Commanding Cellular Fate

As a leader of the cell's proteome, p53 possesses transactivation abilities that can lead the cell down particular pathways including cell cycle arrest, cellular senescence, and apoptosis. The molecular mechanisms behind how p53 functions in each of these pathways are becoming clearer; how p53 chooses a particular pathway is not. Originally, it seemed to be cell type specific as y-irradiation of thymocytes induced p5 3-mediated apoptosis while in normal human fibroblasts it induced a p53-mediated G1 arrest (Clarke et al., 1993; Kuerbitz et al., 1992; Lowe et al., 1993). However, further findings suggested a more complicated scenario involving other factors. Evidence detailed below begins to paint a global picture of p53 function at a post-stabilization point whereby p53 is guided toward a particular pathway by covalent post-translational modifications, co-activators, and promoter selectivity. Though different types of genotoxic events trigger different responses of p53, the net result appears to be a more general guidance toward particular cell fates. Understanding the specifics of this aspect of p53 biology is of utmost interest, as it may yield new ideas for cancer therapy in tumors retaining wildtype p53.

Cell-cycle arrest mediated by p53 occurs largely through the induction of the CDK inhibitor p21Wafl, p21 halts cells in G1 of the cell cycle by inhibiting several cyclin dependent kinases and can also inhibit DNA replication through interactions with PCNA (Fotedar et al., 2004). p53 induces p21 after the occurrence of several types of genotoxic stress, including UV and ionizing irradiation, and may represent the first wave of cellular arsenal p53 utilizes to protect the cell. In addition to p21, GADD45 and 14-3-3a are other genes involved with inhibiting the cell cycle that are capable of being transactivated by p53 (Taylor and Stark, 2001).

Such a robust induction of p21 raises at least two possibilities of explanation. The first is that by evoking a cell cycle arrest, p53 buys time for the cell to assess damaged DNA, attempt DNA repair, transactivate other genes, and decide to revert back to the cell cycle or initiate apoptosis. Indeed, both BRCA1 and WT1 tumor suppressors can selectively coax p53 to induce cell cycle arrest and DNA repair genes (MacLachlan et al., 2002; Maheswaran et al., 1995). In addition, p53 itself has roles in nucleotide excision repair and base excision repair through the induction of p48┬░DB2, XPC, and MSH2 (Sengupta and Harris, 2005). p53 also enhances mismatch repair through associations with APE1/REF1 and DNA polymerase p. The decision of apoptosis isn't to be taken lightly, as this represents an irreversible and terminal effort the cell uses as a last option. By putting the cell in a cell cycle arrest, p53 gives the cell time to assess its options. Alternatively, p21 induction may just be the first wave of defense in a long list of options culminating in apoptosis if everything prior fails.

The first indication that p53 may possess some selectivity toward choosing a particular cell fate came from the analysis of a naturally occurring p53 tumor mutant, p53175P, which retained its ability to induce cell cycle arrest but lost its apoptotic function (Rowan et al., 1996). Though a majority of tumor derived mutations in p53 cause a loss of transactivation function, this mutant was shown to retain its ability to transactivate the cell cycle arrest promoting gene p21WAF1, but not the pro-apoptotic gene box. Others have shown similar phenotypes for an increasing number of these types of mutants (Friedlander et al., 1996; Ludwig et al., 1996; Ryan and Vousden, 1998; Smith et al., 1999). In this regard, p53 may possess the inherent ability, through yet undetermined mechanisms, to selectively choose subsets of genes involved in either cell cycle regulation or apoptosis.

Further evidence of covalent post-translational modifications on p53 driving particular responses supports the idea that p53 has selectivity for choosing a cell fate. Homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates Ser46 after UV irradiation and drives an apoptotic response (D'Orazi et al., 2002; Hofmann et al., 2002). HIPK2-mediated Ser46 phosphorylation, in conjunction with the p53DINPl gene product, is important for the induction of the pro-apoptotic gene p53AIPl (Oda et al., 2000). Ser20 was previously shown to be phosphorylated by the checkpoint kinases

Chkl and Chk2 in response to IR causing the abrogation of the p53-Mdm2 interaction (Chehab et al., 2000; Hirao et al., 2000; Shieh et al., 2000). More recently, however, it has been shown that Chk2 in not needed for p53-mediated cell cycle arrest but is required for an apoptotic response (Jack et al., 2002). The exact mechanism for this is currently unknown, but it does support the notion of p53 having selectivity in determining cell fate. Co-factors, such as JMY and the ASPP family of proteins, also enhance a p53-mediated apoptotic response (Samuels-Lev et al., 2001; Shikama et al., 1999). In the case of JMY, the result may be an indirect effect through interaction with p300 and alteration of p53 acetylation status. The p53 family members p63 and p73 are also required for p53-mediated induction of apoptotic genes, as DNA damaged-induced p53 failed to evoke apoptosis in cells deficient in p63 and p73 (Flores et al., 2002).

