The ARF/p53 pathway

Soon after the realisation that the CDKN2A locus had the capacity to encode an alternative product, ARF, it was demonstrated that ectopic expression of ARF would induce a Gj/Gj phase cell cycle arrest that was dependent on the status of the p53 gene (Quelle et al., 1995; Stott et al., 1998; Zhang et al., 1998; Llanos et al., 2001) (Figure 1.7).

p53 is a DNA-binding transcription factor that is capable of inhibiting cell cycle progression or inducing apoptosis in response to DNA damage or stress (reviewed in Ko and Prives, 1996, Levine, 1997 and Ryan et at., 2001). p53 is also a well characterised tumour suppressor and its loss or mutation occurs in over 50% of human cancers (Hollstein et al., 1994). Numerous p53-dependent target genes have been identified that play a role as downstream effectors of p53 function (Vogelstein et al., 2000). Among those is the CKI p21^'^\ which is the major executor of the Gj cell cycle arrest (el-Deiry et al., 1993; el-Deiry et al., 1994; Xiong et al., 1993a). Another key target is MDM2 (murine double minute), which can both inhibit the transcriptional activity of p53 and target p53 for degradation (Barak et al., 1993; Wu et al., 1993; Haupt et al., 1997; Kubbutat et al., 1997). The MDM2 gene product binds directly to p53 and since the interaction takes place via the transcriptional activation domain of p53, it prevents p53 from acting as a transcription factor (Chen et al., 1995; Momand et al., 1992; Oliner et al., 1993). In addition, MDM2 can function as an E3 ubiquitin ligase, and therefore promotes the turn-over of p53 by targeting it for ubiquitination and proteasome-mediated degradation (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997). Moreover, due to the presence of a nuclear export signal, a feature that is required for MDM2-mediated p53 degradation, MDM2 continuously shuttles between the nucleus and cytoplasm (Freedman and Levine, 1998). Thus, MDM2 and p53 participate in a feedback loop that enables the levels of the two proteins to be tightly balanced (Wu et al., 1993) (Figure 1.7).

Chapter I - Introduction CDK4/CDK6 P Exon l a _{Exon 2} Exon 1p < S ) _MDM2 >►

Figure 1.7 Connection of pRb and p53 pathways through the lNK4a

locus.

The alternative exons and gene products encoded by the INK4a locus are shown. Whereas prevents the phosphorylation and inactivation of pRb by CDK4 and CDK6 , p i4 '^ ^ prevents the MDM2-mediated degradation of p53. The release of E2F by the phosphorylation or ablation of pRb leads to upregulation of ARF, providing a direct connection between the pRb and p53 pathways (adapted from James and Peters (1999)).

In normal cells, p53 has a short half-life and is expressed at very low levels but it is stabilised and accumulates when cells are exposed to genotoxic stress. Interestingly, the balance of p53 and MDM2 can be altered by ARF. Several studies have suggested that ARF physically interacts with MDM2, and in so doing prevents MDM2-mediated p53 degradation (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998; Zhang et al., 1998). However, exactly how ARF impinges on the p53-MDM2 feedback loop remains a matter of debate. For example, it has been proposed that ARF is able to form ternary complexes with both MDM2 and p53 (Zhang et al., 1998), and such complexes are indeed detectable in vitro and in transfected cells (reviewed in James and Peters, 2000). This has proven difficult to reconcile with the consistent observation that ARF is predominantly localised in the nucleolus (Stott et al., 1998; Quelle et al., 1995; Weber et al., 1999; Zhang and Xiong, 1999; Lindstrom et al., 2000; Rizos et al., 2000), whereas MDM2 and p53 are generally nucleoplasmic but shuttle in and out of the nucleus in order to deliver ubiquitinated p53 to the cytoplasmic proteasome (Roth et al., 1998; Freedman and Levine, 1998; Tao and Levine, 1999). Recently, Llanos and co­ workers have clarified these issues by proposing that nucleolar localisation is not essential for p i4 ^ function but may contribute to its availability to antagonise MDM2 (Llanos et al., 2001).

From these considerations, it is evident that ARF has the potential to act as a tumour suppressor. Ectopic expression of ARF results in the stabilisation of p53 by protecting it from MDM2-mediated destruction, leading to up-regulation of the p21^'^^ CDK-inhibitor and cell cycle arrest. As discussed in section 1.8.3, ARF “knockout” mice are indeed tumour prone and there is considerable body of data attesting to a pivotal role for ARF in monitoring oncogenic stress. Perhaps the most persuasive is the fact that ARF is transcriptionnally activated by E2F1. Thus, any agents, such as DNA tumour virus oncoproteins, that disrupt the function of pRb will inevitably induce ARF, resulting in p53 response. Not only does ARF act as a link between the pRb and p53 tumour suppressor pathways, but a number of activated oncogenes also cause up­ regulation of ARF, by as yet undefined mechanisms. Prominent examples are the Myc,

Chapter 1 - Introduction

Abl and Ras oncogenes (Zindy et aL, 1998; Brookes et al., 2002; Ferbeyre et al., 2000; Wei et al., 2001).

Curiously, while Ras activates ARF in mouse embryo fibroblasts, it does not appear to do so in HDFs (Brookes et al., 2002; Huot et al., 2002). As described in section 1.7 and discussed in more detail in subsequent chapters of this thesis, activated Ras also induces expression of plb^^"*^ in both species. It will therefore be interesting to determine whether species or cell type differences in the relative effects of Ras signaling on exon l a and exon ip promoters underlie the relative contributions of p l6iNK4a ARP to tumour suppression.