The p53 Pathway

The p53 pathway is composed of a network of genes and their products that are targeted to respond to a variety of intrinsic and extrinsic stress signals, which impact cellular homeostatic mechanisms that monitor DNA replication, chromosome segregation, and cell division (Vogelstein et al., 2000). The p53 pathway responds to stresses that can disrupt the fidelity of DNA replication and cell division. Mechanistically, a stress signal is transmitted to the p53 protein by post-translational modifications, which activates the p53 protein as a transcription factor that initiates a program of cell cycle arrest, cellular senescence, or apoptosis. The transcriptional network of p53-responsive genes produces proteins that interact with many other signal transduction pathways in the cell, and a number of positive and negative auto-regulatory feedback loops act upon the p53 response. To further elaborate, the p53 circuit communicates with the Wnt-beta-catenin, IGF-1-AKT, Rb-E2F, p38 MAP kinase, cyclin-CDK, p14/19 ARF pathways, and the cyclin G-PP2A and p73 gene products.

p53

p53 is an important tumor suppressor gene. Since the discovery of the p53 gene in 1979, over 80,000 research papers have been reported. Initially, the p53 gene was thought to be an oncogene, but with the deepening of research during the past decades, the function of p53 as a tumor suppressor was gradually revealed. Furthermore, mutations in the p53 gene have been found in more than 50% of human tumor tissues, which is the most common genetic modification in tumors, indicating that the p53 gene is likely to be the major pathogenic factor of tumors.

After the discovery of the p53 gene in humans, monkeys, chickens, and mice, it was mapped. The human p53 gene was located on 17p13, the mouse p53 was located on chromosome 11 and a nonfunctional pseudogene was found on chromosome 14. Among animals with different degrees of evolution, p53 has a very similar gene structure, about 20 KB long, composed of 11 exons and 10 introns. The first exon is non-coded, and exon-2, -4, -5, -7, and -8 encode five evolutionarily highly conserved domains and five highly conserved regions of the p53 gene, namely the 13-19, 117-142, 171-192, 236-258, and 270-286 coding regions. The p53 gene is further transcribed into a 2.5 kb mRNA, encoding 393 amino acid proteins with a molecular weight of 43.7 kDa. The expression of the p53 gene is regulated at least at the transcriptional and post-transcriptional levels.

Human p53 is active as a homotetramer of 4 × 393 amino acids with a complex domain organization, consisting of an intrinsically disordered N-terminal transactivation domain (TAD), a proline (Pro)-rich region, a structured DNA-binding domain (DBD), and tetramerization domain connected via a flexible linker, and an intrinsically disordered C-terminal regulatory domain. The p53 tetramers have a dimer-of-dimers topology, and individual subunits within the human tetramerization domain (residues 325–355) are composed of a short β-strand followed by a sharp turn leading into an α helix.

The p53 pathway activation

Stresses, both intrinsic and extrinsic to the cell, can act upon the p53 pathway. Meanwhile, signals that activate the p53 protein can damage the integrity of the DNA template. Gamma or UV irradiation, alkylation of bases, depurination of DNA, or reaction with oxidative free radicals all contribute to the alteration of DNA in different ways. For each damaging agent, a unique detection and repair mechanism is employed by cells.

Different types of DNA damage appear to activate unique enzyme activities that modify the p53 protein at particular amino-acid residues. As a result, the nature of the stress signal is transmitted to the protein, presumably its activity, by a code inherent to the post-translational modifications that reflect the different types of stress. For example, gamma-radiation activates the ATM kinase and the CHK-2 kinase, which can phosphorylate the p53 protein, while UV-radiation activates ATR, CHK-1, and casein kinase-2, which results in the modification of different amino-acid residues on the p53 protein (Appella and Anderson, 2001). For most of these stress signals, the p53 protein is modified by phosphorylation and acetylation, which appear to alter the p53 protein in two ways: First, the half-life of the protein in a cell increases from 6–20 min to hours, resulting in a 3–10-fold increased concentration of the p53 protein in a cell; Second, enhanced ability of the p53 protein to bind to specific DNA sequences and promotion of gene transcription regulated by DNA sequences.

The activation of the p53 protein in response to stresses is mediated and regulated by protein kinases, histone acetyl-transferases, methylases, ubiquitin, and sumo ligases. As the p53 protein is activated by these protein modifications, it can also be inactivated by phosphatases, histone deacetylases, ubiquitinates, or even inhibitors of ubiquitin ligases. In addition, the activated p53 protein interacts with many important proteins for its transcriptional activity, such as PML bodies (promyelocytic leukemia bodies) and the Werner helicase. Several of these associated proteins are essential for p53 transcriptional activities or even the selective modulation of genes that p53 may regulate in its pathway to carry out its functions.