Another possibility is that p53 responsive elements exist in promoters throughout the genome that have inherently different binding affinities for p53. Binding and transactivaton could again depend on medications and co-factors of p53, or it could conceivably depend on quantitative levels of p53 (Fridman and Lowe, 2003). Engineered systems with conditional p53 expression do indeed show a shift toward Gl arrest when p53 levels are low and an apoptotic response when p53 levels are high. p53 protein levels are in a delicate flux at all times, governed by Mdm2, promoter binding affinity could be one mechanism of several that exists for p53-mediated cell fate selectivity.

A: Apoptosis

Once the apoptotic response is determined and initiated, p53 has been shown to be capable of inducing several apoptotic genes in both the so-called extrinsic and intrinsic apoptotic pathways (Haupt et al., 2003). The role of p53 in the extrinsic pathway includes transactivation of DR5 and Fas/CD95 that encode transmembrane proteins in the TNF-R family (Muller et al., 1998; Owen-Schaub et al., 1995; Wu et al., 1997). These proteins mediate apoptosis upon activation through the downstream activation of several caspases, though they appear to function in a tissue specific manner (Bouvard et al., 2000; Burns et al., 2001). PERP, another transmembrane protein, is induced by p53 after DNA damage and thought to play an as yet ill-defined role in the apoptotic response as well (Attardi et al., 2000). Similarly, PIDD is yet another gene containing a p53 response element that is induced upon shifting to the permissive temperature in a p53 conditional erythroleukemia cell line (Lin et al., 2000).

p53 has several genetic targets within the intrinsic apoptotic pathway, and they represent a majority of the genes mediating the p53-dependent apoptotic response. p53 transactivates several Bcl-2 pro-apoptotic family members including bax, puma, noxa, and bid (Nakano and Vousden, 2001; Oda et al., 2000; Sax et al., 2002). p53 can also induce the expression apaf-1, an essential component of the apoptotic effector machinery and co-activator of caspase 9 (Kannan et al., 2001; Moroni et al, 2001; Robles et al., 2001; Rozenfeld-Granot et al., 2002). Interestingly, p53-mediated induction of apaf-1 is required for Myc-induced apoptosis in mouse embryonic fibroblasts (Soengas et al., 1999). The activation of p53 and subsequent induction of apaf-1 may represent one failsafe mechanism the cell has evolved for inducing apoptosis in response to aberrant oncogenic signals.

Transcription independent mechanisms also exist for the induction of an apoptotic response by p53. In certain contexts or cell types, p53 has been shown to induce apoptosis independently of its transactivation ability. p53 can also directly interact with mitochondrial proteins and permeabilize the outer membrane releasing cytochrome c (Chipuk et al., 2004; Mihara et al., 2003). Further, p53 can directly activate both pro-apoptotic proteins Bax and Bak, with the latter by disruption of the Bak-Mcll complex (Leu et al., 2004). This shift in the balance of pro- and anti-apoptotic Bcl-2 family members may be enough to enhance the apoptotic response in a transcription-independent manner. Under certain conditions, p53 accumulation also induces a biphasic apoptotic response that is first transcriptionally independent followed by one that is transcriptionally dependent (Erster et al., 2004).

p53 also functions as a transrepressor of gene transcription, generally thought to occur through the formation of an mSin3a repressor complex that recruits histone deacetylases to the promoters (Zilfou et al., 2001). The genes to date shown to be repressed by p53 include bcl-2, bcl-X, and survivin, all of which are anti-apoptotic genes (Haldar et al., 1994; Hoffman et al., 2002; Mirza et al., 2002; Miyashita et al, 1994; Sugars et al., 2001). Repression of these genes may therefore be an additional mechanism for p53 to enhance apoptosis, as the net result of inhibition of anti-apoptotic genes is apoptosis.

Taken together, the data emerging suggest an important and centralized role of p53 in the apoptosis pathway that is highly, but not exclusively, dependent on its transactivation function. p53 has been described as a critical 'node' in the cellular circuitry, and as such, has many downstream effector circuits. It is no surprise then that there is no one critical downstream component that governs the apoptotic response; rather, p53 has its "hand" in several death circuit "cookie jars" as to increase the likelihood that apoptosis will occur, as so eloquently put by others (Fridman and Lowe, 2003).

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