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P16 protein is encoded from the CDKN2A locus from alpha mRNA transcript of the p16 gene. The protein acts as a negative regulator of cell cycle progression. The protein inhibits the activation of CDK4 and CDK6 by cyclin D1 and phosphorylation of RB1 (Retinoblastoma Protein). It is a 16.5 kDa protein made up of 156 amino acids and is moderately expressed in organs like thymus, liver, pancreas, prostate, lung and kidney. The main function of the protein is to suppress the tumour cells. The protein is made up of four ankyrin repeats. The tertiary structure contains four helix-turn-helix motifs linked by three loops [12.13].
Fig.1. CDKN2A/P16 Structure (PDB ID: 1A5E)
The gene is activated and encodes the protein due to genotoxic stress like DNA damage and oncogenic stress. The main function of the protein involves regulation of cell cycle progression at the G1/S boundary. For the progression, G1 requires Cdk4 or cyclin D-dependent phosphorylation of retinoblastoma protein. Some related "pocket proteins" like p107 and p130 liberate transcription factors that are required for DNA replication during S phase of the cell cycle. The phosphorylation results in cell cycle arrest at the G1 checkpoint. The unphospharylated Rb-1 prevents the E2F (transcription factor) and prevents progression from the G1 phase to S phase of the cell cycle. Apart from cell cycle regulation, the protein is involved in controlling other cellular processes like angiogenesis, cell senescence, tumour invasion, cell spreading and apoptosis [14, 15, 16].
The apoptosis of cell is induced by TP53 gene, which is activated and phosphorylated by p16 gene to cause programmed cell death on the developing tumour cells and suppress them .
Fig.2. P16 Cell Cycle Regulation
4. Signalling pathway
The main function of the gene is cell cycle regulation and the pathway is known, as p16-cyclin D-pRb Pathway.Rb-1 is the retinoblastoma 110kDa phosphopreotein encoded by Rb gene. Reintroduction of the functional protein in tumour cells results in growth arrest. This protein undergoes cell cycle phosphorylation and is hyperphosphorylated at the G1 phase and the phosphorylation process increases as the cell cycle progresses. When the cell cycle researches the mitotic phase (M phase), the phosphates are removed.
The phosphorylation of Rb protein is done by a group of related kinases cyclin-dependent kinase (CDK's). In the G1 phase, cyclin D is associated with CDK-4 and CDK-6 and form an active cyclin D complex. This complex is responsible for the first phosphorylation of the Rb protein. Subsequently, cyclin E is synthesized that binds to CDK2 and activates it. The complex formed phosphorylates pRb during the restriction point in late G1 phase. In S phase, complex containing cyclin A and CDK2 is formed and later phosphates pRb.
If P16 the tumour suppressor gene binds to the CDK-4 or CDK-6 phosphorylation of Rb protein results in hypophospholyration. Hypophosphorylated Rb binds to E2F (transcription factor) and inactivates it. The inactive E2F binds to the DNA, acts as a dominant repressor complex, and switches off the target genes. Hence, cell cycle progression stops. This function is performed in tumour cells avoiding the development of cancer [18, 19].
Fig. 3. P16-cyclin D-pRb Pathway
5.1 Carcinogens Identified
4-(methylnitrosamino) - 1-(3-pyridyl)-1-butanone
It is a nitrosamine, a potent carcinogen present in tobacco plants. It belongs to the Nicotine-derived nitrosamine ketone family usually activated by CYP1A2 and CYP2A6 (Phase I enzymes) present in liver. These compounds become carcinogenic only after the activation process. These compounds are usually produced during the curing of tobacco leaves. During the curing process nitrosation of nicotine takes place leading to the formation of 4-(methylnitrosamino) - 1-(3-pyridyl)-1-butanone .
Fig.4. 4-(methylnitrosamino) - 1-(3-pyridyl)-1-butanone
5.2 Interaction Mechanism
Promoter Hypermethylation by 4-(methylnitrosamino) - 1-(3-pyridyl)-1-butanone
4-(methylnitrosamino) - 1-(3-pyridyl)-1-butanone (NNK) causes hypermethylation in P16 gene at the CpG islands of the promoter region. This hypermethylation leads to inactivation of the gene leading to tumour development.
DNA (cytosine-5)-mthyltransferase 1 is an enzyme encoded by DNMT-1 gene that is responsible for the regulation of tissue-specific patterns of methylated cytosine residues. The protein contains an intrinsic transcriptional repressor activity that is usually required for transcriptional silencing of transposons and imprinted genes. It is also required for maintaining the chromosome structure and stability .NNK increases the expression of DNTM-1 protein by stimulating the AKT pathway, which plays a major role in cell cycle regulation, apoptosis, cell proliferation, transcription and cell migration. This AKT stabilizes DNTM-1 protein by decreased ubiquitination. The activated AKT inactivates GSK3Î² kinase, which phosphorylates the DNTM-1 protein and activates E-3ubiquitin ligase and Î²-transducin (Î²TrCP). These proteins usually degrade DNTM-1 enzyme since the AKT pathway the DNTM-1 protein accumulates and methylates the promoter region of the P16 gene and inactivates its function by inhibiting its transcription activity. The inactivation of p16 protein results in hyperphosphorylation of Rb-1, which releases E2F (transcription factor) that facilitates cell cycle progression and results in tumour formation. The oestrogen receptor gene is also inactivated promoter hypermethylation [1, 2, 7].
Fig.5. Promoter Hypermethylation of P16 Gene
Fig.6. Phosphorylated RB Releasing E2F for Cell Cycle Progression
The type of cancer caused in lung due to the mutation of P16 gene is Squamous Cell Carcinoma. The main carcinogen causing the cancer is 4-(methylnitrosamino) - 1-(3-pyridyl)-1-butanone which is a type of Nicotine-derived nitrosamine ketone (NNK). It causes methylation in the CpG islands of the promoter region leading to the inactivation of the gene function. Hypermethylation is an epigenetic change that alters the gene expression without changing the actual DNA sequence. Due to the gene inactivation, the tumour cells cannot be suppressed ultimately resulting in tumourogenesis [4, 5, 6].
Smoking causes methylation of the 5' promoter and exon 1 in human breast cells. The methylation results in the loss of expression of mRNA and protein levels. Then the subsequent methylation leads to stepwise inactivation of human mammary epithelial cells (HMEC). The HMEC escapes the M0 level in the cell cycle and results in breast tumourogenesis [4, 9].
Tobacco smoke causes squamous cell carcinoma in the pharynx region. CDKN2A/P16 gene is inactivated due to the hypermethylation of the tobacco smoke in the 5' CpG islands of the promoter region. The inactivation of the P16 gene leads improper phosphorylation of Rb-1 (Retinoblastoma protein) which thereby facilitates cell cycle progression leading to tumour formation [10, 11].
Pancreatic cancer is generally caused by homozygous deletion and loss of second allele of the gene. Only 15% of pancreatic cancer is caused due to smoking due to promoter hypermethylation on the CpG islands in the promoter region. Hypermethylation causes the inactivation of p16 function and facilitates the phosphorylation of Rb-1 protein, which results in cell cycle progression [20, 21].