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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 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 genes 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].
Sub cellular localization
DNA associated transcription factor
2.1 GENE SEQUENCE
2.2 PROTEIN SEQUENCE
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 and 10 introns .
Fig.1. Protein Structure (PDB id: 1TUP)
N-terminal Domain that activates transcription factors (residues 1-50)
Homo-oligomerization domain for tetramerization of the protein(63-92)
Core domain to recognize specific DNA sequence (residues 100-300)
Regulatory domain to recognize damaged DNA(residues 323-393)
Fig.2. TP53 Domains
The tumour protein P53 gene binds directly to the DNA when carcinogens, toxic chemicals, damage it. 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 gene 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 namely 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
3.1 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. Apurinic endonuclease (AP endonuclease) and DNA polymerase binds to P53 gene for DNA excision repair .
3.2 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 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.
Fig.3. Cell Cycle Arrest by P53 Gene
Apart from these functions, it also induces apoptosis.
4. 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
4.1 P53 INDUCING APOPTOSIS
Apoptosis induced by TP53 gene is executed by caspase proteinase. Two pathways activate the caspases
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. DISC activates capase-8 by either directly cleaving and activating the effectors caspases or indirectly activating 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 for the affected cell. 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 induce 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 .
4.1.1 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.
4.1.2 BREAKDOWN OF STRUCTURAL NUCLEAR PROTEINS
Lamins are intranuclear proteins that help in maintaining the shape of the nucleus and mediate the interactions between the chromatin and nuclear membrane. Caspase-6 degrades Lamins and results in chromatin condensation and nuclear fragmentation.
4.1.3 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 [30, 31].
Fig.9. Benzo [a] Pyrene
B. 4-(methylnitroamine)-1-(3-pyridyl)-1-butanone (a type of 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 [28, 29].
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-urothelial cell carcinoma). 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 .
D. 2-Naphthylamine (2-NA)
It is an aryl amine present in tobacco smoke mainly causing bladder cancer. They cause cancer by forming DNA adducts causing transition mutation. These chemicals are mainly used in the production of azo dyes and are known as procarcinogen as it causes cancer only after the activation by the phase I enzymes in the liver. They are deactivated by the conjugation of glucuronic acid .
Fig.12. 2-Naphthylamine (2-NA)
E. N-nitrosonornicotine (NNN)
It is a nitrosamine abundantly found in tobacco smoke and is classified as Group I carcinogen. The chemical is produced by nitrosation of nicotine during the curing process of the tobacco leaves. It is usually yellow oily liquid, which solidifies in cold temperature .
Fig.13. N-nitrosonornicotine (NNN)
5.2 INTERACTION MECHANISM
A. Benzo [a] pyrene
Benzo[a]pyrene is found to be the main chemical in cigarette that is responsible for causing P53 mutation. It is procarcinogen which is activated by enzymes like CYP1A1 or CYP1B1, which converts Benzo[a] Pyrene to its -diol-epoxide, Benzo[a] Pyrene diol epoxide, a P53 mutagen. Enzyme metabolism of Benzo[a] Pyrene leads to the formation of four -7, 8 diol-9, 10-epoxide (BPDE) stereoisomers of which the most abundantly formed is the (+)-anti-BPDE.
Fig.14. B[a] P metabolism
The (+)-anti-BPDE isomer posses high carcinogenic and intrinsic genotoxic characteristics. This isomer forms an adduct with the DNA at the N2 region of deoxyguanosine forming (+)-anti-BPDE-N2-dG complex which is resistant to nuclear excision repair mechanism(NER) and translesional synthesis (TLS) and leads to G-T transversion .
Fig.15. Mechanism of P53 mutation caused by B[a] P
B. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN)
NNK and NNN are potent carcinogens present in tobacco smoke. They enter the body in the vapour state and are activated by Î±-hydroxylation (addition of hydroxyl group) to form intermediates like methanediazohydroxide, which are highly electrophilic that react with DNA to form, adducts. The intermediate formed is said to be a potent carcinogen to induce tumours The NNK intermediates form both methylating and pyridyloxobutylating agents whereas NNN intermediates form only pyridyloxobutylating agents. These methylated intermediated bind to the guanine and adenine region to form DNA adducts. The adducts formed leads to either Aïƒ G or Gïƒ A transition. Gïƒ A transition takes place if the adducts is formed at the O6-guanine region then and Aïƒ G transition takes place if the adducts is formed in the O4-adenine region then takes place .
Fig.16. NNK and NNN forming DNA Adducts
C. 4-Aminobiphenyl (4-ABP)
The chemical enters the body as a procarcinogen which is activated by cytochrome 450 enzymes (Phase I enzymes) present in liver. 4-aminobiphenyl intermediates are highly electrophilic bind to the DNA and form adducts. The major form of complex formed is N-(2'-deoxyguanosin-8-ly)-4-ABP (dG-C8-4-ABP) and other compounds formed are N-(2'-deoxyadenosine-8-ly)-4-ABP and 3-(2'-deoxyguanosine-N2-ly)-4-ABP. These complexes are responsible for either Gïƒ A or Aïƒ G transitions .
Fig.17. 4-ABP Adduct Formation
D. 2-Naphthylamine (2-NA)
2-Naphthylamine enters the body in vapour phase and is metabolically N-hydroxylated and glucuroniated by the phase I enzymes present in liver. The N-glucoronide formed in the liver is transported to the urinary bladder. In the urinary bladder, the glucoronide formed is hydrolyzed to hydroxynaphtalamine, which forms the DNA adduct and leads to bladder cancer. Another metabolite called 2-nitroso-1-naphthol (NO-napthol) is produced from 2-NA. This NO-naphthol is formed by the conversion of N-hydroxy-2-naphthalamine into nitroso compounds, followed by hydroxylation in the presence of NADH and Cu (II). If any one of the two factors is absent, NO-naphthol is not metabolized. The NO-naphthol forms a complex at the O8 position of deoxyguonine. This complex is the DNA adducts leading to transition mutation where G is converted to A .
Fig.18. 2-NA forming DNA Adducts
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 causes 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 causes mispairing and ultimately leading to cancer development .
The type of cancer in pancreas due smoking is adenocarcinoma and the chemicals responsible for the cancer are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (type of NNK) and N-nitrosonornicotine (NNN). The type of mutation caused by these carcinogens is transition mutation where a purine is replaced by a purine or pyrimidine is replaced by pyrimidine. These chemicals cause G: C ïƒ A: T transition in exons (5-8) of the gene [38, 42, 43].
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 .
It is the cancer of the adrenal gland. Smoking directly does not induce the cancer in the adrenal gland but mutates the TP53 gene leading to Li-Fraumeni Syndrome . Li-Fraumeni Syndrome (LFS) known as cancer predisposition syndrome, which is usually associated with soft-tissue sarcoma, breast cancer, adrenocortical carcinoma, leukaemia, pancreatic cancer, colon cancer and brain cancer. The cancer occurs in the adrenal cortex region hence the name. Smoking cause's arginine to proline mutation at the 337-codon region and the type of mutation caused is transversion as the G nucleotide is being converted to C nucleotide. The mutation causes damage to the gene and it loses its function of controlling the cell cycle. Due to the uncontrolled cell, growth tumors are formed leading to cancer [48, 49].
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 9. 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].