Chapter 12 / Role of p53 in Regulation of Death Receptors 201
From: Cancer Drug Discovery and Development: Death Receptors in Cancer Therapy Edited by: W. S. El-Deiry © Humana Press Inc., Totowa, NJ
201
12 Role of p53 in Regulation of Death Receptors
Rishu Takimoto, MD , P h D
INTRODUCTION
p53 functions in the cellular response to DNA damage, thereby preventing accumu- lation of potentially oncogenic mutations and genomic instability (1). Activation of p53 leads to suppression of cell growth or apoptosis to prevent the propagation of mutated genes (1,2). p53 has also been implicated in differentiation (3), senescence (4), and inhibition of angiogenesis (5). Although we do not yet fully understand how p53 elicits its effects upon cells, it is clear that the transcriptional activation function of p53 is a major component of its biological effects. Activated p53 binds to a specific DNA sequence and activates transcription. The importance of the DNA-binding is underscored by the fact that the vast majority of p53 mutations derived from tumors map within the domain required for sequence-specific DNA binding. p53 normally recognizes a 20-base-pair response element that has an internal symmetry. The consensus DNA sequence for p53 binding is 5'-PuPuPuC(A/T-A/T)GPyPyPy- N(0-13)-PuPuPuC(A/T-A/T)GPyPyPy-3', with the third C and seventh G being highly conserved in the 10-base-pair half-sites (6).
Identification of transcriptional targets of p53 has been critical in dissecting pathways by which p53 functions (1,7). A growing number of genes have been found to contain p53- binding sites and/or response elements, and thus to have the potential to mediate the effects of p53 on cells, through upregulation of their expression and function. In this chapter, we review how p53 is activated, and how it emits a signal in response to DNA damage and death receptors induced by p53.
ACTIVATION OF P53
In order to emit the signal of p53, p53 has to be activated, and several activation
mechanisms of p53 have been reported. Most of them are posttranslational modification
of p53. It has been shown that ubiquitin-mediated proteolysis plays a role in the rapid
turnover of p53 protein. But once several stressful conditions have appeared, p53 is
stabilized and activated (Fig. 1). DNA damage, e.g., double-strand DNA breaks follow-
ing ionizing irradiation (IR), thymine dimers produced by ultraviolet irradiation, or chemi-
Fig. 1. Model for activation of p53 in response to DNA damage.
cal damage to DNA bases can lead to p53 activation. Hypoxia, heat-shock, radioactive chemicals, DNA transfection, and expression of viral and cellular oncogenes have also been shown to activate p53. Posttranslational modifications of the C-terminus of p53, including phosphorylation, dephosphorylation, acetylation, antibody binding, deletion of the C-terminus, or addition of a C-terminus peptide can induce sequence-specific DNA binding of p53.
Now two protein kinases, ATM (for ataxia telangiectasia mutated) and Chk2, are found
to play an important role in p53 activation upon DNA damage such as double-strand breaks
induced by IR. Specific phosphorylation, dephosphorylation, and acetylation events have
been reported to activate p53 (1,2,7). MDM2 protein, which was originally found to inter-
act with and inhibit p53-dependent transcriptional activity, has recently been found to
promote rapid degradation of p53. It has become clear that this MDM2-dependent
degradative pathway contributes to the maintenance of low levels of p53 in normal cells
(8,9). DNA damage causes phosphorylation of serine residues in the amino terminus of
p53. ATM, Chk1, and Chk2 phosphorylate p53 at amino termini that are close to the
MDM2 binding site. Upon DNA damage, these kinases phosphorylate the p53 and thereby
inhibit its interaction with MDM2, resulting in stabilization of p53. In particular, serine-
15 has been found to be phosphorylated in response to DNA damage by IR or ultraviolet
(UV) irradiation. Ataxia telangiectasia (AT )cells show delayed phosphorylation of
serine-15 in response to IR, but show normal phosphorylation after UV irradiation,
suggesting that ATM kinase is involved in the serine-15 phosphorylation after IR,
although it is not absolutely required (10). However, recent data suggest that serine-20
Chapter 12 / Role of p53 in Regulation of Death Receptors 203
may be the critical residue regulating p53 stability, whereas serine-15 phosphorylation may be activating transcription by p53 without directly affecting protein stability (11).
ATM also appears to be required for IR-induced dephosphorylation of p53. Serine- 376, which allows specific binding of 14-3-3m proteins to p53 and leads to an increase in the sequence-specific DNA-binding activity of p53 (12). Acetylation of the C-termi- nus of p53 by CREB-binding protein (p300/CBP) was shown to enhance sequence- specific DNA binding by p53 (13). p300/CBP are closely related histone acetyl transferases (HATs) (14) that interact with p53 and function as coactivators for p53- mediated transcription (13–17). The activation of sequence-specific DNA-binding by p53 following DNA damage may involve sequential amino-terminal phosphorylation followed by carboxy-terminal acetylation by the coactivator p300 following DNA dam- age (18). With the use of phosphorylated or acetylated peptide-specific antibodies, p300 has been shown to acetylate Lys-382 while the p300/CBP-associated factor (PCAF) acetylates Lys-320 of p53, and that either acetylation leads to enhance sequence-specific DNA-binding in vitro. p53 was found to be acetylated at Lys-382 and phosphorylated at Ser-33 and Ser-37 in vivo after exposure of cells to UV light or ionizing radiation.
