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The Werner syndrome protein contributes to induction of p53 by DNA damage¹

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel; Veterans Affairs Medical Center, Seattle, Washington 98108, USA; and
^* Departments of Medicine, Neurology and Pharmacology, University of Washington, Seattle, Washington 98195, USA

²Correspondence: E-mail: moshe.oren{at}weizmann.ac.il

SPECIFIC AIMS

We set out to investigate the effects of the Werner syndrome protein (WRN) on p53 and to assess whether WRN contributes to the activation of p53 in response to certain types of DNA damage.

INTRODUCTION

The p53 tumor suppressor is mutated in more than half of all human cancers. These mutations, which compromise the effective response to various stress signals, result in elevated genomic instability and contribute to cancer progression. The p53 protein is usually latent in nonstressed cells, but is activated upon cellular exposure to various conditions of stress, including several types of DNA damage. Activation typically entails changes in the biochemical potency of p53 as well as a marked increase in its overall steady state levels. Upon activation, p53 triggers a biological response that eventually results in resolution of the genomic damage or elimination of cells carrying permanent damage.

Augmented genomic instability and increased cancer risk are also encountered in patients of Werner syndrome (WS), an autosomal recessive disorder associated with various features that mimic aspects of premature aging. WS arises through mutations in the WRN gene, encoding a protein with DNA helicase and exonuclease activities. Recently, p53 and WRN were found capable of engaging in direct protein–protein interactions. Moreover, excess WRN can enhance the transcriptional activity of p53, whereas functional WRN is required for efficient induction of apoptosis by excess p53. These observations raise the possibility that WRN and p53 may participate jointly in a common pathway aimed at preventing the accumulation of cells with defective genomes.

To assess the physiological relevance of the interaction between p53 and WRN, we investigated the contribution of WRN to the induction of p53 by DNA damage. We report here that fibroblasts derived from WS patients exhibit a delayed and attenuated accumulation of p53 after exposure to UV irradiation. In addition, we show that excess WRN can promote the accumulation of p53. These findings support a role for WRN in the signaling events that lead to p53 activation in response to certain types of DNA damage. The failure to induce p53 efficiently may account, in part, for the enhanced genomic instability and elevated cancer risk in WS patients.

PRINCIPAL FINDINGS

1. Overexpression of WRN induces accumulation of p53
Many signals that impinge on the p53 pathway modulate the steady-state cellular concentration of this protein. We therefore asked whether WRN overexpression could affect p53 protein levels. p53-null H1299 cells were transiently transfected with a p53 expression plasmid, either alone or together with a WRN expression plasmid. As seen in Fig. 1A , cotransfection with WRN increased the steady-state levels of p53 in a dose-dependent manner. This increase was not due to a change in the levels of p53 mRNA (Fig. 1B ), implying a translational or post-translational mechanism.

View larger version (35K): [in this window] [in a new window]   Figure 1. Overexpression of WRN increases p53 protein but not p53 mRNA. H1299 cells (5x105 per 6 cm dish) were transfected with the indicated plasmid combinations. The total amount of DNA in each transfection was kept constant by addition of pcFLAG vector control DNA. Cell extracts were prepared 26 h later and subjected to SDS-PAGE and immunoblotting with a mixture of the p53-specific monoclonal antibodies PAb1801 and DO-1. The same membrane was reprobed with a monoclonal antibody specific for -tubulin. B) H1299 cells were transfected with 30 ng of human p53 expression plasmid, either alone or together with 5 µg WRN expression plasmid. Parallel control cultures were transfected with empty vector. Total RNA was extracted from each culture 26 h after transfection. 15 µg of each RNA was subjected to Northern blot analysis with a human p53 cDNA probe. The same blot was subsequently reprobed for GAPDH to normalize for loading variations.

2. WRN contributes to efficient induction of p53 in response to DNA damage
WRN is a member of the RecQ family of DNA helicases, several members of which have been implicated in the response to UV damage and in UV sensitivity. To find out whether WRN plays a role in the regulation of p53 under more physiological conditions, we monitored the induction of p53 by UV in several nonimmortal fibroblastic cultures derived from either control donors or individuals diagnosed as Werner syndrome (WS) patients. UV exposure elicited a pronounced increase in the steady-state levels of p53 the control cultures, GM00380 and AG05247E (Fig. 2A ). In contrast, a much milder increase was evident in the WS patient-derived AG12795 cells. AG05247E originated in an 87-year-old healthy donor, whereas AG12795 came from a 19-year-old WS patient; hence, the impaired p53 induction is not secondary to the senescence-prone phenotype of WS cells. Moreover, all three cultures exhibited similar levels of senescence-associated ß-galactosidase activity (Fig. 2A ).

View larger version (35K): [in this window] [in a new window]   Figure 2. p53 accumulation after UV radiation is attenuated in Werner syndrome fibroblasts. A) Western blot analysis of p53 protein after exposure to 40 J/m2 UV radiation. Cells were harvested at the indicated time points after irradiation and protein extracts were subjected to Western blot analysis as in Fig. 1A . At time 0, a parallel culture of each cell strain was stained for senescence-associated ß-galactosidase activity (SA ß-gal); the percentage of positive cells in each culture is indicated below the appropriate cell strain. AG12795 are nonimmortalized fibroblasts obtained from a WS patient and analyzed after 20 population doubling (PDL). GM00380 and AG05247E are nonimmortalized fibroblasts from donors with normal WRN genes, analyzed after 18 and 24 PDL, respectively. B) Kinetics of p53 accumulation in fibroblasts from control donors and from individuals diagnosed with WS. p53 signals in Western blots were quantified by densitometric scanning; values were normalized for the intensity of the corresponding -tubulin signal. The corrected value obtained for a nonirradiated culture of each strain was taken as 1, and the relative p53 level at each subsequent time point was calculated accordingly. Cultures from patients diagnosed as WS are indicated by empty symbols and dashed lines; filled symbols and full lines relate to healthy controls. GM00380, AG05247E, and AG12795 are as in panel A. AG00780, AG03141B, AG04110A, AG06300A, AG03057, and GM08402 were analyzed at 20, 12, 20, 34, 30, and 11 PDL, respectively.

