The p53 protein is located in the nucleus of cells throughout the body, where it attaches (binds) directly to DNA. (It is actually the TP53 gene that provides instructions for making the tumor protein p53 but for simplicity, I will say p53.) P53 regulates cell division by keeping cells from growing and dividing too fast or in an uncontrolled way.
P53 plays a critical role in determining whether damaged DNA will be repaired or a damaged cell will self-destruct (undergo apoptosis). If the DNA can be repaired, p53 activates other genes to fix the damage. If the DNA cannot be repaired, this protein prevents the cell from dividing and signals it to undergo apoptosis. By stopping cells with mutated or damaged DNA from dividing, p53 helps prevent the development of tumors.
P53 mutations lead to a version of p53 that cannot regulate cell growth and division effectively. Specifically, the altered protein is unable to trigger apoptosis in cells with mutated or damaged DNA.
DNA can be damaged by agents such as toxic chemicals, radiation such as gamma rays, X-rays — even UV light can interact with compounds in the cell generating free radicals which cause chemical damage to DNA.
Since all cells in our body contain DNA, there are lots of places for mutations such as p53 to occur. When p53 does not operate properly, damaged DNA can replicate, producing mutations and DNA rearrangements that contribute to the development of a highly transformed, metastatic cell. Many cancer cells inactivate p53, allowing the cells to evade death and continue proliferating up to becoming a tumor.
Mutant p53 proteins not only lose their tumor suppressive activities but often escalate the development of cancerous tumors by providing them with growth and survival mechanisms. Interestingly, mutations in the p53 gene have been shown to occur at different phases of the cancer process, contributing to tumor initiation, promotion, aggressiveness, and metastasis.
P53 mutations contribute to risk of brain tumors and breast, prostate, colon and other cancers. Mutations in p53 gene usually correlate with poor outcome and early recurrence in cancer.
Enough of the bad news.
p53 has many mechanisms of anticancer function and plays a role in apoptosis, genomic stability, and inhibition of angiogenesis. In its anti-cancer role, p53 works through several mechanisms:
•It can arrest growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle).
•It can initiate apoptosis (i.e., programmed cell death) if DNA damage proves to be irreparable.
•It is essential for the senescence response to short telomeres.
p53 pathway: In a normal cell, p53 is inactivated by its negative regulator, mdm2. Upon DNA damage or other stresses, various pathways will lead to the dissociation of the p53 and mdm2 complex. Once activated, p53 will induce a cell cycle arrest to allow either repair and survival of the cell or apoptosis to discard the damaged cell. How p53 makes this choice is currently unknown.
Activated p53 binds DNA and activates expression of several genes including microRNA miR-34a, WAF1/CIP1 encoding for p21 and hundreds of other down-stream genes. p21 (WAF1) binds to the G1–S/CDK (CDK4/CDK6, CDK2, and CDK1) complexes (molecules important for the G1/S transition in the cell cycle) inhibiting their activity.
When p21(WAF1) is complexed with CDK2, the cell cannot continue to the next stage of cell division. A mutant p53 will no longer bind DNA in an effective way, and, as a consequence, the p21 protein will not be available to act as the “stop signal” for cell division. Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this cell type, p53 activates numerous microRNAs (like miR-302a, miR-302b, miR-302c, and miR-302d) that directly inhibit the p21 expression in hESCs.
Research has also linked the p53 and RB1 pathways, via p14ARF, raising the possibility that the pathways may regulate each other.
p53 expression can be stimulated by UV light, which also causes DNA damage. In this case, p53 can initiate events leading to tanning.
The p21 protein binds directly to cyclin-CDK complexes that drive forward the cell cycle and inhibits their kinase activity thereby causing cell cycle arrest to allow repair to take place. p21 can also mediate growth arrest associated with differentiation and a more permanent growth arrest associated with cellular senescence. The p21 gene contains several p53 response elements that mediate direct binding of the p53 protein, resulting in transcriptional activation of the gene encoding the p21 protein.
Levels of p53 play an important role in the maintenance of stem cells throughout development and the rest of human life.
Embryonic stem cells
p53 is maintained at low inactive levels in human embryonic stem cells (hESCs). This is because activation of p53 leads to rapid differentiation of hESCs. Studies have shown that knocking out p53 delays differentiation and that adding p53 causes spontaneous differentiation, showing how p53 promotes differentiation of hESCs and plays a key role in cell cycle as a differentiation regulator. When p53 becomes stabilized and activated in hESCs, it increases p21 to establish a longer G1. This typically leads to abolition of S-phase entry, which stops the cell cycle in G1, leading to differentiation. p53 also activates miR-34a and miR-145, which then repress the hESCs pluripotency factors, further instigating differentiation.
Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway. This has subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this cell type, p53 activates numerous microRNAs (like miR-302a, miR-302b, miR-302c, and miR-302d) that directly inhibit the p21 expression in hESCs.
Adult stem cells
In adult stem cells, p53 regulation is important for maintenance of stemness in adult stem cell niches. Mechanical signals such as hypoxia affect levels of p53 in these niche cells through the hypoxia inducible factors, HIF-1α and HIF-2α. While HIF-1α stabilizes p53, HIF-2α suppresses it. Suppression of p53 plays important roles in cancer stem cell phenotype, induced pluripotent stem cells and other stem cell roles and behaviors, such as blastema formation. Cells with decreased levels of p53 have been shown to reprogram into stem cells with a much greater efficiency than normal cells. Papers suggest that the lack of cell cycle arrest and apoptosis gives more cells the chance to be reprogrammed. Decreased levels of p53 were also shown to be a crucial aspect of blastema formation in the legs of salamanders. p53 regulation is very important in acting as a barrier between stem cells and a differentiated stem cell state, as well as a barrier between stem cells being functional and being cancerous.
p53 becomes activated in response to myriad stressors, including but not limited to DNA damage (induced by either UV, IR, or chemical agents such as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion, and deregulated oncogene expression. This activation is marked by two major events. First, the half-life of the p53 protein is increased drastically, leading to a quick accumulation of p53 in stressed cells. Second, a conformational change forces p53 to be activated as a transcription regulator in these cells. The critical event leading to the activation of p53 is the phosphorylation of its N-terminal domain. The N-terminal transcriptional activation domain contains a large number of phosphorylation sites and can be considered as the primary target for protein kinases transducing stress signals.