Interestingly, acetylation of p53 by p300 and PCAF was strongly inhibited by phosphopeptides corresponding to the amino terminus of p53 phosphorylated at Ser-37 and/or Ser-33, suggesting that phosphorylation in response to DNA damage may enhance the interaction of p300 and PCAF, thereby driving p53 acetylation. Recent studies also suggest that HDAC1-dependent deacetylation of p53 can reduce its transcriptional activ- ity as well as its growth-suppressive and death-promoting actions (19).
Several viral and cellular oncogenes have been shown to stabilize p53. Viral oncogenes including SV40 T antigen, adenovirus E1A, and human papilloma virus 16 E7 stabilize p53 (20,21). This stabilization does not translate into the activation of p53, but rather a physiologically inactive p53 that is functionally inhibited. On the other hand, adenovirus has other cooperating oncogenes, like E1B and E4, which bind to p53 and inhibit apoptosis and/or growth arrest, thereby leading to successful viral DNA replication and cellular transformation. E1A can also inhibit transcriptional activation by p53 through interaction with CBP/p300, which may inhibit p53-mediated growth arrest and/or apoptosis (22,23).
The E6 gene product encoded by HPV16 binds to p53, which results in degradation of p53 and suppression of negative growth signals from p53.
The mechanism of p53 stabilization in response to viral oncogene expression has not been clearly understood until recently, when p19ARF (p14ARF in human), a product of INK4a/ARF locus translated in an alternate reading frame (24), was identified. It was found that the ability of E1A to stabilize p53 is severely compromised in p19ARF-null cells (25). p19ARF is a tumor suppressor, which can induce cell-cycle arrest in a p53- dependent as well as p53-independent manner (26). It has been shown that p14ARF appears to sequester MDM2 into the nucleolus, keeping MDM2 away from p53.
These mechanisms contribute to p53 stabilization and transactivation of its target genes.
DEATH RECEPTORS OF P53 TARGET MOLECULES
Many target molecules of p53 has been identified. Most of them are related to apoptosis,
cell-cycle arrest, anti-angiogenesis, and DNA repair (Fig. 2). In this issue, three major
death receptors that have been described as p53 target molecules are reviewed.
Fig. 2. p53 targets genes
Fas/APO-1
Fas/APO1 is a potent inducer of apoptosis in hematopoietic or liver cells exposed to Fas ligand (27). Upon Fas ligand binding to the Fas receptor, the Fas receptor trimerizes.
The death domain of Fas/APO1 recruits the Fas-associated death domain (FADD ) adap- tor, which recruits initiator caspase-8 to the death-inducing signaling complex (DISC ), resulting in the activation of the caspase cascade. Fas/APO1 is not absolutely required for p53 to induce apoptosis, because cells that are deficient for Fas/APO1 are proficient in inducing apoptosis upon p53 activation (see Chapter 1). Muller et al. (28) have reported that Fas/APO1 was induced by chemotherapeutic agents in a p53-dependent manner.
They could sensitize the cancer cells to Fas/APO1-mediated apoptosis after DNA damage. Furthermore, they identified a p53-responsive element within the first intron of the Fas/APO1 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53- responsive elements in the promoter of the Fas/APO1 gene. They demonstrated that only wt-p53 protein could bind and transactivate the Fas/APO1, but mt-p53 failed to induce apoptosis through Fas/APO1 activation. They concluded that loss of function of p53 contributes to tumor progression and to resistance of cancer cells to the Fas/APO1- mediated cell-killing signal.
p53-Induced Protein With a Death Domain (PIDD)
p53-induced protein with a death domain (PIDD) was discovered by differential dis-
play methods using Friend-virus-transformed mouse erythroleukemia cells that lack
endogenous p53 expression and express a transfected temperature-sensitive (ts) p53 (29).
Chapter 12 / Role of p53 in Regulation of Death Receptors 205
PIDD was found to be upregulated upon DNA damage in a p53-dependent manner. The size of cDNA of the PIDD was estimated to be 4.2 Kb, and its predicted amino acid content appeared to be 915. An N-terminal seven tandem leucin-rich repeats and a death domain in the C-terminal region were found in the PIDD protein. The p53-consensus binding site was found in a PIDD promoter lesion. Overexpression of PIDD can suppress cell growth and promote apoptosis in p53-null cell lines K562 and Saos2, indicating that PIDD mediates p53-dependent apoptosis. However, its ligand and how PIDD induces apoptosis in the cell remains unclear.
KILLER/DR5
KILLER/DR5 is a death-domain-containing proapoptotic member of a recently discov- ered family of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors (30) (see also Chapter 1). Expression of KILLER/DR5 appears to be increased following exposure of wild-type p53-expressing cells to cytotoxic DNA-damaging agents such as
a-radiation, doxorubicin, or etoposide (31). Indeed, a p53-responsive element was foundwithin intron 1 in the KILLER/DR5 genomic locus (32). Like the Fas/APO1 receptor, signaling through proapoptotic TRAIL receptors involves downstream caspase activa- tion (33,34).
Enhancement of TRAIL sensitivity by p53 overexpression may increase the cancer cell killing. Kim et al. (35) reported that overexpression of p53 by adenovirus could sensitize the cancer cells to TRAIL through induction of KILLER/DR5. This combina- tion cancer therapy may provide a new strategy for cancer cell killing without affecting normal human cells.
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