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The Cell Cycle Mechanism Biology Essay

Cell cycle, is a series of events that take place in a cell and leads to its division and duplication. Cell division is essential for proper control in all living being. This process has many steps and involves many numerous proteins and ordered series of macromolecular events that leads to cell division for production of two identical daughter cells. (Alberts et al., 2008).

The cell cycle consists of distinct phases. It has two sequential processes: Interphase and mitosis (M phase). During interphase the cell prepares itself for the division. Interphase includes G1, S and G2 phases. M phase is a process of nuclear division (Enoch and Nurse, 1991). Stages of mitosis include prophase, metaphase, anaphase and telophase. DNA replication occurs in S phase. S phase is preceded by a gap called G1 phase which the cell prepares for DNA synthesis and is followed by a second gap called G2 which is after S phase. In G2, the cell prepares for entry into mitosis, follows cell division (Figure 1) (Vermeulen et al., 2003)

Figure 1: The stages of the cell cycle. The site of activity of regulatory CDK/cyclin complexes is also indicated (Vermeulen et al., 2003)

Most functional cells irreversibly exit the cell cycle to differentiate such as neurons and myocytes ( Williams and Stoeber 2011) or enter a quiescent state (the G0 phase) such as glial cells and hepatocytes (Williams and Stoeber 2007). Eukaryotic cells normally reside in a quiescent state. For the progression of the cell cycle, the cell reenters G1 to synthesize the necessary factors and prepares itself for the subsequent division (Qu et al., 2003).

There is a restriction point in late G1 which is also called R point. It is a point when cell passes this point, growth factors are no longer necessary to complete the cycle (Campisi et al., 1982). In the cell cycle progression, there are some check points which control the passage through one phase to another (Hartwell and Weinert 1989). At the transition from G1 to S phase, cells pass a checkpoint, which controls entry into the S phase. It is same in G2, there is a second checkpoint which ensure completeness and accuracy of DNA replication before progressing to the M phase. At the end of the G2/M transition, the nucleus and cell divide, and the daughter cells start a new cycle.

Time of the cycle depends on the cell size which vary in higher eukaryotes for example Drosophila, frogs ( Montagne et al., 1999) and yeast (Sveiczer et al., 1996).

More recently, the activities of various protein kinase complexes regulate the cell cycle transitions. Normal cell cycle regulators are cyclin dependent kinases (CDKs). CDKs are a family of protein kinases which controls the cell cycle progression. CDKs protein kinase subunits form a complex with a specific cyclin. Cyclin/CDK complexes play role as a regulator in the progression of cell cycle (Malumbres and Barbacid, 2009). Cell cycle phase transitions are regulated by this cyclin-CDK pairs. In G0/G1, Cyclin D-CDK4, cyclin D-CDK6 and cyclin E-CDK2 are required for the activity of transcription factor E2F. Cyclin A-CDK2 plays role in S phase and cyclin B-CDK1 regulates progression through G2 and entry into mitosis (Sherr and Roberts 2004)

Cell cycle control is critical for progression and proliferation of the cell because uncontrolled proliferation is one of the hallmarks of cancer. Loss of normal cell cycle control underlies tumor growth (Hanahan and Weinberg, 2011).

Checkpoints in the Cell Cycle Control System

In recent years scientist has focused on the cell cycle mechanism known as checkpoints. One of the main role of checkpoints is blocking the cell cycle progression until the damages are repaired which are caused by internal stresses such as defective replication and external stresses such as DNA damaging factors (Medema and Macurek, 2012)

Checkpoints are surveillance mechanisms which regulate the passage through cell cycle phases. Cell cycle transitions are not initiated until the previous process completed ( Yu et al., 1998). Uncompleted cell cycle event sends inhibitory signals to later event. By help of these ‘go’ and ‘stop’ signals, transitions take place. If the cell has appropriate size and proper nutrition, it pass next phase, if any specific threat is detected the cell has to stop dividing. Thus checkpoints function in normal cell cycle progression to provide completion of each phase of the cell cycle and allow for the repair of cellular damage before progression to next phase, to produce two genetically identical daughter cells. (Gabrielli et al., 2012).

The major threat in cell cycle is DNA damage (Walworth, 2000). In case of DNA damage, caused by radiation or mutations, the cell stop dividing until the damage can be repaired. DNA damage induces signaling network and by inactivating the CDKs, cell cycle arrest at G1 phase and cell exit the cell cycle and enter G0 state. In case of irreparable damage, check points promote cell death (Bartek et al., 2001) By this means, dissemination of harmful mutations is prevented. There are three checkpoint mechanisms during cell cycle progression. First one is on at the end of G1, beginning of S phase(G1/S boundary), second one is at the end of G2, beginning of mitosis (G2/M boundary), last one is at the metaphase/anaphase boundary.

