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 gene belongs to the p53 family, which has three main genes: p53, p63 and p73. Out of the three p53 is the main gene in higher organisms. The other two organisms are found in lower organisms. 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 known as "the guardian of the genome" as it maintains the gene stability by preventing genome mutation. This gene is also known as anti-oncogene [20, 21].
The p53 protein is a 53kDa nuclear phosphoprotein made up of 393 amino acids and has four domains. This is encoded by p53 gene, which is made up of 11 exons .
Fig.1. Protein Structure (1TUP)
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N-terminal Domain that activates transcription factors (residues 1-42)
Homo-oligomerization domain for tetramerization of the protein(80-94)
Core domain to recognize specific DNA sequence (residues 100-300)
Regulatory domain to recognize damaged DNA(residues 356-393)
Fig.2. TP53 Domains
The tumour protein p53 gene binds directly to the DNA when carcinogens, toxic chemicals, damage it 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 [24, 25].
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. AP endonuclease and DNA polymerase binds to p53 gene for DNA excision repair .
P53 ACTIVITY IN CELL CYCLE ARREST
Fig.3. Cell Cycle Arrest by p53 Gene
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 of cell cycle from G2 phase to 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.
Apart from these functions, it also induces apoptosis.
5. SIGNALLING PATHWAY
The p53 pathway is activated due to genotoxic stress, as it is a cell stress sensor molecule. It responds to stress factors that damage the DNA. The gene is an effective promoter of apoptosis (programmed cell death) .
Fig.4. P53 Signalling Pathway
The expression of p53 is regulated by both positive feedback loops and negative feedback loops. The positive feedback loop is controlled by MDM2 protein. The protein is encoded by the MDM2 gene, which is usually called the negative regulator. When p53 is unphospharylated, it binds to MDM2 and is degraded through ubiquitin-mediated degradation process. If p53 is phosphorylated then it does not form complex with MDM2 and it continues its function .
Fig.5. P53 Regulation by MDM2
P53 INDUCING APOPTOSIS
Apoptosis induced by Tp53 gene is executed by caspase proteinase. Two pathways activate the caspases
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Extrinsic pathway and
The initiation of extrinsic pathway begins with the 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.6. 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.7. p53 Mediated Apoptosis
Apoptosis is also known as programmed cell death, which is caused by the caspases 3, 6 and 7 present in the apoptosome. 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.8. Apoptosis by Caspases 3, 6 And 7
5.1 CARCINOGEN IDENTIFICATION
A. Benzo [a] Pyrene
Benzo [a] Pyrene belongs to polycyclic aromatics hydrocarbons (PAH) class of chemicals. This chemical is highly carcinogenic which is present in cigarette smoke, tar and in smoked food. It is a pale yellow colour pentacyclic hydrocarbon, which intercalates with DNA to form adducts which later leads to cancer development. Hence, known as a procarcinogen [31, 32].
Fig.9. Benzo [a] Pyrene
B. 4-(methylnitroamine)-1-(3-pyridyl)-1-butanone (NNK)
4-(methylnitroamine)-1-(3-pyridyl)-1-butanone is a nitrosamine mostly present in tobacco smoke. It is procarcinogen which binds with DNA causing transition mutation. This mutation leads to the development of cancer. CYP2A6 gene mainly activates the chemical [29, 30].
Fig.10. 4-(Methylnitroamine)-1-(3-pyridyl)-1-butanone (NNK)
It is an amine derivative of biphenyl found in tobacco smoke. The chemical is similar to benzidine. This is known as a human carcinogen, which is responsible for cancers like bladder cancer (UCC). The chemical is also found in azo compounds, which are aromatic aliphatic compounds. A colourless odourless compound turns into purple colour on exposure to air. 4-aminobiphenyl from cigarette smoke forms DNA adducts and is found in the epithelial lining of the urinary bladder. It also forms protein adducts with albumin and haemoglobin .
5.2 INTERACTION MECHANISM
Benzo [a] pyrene
Benzo[a]pyrene was found to be the main chemical in cigarette is 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.12. B[a] P metabolism
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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 [27,28].
Fig.13. Mechanism of p53 mutation caused by B[a] P
One of the harmful mutagen present in cigarette smoke is Benzo [a] Pyrene, which causes mutation in TP53 gene and leads to lung cancer. The B[a] P entered is absorbed by the epithelial walls of the lungs and metabolizes to benzo (a) pyrene diol epoxide (BPDE). The BPDE directly binds to the gene by forming DNA adducts at the guanine and adenine region . This binding takes place because BPDE is highly electrophilic and guanine is highly nucleophilic. The type of mutation caused by BPDE is G: C ïƒ T: A transversion at 157, 248 and 273 codons, which are known as the hot spot regions of the gene where more number of mutations takes place [15,16,17].
Fig.14. Schematic Representation in Lung Cancer Development
Fig.15. BPDE affecting TP53 gene
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 .
Pancreatic cancer is one of the types of cancer that happens due to disorderness of pancreas. There are many reasons for pancreatic cancer of which smoking plays a major role (about 20-30%). Smoking cigarettes causes pancreas to produce less bicarbonate (a substance used to neutralize stomach acid in digestive system). Pancreatic cancer occurs with the accumulation of genetic changes in the somatic DNA of normal cells and is a multistage process. 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. Papillary and dysplastic papillary ductal lesions contain mutated K-ras gene. The process of conversion of GTP to inactive GDP cannot be carried out by mutated ras oncogene, 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 gene function occurs with the 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 mutation (G: C to A: T) in exon 9. This mutation happens due to tobacco specific N-Nitrosamines (TNAS), mainly NNK and NNN.
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 .
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 the adducts from the O6 position of guanine, that in turn protects the genome from G to A transition . The MGMT are inactivated due to hypermethylation in the promoter gene by methanediazohydroxide. Hypermethylation results in the mispairs between O6- alkyl guanine and thymine during DNA replication thereby resulting in G: C ïƒ A: T transition. This transition occurs at the non-CpG sites of the p53 gene resulting in colorectal cancer .
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.
URINARY BLADDER CANCER
Smoking causes mutation in TP53 gene leading to bladder cancer. The chemicals 4-aminobiphenyl and 2-naphthyline from tobacco smoke alter the gene at exons 5 and 5. The alteration caused by the chemicals is transition mutation. The mutation takes place by the formation of DNA adducts at the C8 position of deoxyguonine and deoxyadenine. Due to the adduct formation A: T ïƒ G: C transition takes place in the CpG islands [17, 18].