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Tp53 is a tumour suppressor gene present in the short arm of the 17th chromosome in Homo sapiens. The gene encodes a protein known as p53 protein through translation process. The main function of the protein is to regulate the cell cycle and act as a tumour suppressor. Hence, this gene is known as the tumour suppressor gene. It is described as "the guardian of the genome" as it maintains the gene stability by preventing genome mutation. This gene is also known as anti-oncogene.
The p53 protein is a 53kDa nuclear phosphoprotein made up of 393 amino acids and has four domains.
N-terminal Domain that activates transcription factors
Homo-oligomerisation domain for tetramerization of the protein
Core domain to recognize specific DNA sequence
Regulatory domain to recognize damaged DNA
Fig.1. p53 Protein Structure
The tumour protein p53 gene binds directly to the DNA when it is damaged by carcinogens, toxic chemicals or UV rays from sunlight. The protein either repairs the DNA or induces cell apoptosis (programmed cell death) when the DNA cannot be repaired. The protein prevents the mutated cell or cell with damaged DNA from dividing thereby suppressing tumour formation.
FUNCTIONS OF P53 GENE
Under normal conditions, the p53 gene is inactive as it is bound to the Mdm2 and does not participate in normal cell cycle progression and cell survival. The gene is activated only when the DNA is damaged causing cell stress and increases the p53 proteins level. This protein does three main functions cell cycle arrest, DNA repair and apoptosis. The gene is activated by other factors like mitotic spindle damage, exposure to nitric oxide, hypoxia, oncogene activation and ribonucleotide depletion. The target genes involved in various functions are
Growth arrest - p21,Gaff45 and 14-3-3s
Apoptosis - Bax,Apaf-1,PUMA,NoxA
DNA repair - p53R2
P53 ACTIVITY IN DNA REPAIR
To repair the DNA p53 activates the DNA repair proteins and p53R2 gene. It is a ribonucleotide reductase induced by the p53 gene to supply deoxynucleotide triphosphates to repair the damaged DNA. P53 also interacts directly with AP endonuclease and DNA polymerase for DNA excision repair .
P53 ACTIVITY IN CELL CYCLE ARREST
To perform cell cycle arrest p53 activates three genes p21, Gaff45 and 14-3-3s by binding to the damaged DNA. The p21 gene binds to G1-S/CDK (CDK2) and S/CDK complexes. These complexes are important for the transition of G1 phase to S phase. When the gene binds to the complex, the activity gets inhibited and the cell does not continue to the next stage in cell division. The progression into the M phase requires Cdc2, which is inhibited by GADD45 or 14-3-3s genes. Hence, p53 regulates cell cycle arrest and prevents tumour formation.
Fig.2. Cell Cycle Arrest by p53 Gene
P53 PATHWAY IN INDUCING APOPTOSIS
Apoptosis induced by Tp53 gene is executed by caspase proteinase. Two pathways activate the caspases
The extrinsic pathway is initiated by ligation of death receptors with their respective ligands. The death receptors are tumour necrosis factors CD95/Fas/Apo-1 and TRAIL receptors. The ligation of the receptors with ligands form the Death Inducing Signalling Complex (DISC) that is composed of the adapter molecule FADD and caspase - 8. Activation DISC activates capase-8 either directly cleaves and activates the effectors caspases or indirectly activated the downstream caspases by cleaving the BH3 protein BID [3, 4]. This leads to the invoking of the intrinsic pathway. In this pathway, anti-apoptotic Bcl-2 protein family regulates caspase. These proteins induce the release of apoptogenic factors like cytochrome c or Smac from the mitochondria to the cytosol. The intrinsic pathway is usually triggered due to stress or DNA damage. The release of cytochrome c facilitates the formation of apoptosome complex, which is composed of Apaf-1 and caspase-9 .
Fig.3. Apoptosome formation
These apoptosomes activates the effector caspases 3, 6 and 7 that execute the death program.p53 response elements are found in the promoters of Bcl-2, Bax and BH3. The Bax protein family contains two important proteins PUMA and NOXA, which are up regulated during P53 apoptosis. During the up regulation process PUMA encodes PUMA-Î± and PUMA-Î², which promotes mitochondrial translocation, initiating apoptosis. Effector caspases 3, 6 and 7 digest essential targets of the cell and inducing apoptosis activity. Caspase-6 activity is induced by DNA damage through a response element present in the third intron.
Fig.4. p53 Mediated Apoptosis
Apoptosis is also known as programmed cell death, which is caused by the caspases 3, 6 and 7 present in the apotosome. They cause cell death by activating DNase, inhibiting DNA repair enzymes and breaking down the structural units in the nucleus .
INACTIVATION OF ENZYMES
Enzyme Poly (ADP-ribose) polymerase (PARP) is used to repair the damaged DNA. Caspase-3 cleaves PARP and prevents it from doing its function and the damaged DNA remains unrepaired.