The protein kinases that are known to target this transcriptional activation domain of p53 can be roughly divided into two groups. A first group of protein kinases belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. A second group of protein kinases (ATR, ATM, CHK1 and CHK2, DNA-PK, CAK, TP53RK) is implicated in the genome integrity checkpoint, a molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress. Oncogenes also stimulate p53 activation, mediated by the protein p14ARF.
In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A protein called Mdm2 (also called HDM2 in humans), binds to p53, preventing its action and transports it from the nucleus to the cytosol. Also Mdm2 acts as ubiquitin ligase and covalently attaches ubiquitin to p53 and thus marks p53 for degradation by the proteasome. However, ubiquitylation of p53 is reversible.
MI-63 binds to MDM2 making the action of p53 again possible in situations were p53’s function has become inhibited.
A ubiquitin specific protease, USP7 (or HAUSP), can cleave ubiquitin off p53, thereby protecting it from proteasome-dependent degradation via the ubiquitin ligase pathway. This is one means by which p53 is stabilized in response to oncogenic insults. USP42 has also been shown to deubiquitinate p53 and may be required for the ability of p53 to respond to stress.
Recent research has shown that HAUSP is mainly localized in the nucleus, though a fraction of it can be found in the cytoplasm and mitochondria. Overexpression of HAUSP results in p53 stabilization. However, depletion of HAUSP does not result to a decrease in p53 levels but rather increases p53 levels due to the fact that HAUSP binds and deubiquitinates Mdm2. It has been shown that HAUSP is a better binding partner to Mdm2 than p53 in unstressed cells.
USP10 however has been shown to be located in the cytoplasm in unstressed cells and deubiquitinates cytoplasmic p53, reversing Mdm2 ubiquitination. Following DNA damage, USP10 translocates to the nucleus and contributes to p53 stability. Also USP10 does not interact with Mdm2.
Phosphorylation of the N-terminal end of p53 by the above-mentioned protein kinases disrupts Mdm2-binding. Other proteins, such as Pin1, are then recruited to p53 and induce a conformational change in p53, which prevents Mdm2-binding even more. Phosphorylation also allows for binding of transcriptional coactivators, like p300 and PCAF, which then acetylate the carboxy-terminal end of p53, exposing the DNA binding domain of p53, allowing it to activate or repress specific genes. Deacetylase enzymes, such as Sirt1 and Sirt7, can deacetylate p53, leading to an inhibition of apoptosis. Some oncogenes can also stimulate the transcription of proteins that bind to MDM2 and inhibit its activity.
Role in disease
Overview of signal transduction pathways involved in apoptosis.
If the TP53 gene is damaged, tumor suppression is severely compromised. People who inherit only one functional copy of the TP53 gene will most likely develop tumors in early adulthood, a disorder known as Li-Fraumeni syndrome.
The TP53 gene can also be modified by mutagens (chemicals, radiation, or viruses), increasing the likelihood for uncontrolled cell division. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. Loss of p53 creates genomic instability that most often results in an aneuploidy phenotype.
Increasing the amount of p53 may seem a solution for treatment of tumors or prevention of their spreading. This, however, is not a usable method of treatment, since it can cause premature aging.
Restoring endogenous normal p53 function holds some promise. Research has shown that this restoration can lead to regression of certain cancer cells without damaging other cells in the process. The ways by which tumor regression occurs depends mainly on the tumor type. For example, restoration of endogenous p53 function in lymphomas may induce apoptosis, while cell growth may be reduced to normal levels. Thus, pharmacological reactivation of p53 presents itself as a viable cancer treatment option. The first commercial gene therapy, Gendicine, was approved in China in 2003 for the treatment of head and neck squamous cell carcinoma. It delivers a functional copy of the p53 gene using an engineered adenovirus.
Certain pathogens can also affect the p53 protein that the TP53 gene expresses. One such example, human papillomavirus (HPV), encodes a protein, E6, which binds to the p53 protein and inactivates it. This mechanism, in synergy with the inactivation of the cell cycle regulator pRb by the HPV protein E7, allows for repeated cell division manifested clinically as warts. Certain HPV types, in particular types 16 and 18, can also lead to progression from a benign wart to low or high-grade cervical dysplasia, which are reversible forms of precancerous lesions. Persistent infection of the cervix over the years can cause irreversible changes leading to carcinoma in situ and eventually invasive cervical cancer. This results from the effects of HPV genes, particularly those encoding E6 and E7, which are the two viral oncoproteins that are preferentially retained and expressed in cervical cancers by integration of the viral DNA into the host genome.
The p53 protein is continually produced and degraded in cells of healthy people, resulting in damped oscillation. The degradation of the p53 protein is associated with binding of MDM2. In a negative feedback loop, MDM2 itself is induced by the p53 protein. Mutant p53 proteins often fail to induce MDM2, causing p53 to accumulate at very high levels. Moreover, the mutant p53 protein itself can inhibit normal p53 protein levels. In some cases, single missense mutations in p53 have been shown to disrupt p53 stability and function.
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