Factores de la producción

The p53 gene was the fi rst tumor suppressor gene to be identifi ed and, since its discovery, scientists have found that the p53 pathway is altered in most human cancers. Two p53 homologs, p73 and p63, have also been identi-fi ed but mutations in cancer cells are rare. Its protein product, p53, is at the heart of the cell’s tumor suppressive mechanism and thus has been nicknamed the “guardian of the genome.” In the absence of cellular stress, low levels of p53 induce antioxidant activity which decreases the levels of reactive oxygen species (ROS) and subsequent DNA damage (Sablina et al., 2005). As mentioned in Chapter 2, normal cell metabolism produces ROS

PAUSE AND THINK

As a tumor suppressor protein, do you suppose RB inhibits or activates the transcription fac-tors needed for cell prolifera-tion? It inhibits the transcriptional activity of factors needed for cell cycle progression. Therefore, tar-get genes important for cell growth are not expressed. How-ever, loss of the tumor suppres-sor protein RB results in the loss of inhibition and, consequently, uncontrolled cell cycle progres-sion and diviprogres-sion. Think about the role of RB in differentiation.

Do you suppose it inhibits or acti-vates transcription factors that are responsible for turning on cell-type specifi c genes? As a tumor suppressor it stimulates the activity of transcription fac-tors, such as Myo D, that activate genes involved in differentiation.

Loss of RB leads to an increase in cell number and to the failure of differentiation.





that can react with DNA. It has been estimated that endogenous ROS modify approximately 20,000 bases of DNA per day in a single cell (Sablina et al. 2005). p53 protects against ROS by upregulating genes whose products have antioxidant functions, such as glutathione peroxidase 1 and sestrins—proteins involved in hydrogen peroxide metabolism. This antioxidant activity guards against mutation and may help prevent cancer.

Many types of “danger signals,” such as cell stress and DNA damage, can activate p53 and trigger several crucial cellular responses that suppress tumor formation (Figure 6.3). Upstream stress activators include radia-tion-, drug-, or carcinogen-induced DNA damage, oncogenic activation, hypoxia, and low ribonucleotide pools. These conditions may nurture tumor initiation. In response to these stress signals p53 can elicit down-stream cellular effects, including transient or permanent cell cycle arrest, DNA repair, apoptosis, and inhibition of angiogenesis (see section 9.7). The ability to cause the cell cycle to pause allows for the repair of mild DNA damage and prevents the propagation of mutations within the genome.

More severe DNA damage induces irreversible cell cycle arrest called senes-cence. Apoptosis is another means of preventing propagation of mutations;

cell suicide benefi ts the organism as a whole if DNA damage cannot be repaired. Mutated cells are better dead. Apoptosis is the critical biological function mediating the tumor suppressor function of p53. Under certain stress conditions p53 may play a pro-oxidant role that may contribute to the cellular effect of apoptosis. It should be mentioned that p53 may also play a role in regulating metabolism (discussed in Chapter 11).

DNA damage Aberrant growth signals Oncogene activation

Cell stress - Hypoxia - Nucleotide depletion

Upstream activators of p53

Downstream cell effects of p53

p53

Cell cycle arrest or senescence

Apoptosis

DNA repair Inhibition of angiogenesis

Figure 6.3 Upstream activators and downstream effects of p53.

Self test Close this book and try to redraw Figure 6.3. Check your answer. Correct your work. Close the book once more and try again.

The overall regulation of the p53 pathway possesses an extraordinary complexity that compels us to try to unravel each layer. Let us begin by examining the structure of the p53 protein and its interactions with its inhibitors, and then move on to dissecting how its activity is switched on and how it exerts its effects.

Structure of the p53 protein

The p53 gene, located on chromosome 17p13, contains 11 exons that encode a 53 kDa phosphoprotein. The p53 protein is a transcription factor containing four distinct domains: the amino-terminal transactiv-ation domain, the DNA-binding domain containing a Zn²⁺ ion, an oligomerization domain, and a carboxy-terminal regulatory domain (Figure 6.4). The p53 protein binds as a tetramer to a DNA response element containing two inverted repeats of the sequence 5′-PuPuPu C(A/T)-3′ (Pu symbolizes either purine base A or G) in order to regulate transcription of its target genes. Oligonucleotide array experiments have demonstrated that p53 binds to approximately 300 different gene promoter regions, thus suggesting that p53, has a powerful regulatory role. The p53 target genes code for proteins and microRNAs. Phospho-rylation patterns of p53, as well as interactions with binding partners, are correlated with distinct transcriptional programs. Several specifi c p53 target genes and the mechanism of how they exert their effect will be discussed later in the chapter.

Transactivation domain and MDM2 binding site

N – – C

Amino

acids 1 42 102

175

292 324 355 393

Mutational hotspots DNA binding domain

Oligomerization domain

Regulatory domain

248

245 249 282 273

Figure 6.4 Domains of the p53 protein and location of mutational hotspots (marked in red).

