein contribute to replicative senescence (Sugrue et al., 1997; Webley et al., 2000). It has been demonstrated that enhanced levels of p53 in p53 gene-targeted mice resulted in animals with a reduced susceptibility to cancer but with symptoms of premature ageing (Tyner et al., 2002). Mice with a wildtype copy of p53 and a copy of an N-terminal deleted form had decreased longevity and pathological symptoms similar to ageing. The authors concluded that the enhanced levels of p53 in mice led to a decrease in proliferative capacity of the stem cells. This provoked the speculation that higher than normal levels of p53 might contribute to the pathology of human premature ageing disorders (Ferbeyre and Lowe, 2002). HGP is a very rare premature ageing disorder; symptoms include growth retardation, absence of sub-
* Corresponding author. Tel.: /44-131-651-1077; fax: /44-131651-1072. E-mail address: [email protected] (M. O’Neill).
cutaneous fat, arteriosclerosis, osteoarthritis, skeletal abnormalities and aged appearance. Life expectancy is short ( :/13 yr) with death invariably from cardiac failure (Jansen and Romiti, 2000). There is no evidence of increased susceptibility to cancer, however given the rarity of the syndrome and the low life expectancy this may not be significant. Fibroblasts from these patients show a decreased rate of DNA synthesis after UV irradiation suggesting an impaired capacity to repair DNA (Wang et al., 1991). It is believed to be due to an autosomal dominant mutation that spontaneously arises in the germ line and telomeres are shorter than age matched controls (Allsopp et al., 1992). HGP fibroblasts undergo fewer cell doublings and the rate of doubling is extremely slow (1 division per week). Patterns of gene expression in HGP are consistent with that of old age, with genes involved in cell division and DNA or RNA synthesis being down-regulated in these two situations (Ly et al., 2000). As cells undergo senescence or are subjected to UV or ionising radiation (IR), p53 becomes post-translationally modified at specific residues. These modifications render p53 resistant to degradation by Mdm2, therefore levels of p53 in the cell increase (Steele et al., 1998). Different assaults instigate modification at different residues, with UV and cellular senescence showing similar modification
0047-6374/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0047-6374(03)00007-1
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patterns that are distinct from those caused by IR (Webley et al., 2000). Here we investigate the hypothesis that an enhanced p53 response to DNA damaging agents, leading to the activation of downstream pathways that might regulate cell cycle arrest and entry into senescence, is the basis for the premature ageing disorder Hutchinson-Gilford Progeria.
2. Materials and methods Human fibroblasts from progeria patients (AG03199, AG06917 and AG08466) and their unaffected family members (AG03257, AG06299 and AG08470) were obtained from the NIA Aging Cell Repository (Coriell Cell Repository, Camden, NJ). Fibroblasts were cultured in Glasgow modified Eagle’s Medium, supplemented with 20% fetal calf serum. Fibroblasts cultures that were 50% confluent in 60 mm dishes were exposed to 30 J m 2 UVC light, returned to the incubator and harvested at the time intervals indicated (Fig. 1). Similarly for treatment with IR, cells were exposed to 25 Gy in a Torrex Faxitron X-ray and then returned to
the incubator and harvested at the time intervals indicated (Fig. 2). Total cell protein was isolated from fibroblasts that were first washed in 1 / PBS then resuspended in a 50 ml volume of lysis buffer (125 mM Tris pH 7, 2 mM EDTA, 2% SDS, 33 mM DTT, 2 mM PMSF) and boiled for 5 min before centrifuging to remove cell debris. Cell lysates were stored at /20 8C. Equal amounts (80 mg) of protein were loaded on a SDS PAGE gel and run, with cooling at 50 mA. Protein was transferred to a PVDF membrane using a Bio-Rad Transblot semi-dry transfer system. After blocking in 5% 1 / TBST (50 mM Tris (pH 7.5) 150 mM NaCl and Tween 0.1%) the membranes were hybridised to the primary antibody for a minimum of 1 h. Antibodies: p53 (Calbiochem) was used at a dilution of 1/2500 and incubated for 1 h at room temperature; p21WAF1 (Calbiochem) was used at a dilution of 1/66 and incubated at 4 8C overnight. After washing, the membranes were incubated with the HRP tagged secondary antibody and finally detected with ECL Plus (Amersham). Intensity of signal was determined by scanning densitometry using ImageJ.