Downstream events of the p53 pathway

Once the p53 protein is activated, it initiates a transcriptional program. The activated p53 protein binds to a specific DNA sequence, termed the p53-responsive element (RE), composed of RRRCWWGYYY (spacer of 0–21 nucleotides), where R is a purine, W is A or T, and Y is a pyrimidine. Thus, two degenerate 10 bp sequences separated in the genome by a variable length spacer are required to regulate the p53-responsive genes. Genes in this p53 network initiate one of three programs that result in cell cycle arrest (G-1 or G-2 blocks are observed), cellular senescence, or apoptosis.

1. Cell-cycle arrest

Cell-cycle arrest by p53 is mainly mediated by the transcriptional activation of p21/WAF1. p53 can activate the transcriptional upregulation of CDKN1A, which encodes for cell cycle inhibitor p21. The p21 mRNA is highly induced after p53 activation and is the first p53 target gene. p21 binds to cyclin E/Cdk2 and cyclin D/Cdk4 complexes to cause G1 arrest in the cell cycle (Harper et al. 1993). The inhibition of Cdk2 and Cdk4 by p21 blocks pRb phosphorylation, promotes pRb binding to E2F1, and promotes transcription silencing of E2F1 targets critical for DNA replication and cell-cycle progression.

In addition, p53 activation also arrests cells at the G2/M phases. Although p21 can also inhibit cyclin B/Cdc2 from inhibiting cell-cycle progression through mitosis, other p53 target genes, such as 14-3-3σ, may participate in blocking the G2/M transition.

2. Cellular senescence

Cellular senescence is defined as a cell state characterized by prolonged and generally irreversible cell-cycle arrest and the acquisition of different phenotypic alterations, including morphological changes, chromatin remodeling, metabolic reprogramming, and secretion of pro-inflammatory factors or SASP (senescence-associated secretory phenotypes).

Cellular senescence mediates via p53-induced transcriptional activation of the cyclin-dependent kinase (CDK) inhibitors P21CIP1 (CDKN1A) and P16 INK4A (CDKN2A). The role of P21CIP1 may be to initiate senescence, whereas P16INK4A may be responsible for durable growth arrest. Apart from P21INK4A, there are other target genes like PML nuclear body scaffold (PML) and SERPINE1 (Serpin family E member 1), also known as Plasminogen activator inhibitor-1 (PAI-1), which are transcriptionally activated by p53 and plays a role in senescence.

3. Apoptosis

Many apoptosis-related genes transcriptionally regulated by p53 have been identified, which are candidates for implementing p53 effector functions. In response to oncogene activation, p53 mediates apoptosis through a linear pathway involving Bax transactivation, Bax translocation from the cytosol to membranes, cytochrome c release from mitochondria, and caspase-9 activation, followed by the activation of caspase-3, -6, and -7. p53-mediated apoptosis can be blocked at multiple death checkpoints by inhibiting p53 activity directly, by Bcl-2 family members regulating mitochondrial function, by E1B 19K blocking caspase-9 activation, and by caspase inhibitors.

p53 cancer mutations

In over 50% of human cancer types, p53 is directly inactivated by mutation. The prevalence of p53 mutation varies significantly by cancer type and also depends on the developmental stage of a tumor, ranging from less than 5% in cervical cancer and 10% in leukemia to 80% in small-cell lung cancer and 90% in ovarian cancer. Most cancer-associated mutations are located in the DBD, which involves several mutational hotspots, with the most frequent somatic cancer mutations being R175H, Y220C, G245S, R248Q/W, R249S, R273C/H, and R282W.

p53 in cancer therapy

Dysfunction of the p53 gene occurs in most, if not all, human malignancies. Two principal mechanisms are responsible for this dysfunction: mutation and down-regulation of wild-type p53 mediated by MDM2/MDM4. Because of its almost universal inactivation in malignancy, p53 is a highly attractive target for the development of new anticancer drugs. There have been at least seven different approaches to developing translational therapies for the p53 protein and pathway.

• Small-Molecule Stabilizers of p53 – the Y220C Paradigm
• Rescue of Zinc-Binding – Deficient Mutants with Metallochaperones
• Alkylating Agents and DNA Intercalators
• Rescue of Nonsense Mutated p53
• Targeting the Tetramerization Domain
• Inhibitors of the p53 – MDM2/MDMX Interaction
• Targeting Other p53 Modulators

Despite nearly 40 years of research on p53, resulting in a staggering 80,000 publications, there are only beginning to grasp the full complexity of the p53 pathway, and the future will undoubtedly bring many novels, sometimes surprising, or even paradigm-shifting discoveries. Nevertheless, Creative BioMart is ready to provide a list of related protein products to support your ongoing research. Please feel free to contact us if you’re interested.