Regulation of p53 protein by MDM2

Normally, the level of p53 protein in a cell is low. The activity of p53 in a cell is regulated at the level of protein degradation, not at the level of expression of the p53 gene. The MDM2 protein, a ubiquitin ligase, is its main regulator. Ubiquitin ligases are enzymes that attach a small peptide called ubiquitin to proteins, fl agging it for proteolysis (enzymatic protein degradation involving cleavage of peptide bonds) in proteosomes.

MDM2 modifi es the carboxy-terminal domain of p53 and thus targets it for degradation by proteosomes in the cytoplasm. In addition, MDM2 modifi es the activity of p53 as it binds to and inhibits the p53 transacti-vation domain at the amino-terminal and transports the protein into the cytoplasm, away from nuclear DNA. Thus, the activity of p53 as a tran-scription factor is out of reach. The binding of MDM2 to p53 is part of an autoregulatory feedback loop (Figure 6.5, shown by red arrows) as the MDM2 gene is a transcriptional target of p53. Therefore, p53 stimu-lates the production of its negative regulator MDM2 that causes the deg-radation of p53. Small amounts of p53 will reduce the amount of MDM2 protein and this will result in an increase of p53 activity, thus completing the loop.

Upstream: molecular pathways of p53 activation

The mechanism by which p53 becomes activated depends on the nature of the stress signal. Stress is “sensed” by cellular proteins, many of which are kinases that convey the danger signals to p53 via phosphorylation.

Active p53

p53

p53

p53 MDM2

mdm2 Nucleus

Ubiquitin

p53 inactivation: Degradation Low MDM2

Low p53

Nuclear export

Inhibition of p53 transcriptional activity Figure 6.5 Regulation of p53 by

MDM2. See text for details.

Disruption of the p53–MDM2 interaction is fundamental to the activa-tion of p53 by its upstream factors.

The upstream activators of p53 utilize three main independent molec-ular pathways to signal cellmolec-ular distress (Figure 6.6). DNA damage caused by ionizing radiation is signaled by two protein kinases. The fi rst kinase, ATM, stimulated by DNA double-strand breaks, phosphorylates and activates a second kinase Chk2. Both ATM and Chk2 kinases phos-phorylate amino-terminal sites of p53 and this phosphorylation inter-feres with binding of MDM2. A second molecular pathway that signals cellular distress to p53 is executed by two different kinases, ATR and casein kinase II. These also phosphorylate p53 and disrupt its interaction with MDM2. Lastly, activated oncogenes, such as Ras, induce the activity of the protein p14arf, another modulator of the p53–MDM2 complex.

P14arf is one of two translational products of the INK4a/CDKN2A gene (p16, a cyclin kinase inhibitor, is the other product). P14arf does not bind to the interface of p53–MDM2, but functions by sequestering MDM2 to the nucleolus of the cell. All three pathways prevent degradation of p53 by MDM2.

Downstream: molecular mechanisms of p53 cellular effects

The main mechanism by which p53 exerts its tumor suppressing effects is by inducing the expression of specifi c target genes. Let us examine how the resulting network of proteins triggers these responses (Figure 6.7).

DNA damage

Oncogene

activation Cell stress

ATM p14arf ATR

ChK2 MDM2 Casein

kinase II Upstream factors

P P

Active P53

Figure 6.6 Upstream activators of p53. The fi rst and the last pathways involve kinases and result in the phosphorylation of p53 (shown as P). All pathways disrupt the interaction of p53 with MDM2.

Inhibition of the cell cycle

One of the central functions of p53 is to cause either transient cell cycle arrest or senescence in response to DNA damage so that there is either an opportunity to repair the damage prior to the next round of replication or a complete restraint of cell division, respectively; thus, damaged DNA will be prevented from being replicated and passed on to daughter cells and maintenance of the genome will be facilitated. The molecular mechanism responsible for this cellular response involves the transcriptional induc-tion of the p21 gene. Its product, the p21 protein, inhibits several cyclin–

cdk complexes and causes a pause in the G₁ to S (and G₂ to M) transition of the cell cycle (see Pause and Think).

In addition, p21 also binds PCNA (proliferating cell nuclear antigen), a protein that has a role in DNA synthesis and DNA repair. The interaction with p21 is such that it inhibits PCNA’s role in DNA replication but not in DNA repair. Therefore, p21 is an important part of the molecular mecha-nism that facilitates the ability of p53 to bring about a pause in the cell cycle and at the same time allow DNA repair. A microRNA regulated by p53 and miR-34a can also induce cell cycle arrest and senescence (not shown in Figure 6.7).