Fig. 1. Western blot analyses of p53 after UV irradiation of HGP fibroblasts. (A) HGP (AG08466) and unaffected control (AG08470), (B) HGP (AG06917) and unaffected control (AG06299), (C) HGP (AG03199)* and unaffected control (AG03257). Fibroblasts were irradiated with UVC 30 J m 2 and samples collected at time intervals indicated. nr, Non-irradiated cells. After initial reaction with p53, filters were stripped and reprobed for actin. The values below each lane indicate p53 response levels, obtained by densitometry, after normalization for actin, expressed relative to the nonirradiated culture. The doublet of p53 in samples AG08466, AG06917 and AG06299 is due to the polymorphism at amino acid 72 (Matlashewski et al., 1987). Arg72 moves faster than Pro72. *The basal level of p53 in AG03199 was extremely low and so the p53 actin ratios for this cell line are expressed relative to the non-irradiated sample for the control (AG03257).
M. O’Neill et al. / Mechanisms of Ageing and Development 124 (2003) 599 /603
Fig. 2. Analysis of p53 response after exposure of HGP (AG08466) and control (AG08470) fibroblasts to IR. Fibroblasts were exposed to 25 Gy of IR and treated as in Fig. 1. Filters were probed first with p53 then stripped and reprobed for actin. The p53: actin values were calculated in the same way as Fig. 1.
Total RNA was prepared from all three progeria cell lines and familial controls using RNA-BeeTM (Biogenesis, Poole, UK). The p53 coding region was amplified by rtPCR as described in Bentley et al. (2002) and the sequence determined using the PCR primers.
3. Results 3.1. p53 response to UVC We first of all examined levels of p53 in fibroblasts from three unrelated HGP patients and unaffected family members after irradiation with UV (30 J m 2). Total protein was isolated from non-irradiated cells and 3, 6, 24 and 48 h after irradiation; Fig. 1(A /C) shows western blots for the three HGP cell line and their controls.
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Contrary to the situation in the p53 gene-targeted mice (Tyner et al., 2002), where expression of the expected N-terminal truncated protein from the targeted allele could not be detected, the existence of a p53 protein polymorphism in two out of the three HGP cell lines and one of the controls, demonstrated that both p53 alleles in the HGP and the control samples produced normal amounts of p53 protein. The levels of p53 protein increased in all the HGP and control samples 3 h after UV exposure, demonstrating that there was a response to the stimulus. Samples from the AG06917/AG06299 pairing demonstrated a maximal 6-fold increase in p53 levels while the AG08466/ AG08470 indicated a 19- and 11-fold increase, respectively. The highest increase was with AG03199/ AG03257, which showed an approximate 35 /45-fold increase. This heterogeneity can be attributed, at least in part, to variation between lines in the initial p53 levels prior to irradiation. The non-irradiated samples indicate the basal level of p53 in the cells prior to damage. Although those levels were low for each cell line, they did vary from sample to sample. We can assume that such low levels of p53 are a result of normal regulation of p53 by Mdm2. When comparing each progeria sample with its unaffected control there was little difference in the overall p53 response. AG08470 and AG08466 had a similar response pattern reaching highest levels of p53 24 h after irradiation. AG06917 and its control AG06299 also had similar p53 responses, but reached a maximum increase within 6 h. The p53 levels then fell. AG03257 and AG03199 also showed a similar overall increase in p53 levels. The p53 response in AG03199 would appear to be 10 times stronger than that of its control. However, this is due to the very low p53 level detected in the progeria sample prior to irradiation. We conclude that there is no difference in the strength and kinetics of the p53 response to UV between progeria and control cell lines. 3.2. p53 response to ionising radiation We also examined levels of p53 after treatment with 25 Gy of IR. Fig. 2 shows a western blot of AG08466 and AG08470. There was an increase in p53 levels in response to IR but there was no noticeable difference between the progeria cell line and its control. Both showed a 5/6-fold increase in p53 levels after 2 h, which started falling by 18 h. 3.3. p21WAF1 response to UV The progeria fibroblasts showed a similar p53 response to UV and IR when compared to control fibroblasts. Stabilisation of p53 in the cell allows for activation of other genes involved in cell cycle arrest and
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apoptosis. One such p53 effector, p21WAF1, is transcriptionally activated by p53 upon exposure to damage such as UV and IR and instigates cell cycle arrest in G1/S by inhibiting cyclin dependent kinase and ultimately resulting in the dephosphorylation of Rb (Wynford-Thomas, 1999). We therefore investigated levels of p21WAF1 after UV irradiation in progeria and controls. Fig. 3 shows the western data for AG08466 and AG08470. Both showed an increase in levels of p21WAF1 6 /12 h after UV exposure although the response was greater in progeria cells (17-fold increase compared to 5-fold in the control). Consistent with the p53 response levels, p21WAF1 remained high 24 h after irradiation. Such an increase in p21WAF1 levels could perhaps be enough to stimulate the onset of senescence in progeria. However, a similar difference in the p21WAF1 response was not seen when the other two progeria lines and their controls were compared. Thus, we have no evidence that the p21WAF1 response to UV is altered in progeria cell lines compared to their controls.