A quantitative analysis of the kinetics of p53 induction in cultures from five individuals diagnosed as WS patients and four controls revealed that whereas there was some heterogeneity in the extent of p53 induction among control cultures, all WS cells were severely impaired with regard to induction of p53 by UV (Fig. 2B ). In some of the WS cultures, these differences became far less pronounced 24 h after exposure to UV (data not shown). Hence, the induction of p53 in WS patients’ cells is severely attenuated and delayed.

Two of the cultures used in our study (AG4110A and AG06300A) were recently reported to express apparently wild-type WRN protein. The corresponding patients may thus represent other disorders with clinical features reminiscent of WS. In fact, although the official diagnosis of the donor of AG06300A was WS, the donor of AG4110A was diagnosed as suffering from severe WS or possibly mild progeria. We therefore compared WRN expression in AG06300A and AG4110A relative to AG00780, derived from a bona fide WS patient, and to cultures from control donors. As expected, WRN protein was undetectable in the AG00780 cells (not shown). On the other hand, AG04110A fibroblasts expressed levels of WRN comparable to those seen in the controls, GM00380 and AG05247E, suggesting that the donor may have suffered from mild progeria rather than WS. A rather different picture was revealed with AG06300. Though a protein migrating as wild-type WRN was indeed detectable, its levels were significantly lower than in control cells (not shown). Such reduced expression, whose cause remains unknown, may conceivably give rise to clinical features of WS and to an attenuated p53 response.

CONCLUSIONS AND SIGNIFICANCE

The data presented here demonstrate that overexpression of WRN results in accumulation of p53 protein. WS cells are compromised with regard to induction of p53 by DNA damage. These findings are consistent with a model where WRN participates in activation of p53 by at least certain types of genotoxic stress. Although the exact functions of WRN within the cell remain to be better elucidated, its biochemical features imply an involvement in one or more types of DNA transactions. These may include DNA replication, recombination, transcription, or some sort of DNA quality control. Of note, WRN-related members of the RecQ family of helicases have been implicated in the suppression of illegitimate recombination in UV-treated cells, as well as the coupling of cell cycle checkpoints to DNA damage.

Our findings suggest that WRN may help maintain genomic integrity by facilitating p53 activation in response to particular types of DNA damage. Such facilitation may be mediated through direct physical association between the two proteins (Fig. 3 ). Presumably, WRN-dependent activation of the p53 response may prevent the proliferation of cells with damaged genomes and the propagation of the damage (Fig. 3) . Furthermore, p53 may contribute to DNA repair processes, either directly or through transcriptional activation of appropriate target genes (Fig. 3) .

View larger version (33K): [in this window] [in a new window]   Figure 3. Model for the putative involvement of WRN in the activation of p53 by DNA damage. WRN may relay DNA damage signals to p53 either by direct interaction with p53 or through interaction with other regulators of p53, such as ATM or Chk1 and Chk2. This leads to p53 activation, enhanced transcription of target genes such as Bax, p21, p53R2, and eventual phenotypic outcomes.

A remarkable similarity exists between WRN and the ATM protein, mutated in ataxia telangiectasia (AT). ATM activity is triggered in response to DNA strand breaks and its dysfunction confers enhanced sensitivity to ionizing radiation. Moreover, ATM couples DNA damage with multiple cell cycle checkpoints. Like WS cells, AT cells also exhibit attenuated p53 induction. Sgs1p, a yeast homologue of WRN, acts upstream of and may be engaged in interactions with Rad53p, the yeast homologue of the DNA damage checkpoint kinase Chk2/Cds1. Chk2, along with the related Chk1, is regulated by the yeast ATM homologue Mec1/Rad3. This raises the intriguing possibility that WRN may contribute to p53 induction through direct or indirect interactions with ATM, the ATM-related ATR kinase, or the ATM-regulated kinases Chk1 and Chk2, all of which play important roles in the activation of p53 in response to various types of stress (Fig. 3) .

In the absence of WRN, it is conceivable that the failure to orchestrate an effective p53 response may result in enhanced accumulation of genomic damage. This is likely to underlie, at least in part, the elevated cancer risk of WS patients. Moreover, it may play a role in enabling a faster functional decay of critical systems within the body, thereby possibly contributing to features characteristic of premature aging.

We observed a failure to induce p53 properly also in cells of patients exhibiting WS-like features while maintaining the expression of wild-type WRN protein. Hence, defects in the activation of p53 after exposure to UV may be caused by deficiencies in WRN itself as well as in proteins whose dysfunction gives rise to other disorders with WS-like features. This may indicate the existence of a signaling pathway involving WS along with one or more other proteins, whose functionality contributes to the maintenance of genomic stability. Defects in various components of this pathway may give rise to a failure to activate p53 in response to DNA damage (Fig. 3) , as well as to features of premature aging. It is tempting to speculate that these two latter outcomes are causally related.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0171fje To cite this article, use (September 8, 2000) FASEB J. 10.1096/fj.00-0171fje

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