BREAKDOWN OF STRUCTURAL NUCLEAR PROTEINS
Lamins are intranuclear proteins that help in maintaining the shape of the nucleus and also mediate the interactions between the chromatin and nuclear membrane. Caspase-6 degrades Lamins and results in chromatin condensation and nuclear fragmentation.
Fragmentation of DNA
The fragmentation of DNA to nucleosomal units is caused by CAD (Caspase activated DNase) enzyme which normally exists in inactive ICAD form. Caspase-3 cleaves ICAD to CAD and results in rapid fragmentation of nuclear DNA to nucleosomal units .
Fig.4. Apoptosis by Caspases 3, 6 And 7
MUTATION ON P53 GENE
Smoking causes mutation in p53 gene, which ultimately causes colorectal cancer. The cigarette smoke contains many mutagenic compounds like nitrosamines, aromatic amines and polynuclear hydrocarbons . Among these the tobacco specific nitrosamine 4-(methylnitroamine)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to methanediazohydroxide. That compound methylates DNA and forms different adducts like 7-methylguanine and O6-methylguanine. O6-methylguanine-DNA mthyltransferase (MGMT) is a DNA repair protein that removes adducts from O6 position of guanine, thereby protecting the genome from G to A transition . The MGMT are inactivated due to hypermethylation in the promoter gene by methanediazohydroxide. Because of hypermethylation O6- alkyl guanine mispairs with thymine during DNA replication and results in G: C to A: T transition at the non-CpG sites of the p53 gene causing colorectal cancer .
The type of cancer caused in oesophagus due to smoking is squamous cell carcinoma. One of the genes mutated in this type of cancer is Tp53. The mutation takes place in the exon 5 causing transversion and transition mutations [2, 8, 10]. The chemicals in cigarette causing mutations are N-nitrosodiethylamine and N-nitrosopiperidine. These cause transition mutations and benzo-a-pyrene cause transversion mutation . G: C to T: A transversion account only 16% whereas G: C to A: T and A: T to G: C transition account about 22%. The mutations take place in the CpG islands. The chemicals usually damage the DNA, which leads to cancer development .
P53 is a tumour suppressor gene that is involved in the cascade of events leading to toxicity of the diverse xenobiotics. Smoking contains about 4,000 chemicals in which about 60 chemicals are carcinogenesis. Among them mainly PAH (poly aromatic hydrocarbon) is the responsible for liver cancer .
Pancreatic cancer is one of the types of cancer that happens due to disorderness of pancreas. There are many factors for pancreatic cancer; smoking is one of them (about 20-30%). Smoking cigarettes causes pancreas to produce less bicarbonate (a substance used to neutralize stomach acid in digestive system). Pancreatic cancer is a multi-stage process resulting from the accumulation of genetic changes in the somatic DNA of normal cells. Pancreatic cancer is mainly a genetic disease and the chance of this cancer due to smoking is about only 20-30%. The tumour promoter in pancreatic cancer is not regulated by single gene. K-ras is the highly mutable gene in pancreatic cancer in first stage. Mutated K-ras has been found in papillary and dysplastic papillary ductal lesions. The mutated ras oncogene is not able to convert GTP to inactive GDP, resulting in a constitutively active ras protein product, unregulated cellular proliferation signals, and susceptibility to transformation. Tp53 mutation happens in later-stage of PanINs that have acquired significant features of dysplasia. Seventy percent of pancreatic adenocarcinomas have loss of p53 function. Inactivation of p53 function occurs through loss of one p53 allele and mutational inactivation of the other. TP53 mutation is the major cause of pancreatic cancer. In pancreatic cancer this mutation is transition (G:C to A:T) in exon 9. This mutation happens due to tobacco specific N-Nitrosamines (TNAS), mainly NNK and NNN.
The TP53 gene gets mutated in adrenal gland causing Li-Fraumeni Syndrome which increases a person's risk for a wide range of tumours and adrenocortical tumours are one among them. The type of mutation caused is pR337P which leads to the development of adrenocortical carcinoma. Smoking is one of the main risks associated with the syndrome.
Chemical mutations on p53 gene
Benzo[a]pyrene was found to be the main chemical in cigarette's responsible for causing p53 mutation. It is procarcinogen which means that there is the involvement of enzymes (CYP1A1/CYP1B1) in converting Benzo[a] Pyrene to its -diol-epoxide, Benzo[a] Pyrene diol epoxide, which is the p53 mutagen. Enzyme metabolism of Benzo[a] Pyrene leads into four -7, 8 diol-9,10-epoxide stereoisomers of which the most abundantly formed is the (+)-anti-BPDE.
Fig.5. B[a] P metabolism
Also of all other isomers the (+)-anti-BPDE as got more carcinogenic and intrinsic genotoxic characteristics. This isomer forms an adduct with the DNA forming (+)-anti-BPDE-N2-dG which is resistant to nuclear excision repair mechanism and translesional synthesis (TLS) which leads to G-T transversion.