Fig. 3. Western blot of p21 on HGP (AG08466) and control (AG08470) fibroblasts after exposure to UV. Fibroblasts were exposed to UVC (30 J m 2) as described in Fig. 1. Probed with p21WAF1 and then with actin. The p53: actin values were calculated in the same way as Fig. 1.
3.4. p53 coding sequence in progeria cell lines The sequence of p53 from all three progeria fibroblast cultures was determined via rtPCR and compared with the sequence of an unaffected family member. No alterations were identified within the coding sequence other than the change at the codon for proline at residue 72. This is a common polymorphism resulting in the substitution of arginine 72 (Matlashewski et al., 1987) and does not affect the p53 response to UV. Two of the progeria cultures (AG06917 and AG08466) and one control (AG06299) were heterozygous for this polymorphism and the remaining samples were homozygous for the allele coding for arginine.
4. Discussion The concept of a balance between cellular ageing and cancer susceptibility maintained by levels of p53 has its attractions. However mounting evidence would suggest that it is not as simple as it would first appear. Mice possessing a truncated form of p53 alongside a fully functional p53 demonstrated a resistance to tumour development and premature ageing (Tyner et al., 2002). However, mice that had a p53 transgene as well as two functional copies of p53 showed increased tumour resistance but no premature ageing (Garcia-Cao et al., 2002). The authors attributed this finding to the fact that p53 is regulated normally in these transgenic mice and is able to induce high levels of p53 when subjected to assaults such as IR, which would then lead to cancer. They argued that the mice of Tyner et al. (2002) probably demonstrated constitutive expression of p53 and it was the continual presence of high levels of p53 that might trigger the early onset of ageing (Garcia-Cao et al., 2002). Our data on this rare human premature ageing disorder suggested that the drastic progeria phenotype did not result from an elevated p53 response to DNA damage. Basal levels of p53 remained low in progeria lines implying that regulation of p53 is normal. There were certainly increased levels of p53 on exposure to UV and IR, but there was no unusual response in any of the progeria fibroblasts studied compared to their familial controls. The p21WAF1 response to UV was elevated in one of the samples (AG08466) compared to its control, but there was no evidence of a similar elevated response in the other progeria samples. Therefore we conclude that it was unlikely that high levels of p21WAF1 were triggering a senescent response in the progeria cell lines. It is also too simplistic to extrapolate data from fibroblasts and apply them to the patient, but there are currently no data relating to the levels of p53 or p21WAF1 in tissues of progeria patients. Certainly levels of p53 can vary greatly within mouse tissues post-
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irradiation implying that p53 is regulated in a tissue specific manner (Midgley et al., 1995). Therefore it remains possible that an enhanced p53 response may occur in only certain tissues of progeria patients. We conclude that it is extremely unlikely that these cell cycle proteins are directly causative of the progeria phenotype; although they will inevitably be involved in the senescent phenotype. DNA micro array data has thrown up several other lines of enquiry and it was suggested by Ly et al. (2000) that likely candidates for the genes causing ageing were perhaps those associated with mitosis. In both elderly patients and in patients with progeria there is a decrease in levels of gene expression associated with the mitotic regulation. The specific mutation causing HGP is unknown but identifying the nature of the response it triggers may bring us closer to understanding the normal human ageing condition.
Acknowledgements This work was supported by a programme grant (SP2095/0301) from Cancer Research UK to DWM. Sequencing of the p53 coding region was funded by Research into Ageing.