PAUSE AND THINK Why would an inhibitor of cyc-lin–cdk complexes cause a pause in the G₁–S transition? Recall the role of cyclin–cdk complexes in the cell cycle; importantly they act as kinases. As kinases they phosphorylate. What do they phosphorylate? RB. Failure of the cdk complex to phosphorylate RB prevents the release of the transcription factor E2F and blocks the transition into S phase.

Figure 6.7 Downstream effects of p53. p53 exerts many of its effects by regulating target genes as shown.

Apoptosis

The expression of several mediators of apoptosis is transcriptionally regu-lated directly by p53 (Table 6.2). The targets include genes that code for proteins involved in two apoptotic pathways that respond to external and internal signals, respectively. (Apoptosis will be described in Chapter 7.) In general, genes encoding proteins that promote apoptosis, pro-apop-totic proteins, are induced while genes encoding proteins that antagonize apoptosis, anti-apoptotic proteins, are repressed. The mitochondrial pro-apoptotic proteins NOXA, PUMA, and p53AIP1, that cause the release of cytochrome c and activate the apoptosome, are induced. Also, p53 tips the balance regulated by the Bcl-2 protein family towards apoptosis by inducing gene expression of the pro-apoptotic protein Bax and repressing the expression of anti-apoptotic protein Bcl-2. Fas receptor (FASR) is a transmembrane receptor that receives extracellular stimuli to stimulate apoptosis. Expression of the Fas receptor gene is induced by p53. Apopto-sis is also triggered when survival signaling is blocked by p53’s induction of IGF-BP3 (insulin-like growth factor-binding protein 3). IGF-BP3 blocks the signaling of IGF-1 to its receptor. Activation of these different path-ways in concert is required for a full apoptotic response. Transcription-independent mechanisms for the induction of apoptosis by p53 also exist and will be discussed in Chapter 7.

DNA repair and angiogenesis

Both DNA repair and angiogenesis are covered in depth elsewhere in this volume (Chapters 2 and 9, respectively). In general, a role for the transcriptional regulation of important genes in these processes by p53

Table 6.2 p53-inducible apoptotic target genes

Gene Location of gene product

Bax Intrinsic pathway

NOXA Intrinsic pathway

PUMA Intrinsic pathway

P53AIP1 Intrinsic pathway

FAS Extrinsic pathway

IGF-BP3 Extrinsic pathway

DR5 Extrinsic pathway

PIDD Extrinsic pathway

PERP Endoplasmic reticulum

has been established. For example, the gene XPC that is involved in nucleotide excision repair is regulated by p53 through a p53 response element in its promoter. Thrombospondin, an inhibitor of angiogenesis, is also transcriptionally regulated by p53. This further supports the role of p53 as a transcriptional regulator in different biological responses.

Decision making

As the guardian of the genome, p53 prevents damaged DNA from being passed on to daughter cells either by inhibiting the cell cycle or by induc-ing apoptosis. Cell cycle inhibition and apoptosis are two independent effects of p53. The molecular factors that determine the biological out-come of whether inhibition of the cell cycle or apoptosis takes place are just being elucidated. One model that has been put forth is that different combinations of transcription factors that act as dimers infl uence the bio-logical response. Oncogene activation (e.g. Myc) is an upstream inducer of p53 that triggers apoptosis. The mechanism of this stress signal acts via the cyclin–cdk inhibitor p21, the main effector of cell cycle inhibition but also an inhibitor of cell death. The regulation of the p21 gene is a pivotal point in the p53 decision-making process. Both p53 and a transcription factor called Miz-1 are required for p21 gene expression. Now enter the oncogene, Myc, which competes with p53 for binding with Miz-1. Myc interacts with Miz-1 and inhibits the transcription of p21. Through this mechanism of preventing expression of p21, Myc not only overrides the p53-regulated block to cell cycle progression but also blocks the p21-mediated inhibition of apoptosis (Figure 6.8). p53 is not altered and is free to induce the expression of pro-apoptotic targets. Additional events are also required for full activation of apoptosis, as p53 phosphorylation and apoptotic co-factors are required for the induction of some apoptotic genes. Revealing the mechanisms behind other modes of upstream stress inducers of p53, such as oxidative stress, requires further studies.

The apoptosis-stimulating proteins of p53 (ASPP) family also plays a role in p53 decision making (Slee and Lu, 2003). These proteins bind to the p53 DNA-binding domain and have been shown specifi cally to enhance the ability of p53 to activate genes involved in apoptosis and not cell cycle arrest. The selection of apoptotic genes versus growth arrest genes could potentially be accomplished by specifi c promoter sequences that serve to distinguish the functionally distinct classes of genes. The regu-lation of ASPP itself requires further study. Mutations in the ASPP binding site of the p53 gene and epigenetic silencing of the ASPP gene have been identifi ed in tumor cells. These tumor cells may have been initiated because they escaped from the apoptotic program normally augmented by ASPP. Other co-activators may also enhance the selectivity of p53 to activate apoptotic genes.