References Allsopp, R.C., Vazirir, H., Patterson, C., Goldstein, S., Younglai, E.G., Futcher, A.B., Greider, C.W., Harley, C.B., 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 10114 /10118. Bentley, D.J., Harrison, C., Ketchen, A., Redhead, N.J., Samuel, K., Waterfall, M., Ansell, J.D., Melton, D.W., 2002. DNA ligase I null
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mouse cells show normal DNA repair activity but altered DNA replication and reduced genome stability. J. Cell Sci. 115, 1551 / 1561. Ferbeyre, G., Lowe, S.W., 2002. The price of tumor suppression. Nature 415, 26 /27. Garcia-Cao, I., Garcia-Cao, M., Martin-Caballero, M., Criado, L.M., Klatt, P., Flores, J.M., Veill, J.C., Blasco, M.A., Serrano, M., 2002. ‘Super p53’ mice exhibit enhanced DNA damage response, are tumour resistant and age normally. EMBO J. 21, 6225 /6235. Jansen, T., Romiti, R., 2000. Progeria Infantum (Hutchinson-Gilford Syndrome) associated with scleroderma-like lesions and acroosteolytis: a case report and brief review of the literature. Pediatr. Dermatol. 17, 282 /285. Ly, D.H., Lockhart, D.J., Lerner, R.A., Schultz, P.G., 2000. Mitotic misregulation and human aging. Science 287, 2486 /2492. Matlashewski, G.J., Tuck, S., Pim, D., Lamb, P., Scheider, J., Crawford, L.V., 1987. Primary structure polymorphism at amino acid residue 72 of human p53. Mol. Cell Biol. 7, 961 /963. Midgley, C.A., Owens, B., Briscoe, C.V., Thomas, D.B., Lane, D.P., Hall, P.A., 1995. Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo. J. Cell Sci. 108, 1843 /1848. Steele, R.J.C., Thompson, A.M., Hall, P.A., Lane, D.P., 1998. The p53 tumour suppressor gene. Br. J. Surg. 85, 1460 /1467. Sugrue, M.M., Shin, D.Y., Lee, S.W., Aaronson, S.A., 1997. Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53. Proc. Natl. Acad. Sci. USA 94, 9648 /9653. Tyner, S.D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H., Lu, X., Soron, G., Cooper, B., Brayton, C., Park, S.H., Thompson, T., Karsenty, G., Bradley, A., Donehower, L.A., 2002. p53 mutant mice that display early ageing-associated phenotypes. Nature 415, 45 /53. Wang, S., Nishigori, C., Yagi, T., Takebe, H., 1991. Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells. Mutat. Res. 256, 59 /66. Webley, K., Bond, J.A., Jones, C.J., Blaydes, J.P., Craig, A., Hupp, T., Wynford-Thomas, D., 2000. Posttranslational modifications of p53 in replicative senescence overlapping but distinct from those induced by DNA damage. Mol. Cell Biol. 20 (8), 2803 /2808. Wynford-Thomas, D., 1999. Cellular senescence and cancer. J. Pathol. 187, 100 /111.
p53 and a human premature ageing disorder Mary O’Neill a,*, Fatima Nu´n˜ez b, David W. Melton a a
Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK b Centro de Investigac¸ion del Cancer, Campus Unamuno, 37007 Salamanca, Spain Received 17 April 2002; received in revised form 19 December 2002; accepted 30 January 2003
Abstract It has been shown that enhanced levels of p53 activity contribute to reduced cancer susceptibility in mice, however longevity is compromised due to the onset of an early-ageing phenotype. The effects of enhanced levels of p53 in these in mice could therefore have implications for human premature ageing disorders. We examined the DNA damage response of p53 and its target p21WAF1 to UV and ionising radiation in fibroblasts from patients with the premature ageing disorder Hutchinson-Gilford Progeria (HGP). We report a normal p53 response to these DNA damaging agents suggesting that, in this particular human disorder, the premature ageing phenotype does not arise from an enhanced p53 response. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Progeria; p53; DNA damage; p21WAF1; Senescence
1. Introduction The p53 tumour suppressor protein plays a vital role in maintaining the control of the cell cycle. A defective p53 protein leads to increased cancer susceptibility while higher than normal levels of the protein contribute to replicative senescence (Sugrue et al., 1997; Webley et al., 2000). It has been demonstrated that enhanced levels of p53 in p53 gene-targeted mice resulted in animals with a reduced susceptibility to cancer but with symptoms of premature ageing (Tyner et al., 2002). Mice with a wildtype copy of p53 and a copy of an N-terminal deleted form had decreased longevity and pathological symptoms similar to ageing. The authors concluded that the enhanced levels of p53 in mice led to a decrease in proliferative capacity of the stem cells. This provoked the speculation that higher than normal levels of p53 might contribute to the pathology of human premature ageing disorders (Ferbeyre and Lowe, 2002). HGP is a very rare premature ageing disorder; symptoms include growth retardation, absence of sub-
* Corresponding author. Tel.: /44-131-651-1077; fax: /44-131651-1072. E-mail address: [email protected] (M. O’Neill).
cutaneous fat, arteriosclerosis, osteoarthritis, skeletal abnormalities and aged appearance. Life expectancy is short ( :/13 yr) with death invariably from cardiac failure (Jansen and Romiti, 2000). There is no evidence of increased susceptibility to cancer, however given the rarity of the syndrome and the low life expectancy this may not be significant. Fibroblasts from these patients show a decreased rate of DNA synthesis after UV irradiation suggesting an impaired capacity to repair DNA (Wang et al., 1991). It is believed to be due to an autosomal dominant mutation that spontaneously arises in the germ line and telomeres are shorter than age matched controls (Allsopp et al., 1992). HGP fibroblasts undergo fewer cell doublings and the rate of doubling is extremely slow (1 division per week). Patterns of gene expression in HGP are consistent with that of old age, with genes involved in cell division and DNA or RNA synthesis being down-regulated in these two situations (Ly et al., 2000). As cells undergo senescence or are subjected to UV or ionising radiation (IR), p53 becomes post-translationally modified at specific residues. These modifications render p53 resistant to degradation by Mdm2, therefore levels of p53 in the cell increase (Steele et al., 1998). Different assaults instigate modification at different residues, with UV and cellular senescence showing similar modification
0047-6374/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0047-6374(03)00007-1
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patterns that are distinct from those caused by IR (Webley et al., 2000). Here we investigate the hypothesis that an enhanced p53 response to DNA damaging agents, leading to the activation of downstream pathways that might regulate cell cycle arrest and entry into senescence, is the basis for the premature ageing disorder Hutchinson-Gilford Progeria.
2. Materials and methods Human fibroblasts from progeria patients (AG03199, AG06917 and AG08466) and their unaffected family members (AG03257, AG06299 and AG08470) were obtained from the NIA Aging Cell Repository (Coriell Cell Repository, Camden, NJ). Fibroblasts were cultured in Glasgow modified Eagle’s Medium, supplemented with 20% fetal calf serum. Fibroblasts cultures that were 50% confluent in 60 mm dishes were exposed to 30 J m 2 UVC light, returned to the incubator and harvested at the time intervals indicated (Fig. 1). Similarly for treatment with IR, cells were exposed to 25 Gy in a Torrex Faxitron X-ray and then returned to
the incubator and harvested at the time intervals indicated (Fig. 2). Total cell protein was isolated from fibroblasts that were first washed in 1 / PBS then resuspended in a 50 ml volume of lysis buffer (125 mM Tris pH 7, 2 mM EDTA, 2% SDS, 33 mM DTT, 2 mM PMSF) and boiled for 5 min before centrifuging to remove cell debris. Cell lysates were stored at /20 8C. Equal amounts (80 mg) of protein were loaded on a SDS PAGE gel and run, with cooling at 50 mA. Protein was transferred to a PVDF membrane using a Bio-Rad Transblot semi-dry transfer system. After blocking in 5% 1 / TBST (50 mM Tris (pH 7.5) 150 mM NaCl and Tween 0.1%) the membranes were hybridised to the primary antibody for a minimum of 1 h. Antibodies: p53 (Calbiochem) was used at a dilution of 1/2500 and incubated for 1 h at room temperature; p21WAF1 (Calbiochem) was used at a dilution of 1/66 and incubated at 4 8C overnight. After washing, the membranes were incubated with the HRP tagged secondary antibody and finally detected with ECL Plus (Amersham). Intensity of signal was determined by scanning densitometry using ImageJ.
Fig. 1. Western blot analyses of p53 after UV irradiation of HGP fibroblasts. (A) HGP (AG08466) and unaffected control (AG08470), (B) HGP (AG06917) and unaffected control (AG06299), (C) HGP (AG03199)* and unaffected control (AG03257). Fibroblasts were irradiated with UVC 30 J m 2 and samples collected at time intervals indicated. nr, Non-irradiated cells. After initial reaction with p53, filters were stripped and reprobed for actin. The values below each lane indicate p53 response levels, obtained by densitometry, after normalization for actin, expressed relative to the nonirradiated culture. The doublet of p53 in samples AG08466, AG06917 and AG06299 is due to the polymorphism at amino acid 72 (Matlashewski et al., 1987). Arg72 moves faster than Pro72. *The basal level of p53 in AG03199 was extremely low and so the p53 actin ratios for this cell line are expressed relative to the non-irradiated sample for the control (AG03257).
M. O’Neill et al. / Mechanisms of Ageing and Development 124 (2003) 599 /603
Fig. 2. Analysis of p53 response after exposure of HGP (AG08466) and control (AG08470) fibroblasts to IR. Fibroblasts were exposed to 25 Gy of IR and treated as in Fig. 1. Filters were probed first with p53 then stripped and reprobed for actin. The p53: actin values were calculated in the same way as Fig. 1.
Total RNA was prepared from all three progeria cell lines and familial controls using RNA-BeeTM (Biogenesis, Poole, UK). The p53 coding region was amplified by rtPCR as described in Bentley et al. (2002) and the sequence determined using the PCR primers.
3. Results 3.1. p53 response to UVC We first of all examined levels of p53 in fibroblasts from three unrelated HGP patients and unaffected family members after irradiation with UV (30 J m 2). Total protein was isolated from non-irradiated cells and 3, 6, 24 and 48 h after irradiation; Fig. 1(A /C) shows western blots for the three HGP cell line and their controls.
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Contrary to the situation in the p53 gene-targeted mice (Tyner et al., 2002), where expression of the expected N-terminal truncated protein from the targeted allele could not be detected, the existence of a p53 protein polymorphism in two out of the three HGP cell lines and one of the controls, demonstrated that both p53 alleles in the HGP and the control samples produced normal amounts of p53 protein. The levels of p53 protein increased in all the HGP and control samples 3 h after UV exposure, demonstrating that there was a response to the stimulus. Samples from the AG06917/AG06299 pairing demonstrated a maximal 6-fold increase in p53 levels while the AG08466/ AG08470 indicated a 19- and 11-fold increase, respectively. The highest increase was with AG03199/ AG03257, which showed an approximate 35 /45-fold increase. This heterogeneity can be attributed, at least in part, to variation between lines in the initial p53 levels prior to irradiation. The non-irradiated samples indicate the basal level of p53 in the cells prior to damage. Although those levels were low for each cell line, they did vary from sample to sample. We can assume that such low levels of p53 are a result of normal regulation of p53 by Mdm2. When comparing each progeria sample with its unaffected control there was little difference in the overall p53 response. AG08470 and AG08466 had a similar response pattern reaching highest levels of p53 24 h after irradiation. AG06917 and its control AG06299 also had similar p53 responses, but reached a maximum increase within 6 h. The p53 levels then fell. AG03257 and AG03199 also showed a similar overall increase in p53 levels. The p53 response in AG03199 would appear to be 10 times stronger than that of its control. However, this is due to the very low p53 level detected in the progeria sample prior to irradiation. We conclude that there is no difference in the strength and kinetics of the p53 response to UV between progeria and control cell lines. 3.2. p53 response to ionising radiation We also examined levels of p53 after treatment with 25 Gy of IR. Fig. 2 shows a western blot of AG08466 and AG08470. There was an increase in p53 levels in response to IR but there was no noticeable difference between the progeria cell line and its control. Both showed a 5/6-fold increase in p53 levels after 2 h, which started falling by 18 h. 3.3. p21WAF1 response to UV The progeria fibroblasts showed a similar p53 response to UV and IR when compared to control fibroblasts. Stabilisation of p53 in the cell allows for activation of other genes involved in cell cycle arrest and
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apoptosis. One such p53 effector, p21WAF1, is transcriptionally activated by p53 upon exposure to damage such as UV and IR and instigates cell cycle arrest in G1/S by inhibiting cyclin dependent kinase and ultimately resulting in the dephosphorylation of Rb (Wynford-Thomas, 1999). We therefore investigated levels of p21WAF1 after UV irradiation in progeria and controls. Fig. 3 shows the western data for AG08466 and AG08470. Both showed an increase in levels of p21WAF1 6 /12 h after UV exposure although the response was greater in progeria cells (17-fold increase compared to 5-fold in the control). Consistent with the p53 response levels, p21WAF1 remained high 24 h after irradiation. Such an increase in p21WAF1 levels could perhaps be enough to stimulate the onset of senescence in progeria. However, a similar difference in the p21WAF1 response was not seen when the other two progeria lines and their controls were compared. Thus, we have no evidence that the p21WAF1 response to UV is altered in progeria cell lines compared to their controls.
Fig. 3. Western blot of p21 on HGP (AG08466) and control (AG08470) fibroblasts after exposure to UV. Fibroblasts were exposed to UVC (30 J m 2) as described in Fig. 1. Probed with p21WAF1 and then with actin. The p53: actin values were calculated in the same way as Fig. 1.
3.4. p53 coding sequence in progeria cell lines The sequence of p53 from all three progeria fibroblast cultures was determined via rtPCR and compared with the sequence of an unaffected family member. No alterations were identified within the coding sequence other than the change at the codon for proline at residue 72. This is a common polymorphism resulting in the substitution of arginine 72 (Matlashewski et al., 1987) and does not affect the p53 response to UV. Two of the progeria cultures (AG06917 and AG08466) and one control (AG06299) were heterozygous for this polymorphism and the remaining samples were homozygous for the allele coding for arginine.
4. Discussion The concept of a balance between cellular ageing and cancer susceptibility maintained by levels of p53 has its attractions. However mounting evidence would suggest that it is not as simple as it would first appear. Mice possessing a truncated form of p53 alongside a fully functional p53 demonstrated a resistance to tumour development and premature ageing (Tyner et al., 2002). However, mice that had a p53 transgene as well as two functional copies of p53 showed increased tumour resistance but no premature ageing (Garcia-Cao et al., 2002). The authors attributed this finding to the fact that p53 is regulated normally in these transgenic mice and is able to induce high levels of p53 when subjected to assaults such as IR, which would then lead to cancer. They argued that the mice of Tyner et al. (2002) probably demonstrated constitutive expression of p53 and it was the continual presence of high levels of p53 that might trigger the early onset of ageing (Garcia-Cao et al., 2002). Our data on this rare human premature ageing disorder suggested that the drastic progeria phenotype did not result from an elevated p53 response to DNA damage. Basal levels of p53 remained low in progeria lines implying that regulation of p53 is normal. There were certainly increased levels of p53 on exposure to UV and IR, but there was no unusual response in any of the progeria fibroblasts studied compared to their familial controls. The p21WAF1 response to UV was elevated in one of the samples (AG08466) compared to its control, but there was no evidence of a similar elevated response in the other progeria samples. Therefore we conclude that it was unlikely that high levels of p21WAF1 were triggering a senescent response in the progeria cell lines. It is also too simplistic to extrapolate data from fibroblasts and apply them to the patient, but there are currently no data relating to the levels of p53 or p21WAF1 in tissues of progeria patients. Certainly levels of p53 can vary greatly within mouse tissues post-
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irradiation implying that p53 is regulated in a tissue specific manner (Midgley et al., 1995). Therefore it remains possible that an enhanced p53 response may occur in only certain tissues of progeria patients. We conclude that it is extremely unlikely that these cell cycle proteins are directly causative of the progeria phenotype; although they will inevitably be involved in the senescent phenotype. DNA micro array data has thrown up several other lines of enquiry and it was suggested by Ly et al. (2000) that likely candidates for the genes causing ageing were perhaps those associated with mitosis. In both elderly patients and in patients with progeria there is a decrease in levels of gene expression associated with the mitotic regulation. The specific mutation causing HGP is unknown but identifying the nature of the response it triggers may bring us closer to understanding the normal human ageing condition.
Acknowledgements This work was supported by a programme grant (SP2095/0301) from Cancer Research UK to DWM. Sequencing of the p53 coding region was funded by Research into Ageing.
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