Eukaryotic cells have signaling pathways to coordinate cell cycle transitions. Cell-cycle progression is stimulated by protein kinase complexes, (cyclin-dependent kinases-CDK) (Sherr and Robert, 1999)

In addition, loss of checkpoint controls also increases genomic instability and alterations of checkpoint components occur in many human tumors, in cancers the cell cycle checkpoints are commonly defective (Gabrielli et al., 2012)

Many tumor suppressors are components of cell cycle check points, for example p53, p16CDKN2A, and BRCA1, which play important role in checkpoint to respond different stresses (Stewart et al., 2003). For example the tumour suppressor protein p53 is a specific DNA-binding protein, which has ability to induce cell cycle arrest or apoptosis at the cell cycle checkpoints (Vermeulen et al., 2003) Mutation of p53 is the most frequently observed genetic lesion in human tumors (Stewart and Pietenpol, 2001).

CDKs and Cylincs

Cyclin dependent kinases (CDK) are protein kinase family which control and regulate the cell cycle progression (Sherr and Roberts, 1999). CDKs are serine/threonine kinases (Chang et al., 2007). CDKs are small proteins, their molecular weights ranging from 34 to 40 kDa. They are major proteins regulating the cell cycle (Moore et al., 2002). There are many activators (cyclins) and CDK inhibitors (INK4, and Cip and Kip inhibitors) which regulate the CDK activity ( Park and Lee, 2003). CDK activation requires the binding of cyclins (Malumbres and Barbacid, 2009). Cyclins are regulatory subunits of CKDs. CDK and cyclins make a complex besides CDK/cyclin combinations organizes and orders the molecular events in the cell cycle (Chang et al., 2007). Different Cyclin/CDK complexes are expressed in different phases of the cell cycle. Cyclins are synthesized and destroyed at specific times during the cell cycle and regulate it. In human cells, there are multiple loci encoding CDKs and cyclins (13 and 25 loci, respectively) (Malumbres and Barbacid, 2005). However, only certain subset of CDK/cyclin complex is directly involved in driving the cell cycle. There are four CDKs which take part in cell cycle. These are; CDK2, CDK4, CDK6 and a mitotic CDK (CDK1) and ten cyclins with four different classes. These are; A-, B-, D- and E-type cyclins (Malumbres and Barbacid, 2009). D-type cyclins bind to CDK4 and CDK6, cyclins A and E bind to CDK2, and cyclin B binds to CDC2 (also known as CDK1) (Shapiro et al., 2000)

In mammalian cells, CDK4 and CDK6 make a complex with cyclins D and E which form active protein kinase complexes that function in early in G1 progression (Shackelford et al., 1999). Cyclin A activity contributes the G1/S transition, S phase progression, and G2 to M phase transition (Girard et al., 1991; Lehner et al., 1990) Cyclin A associates with CDK2 and CDC2 (or CDK1) (Pines and Hunter, 1990; Rosenblatt et al., 1992). Cyclin A/CDK2 activity is essential for S phase progression, while cyclin A/CDC2 (CDK1) activity is necessary for G2/M progression (Shackelford et al., 1999). In short CDK2 make a complex with E and A which is essential for G1 to S transition (Pagano et al., 1993; Tsai et al.,1993). CDK1 binds to cyclin B which is required for mitosis.

Cyclin D and CDK4/CDK6

As it is mentioned above, cyclin/CDK activities characterize the each phase of the cell cycle. In early G1, a series of molecular events run the cell cycle and division. One of them is induction of D type cyclins in response to growth factors or mitogenic stimuli and the following phosphorylation of the retinoblastoma protein (pRb) by G1 cyclin/CDK protein kinase complexes (Shackelford et al., 1999). Cyclin D rapidly disappears with the removal of mitogenic stimuli (Cocks et al., 1992; Matsushime et al., 1991). There are three isoforms of Cyclin D, these are; D1, D2, and D3 (Sherr, 1993). In mammalian cells, Cyclin D-CDK4/6 is essential for the progression of cells through G, into S phase. D type cyclins are required for the activation of CDK4/6. Active Cyclin D- CDK4/6 complex mediates the phosphorylation of retinoblastoma (pRb) family protein (Shackelford et al., 1999). Cyclin D kinase activity is maximal in early to mid-G1 (Matsushime et al., 1994). Levels of D type cyclins are important, lower level of D cyclins lead the cell exit from G1 to G0 state (Matsushime et al., 1991). In Go cells, cyclin D levels are low (Won et al., 1992). In a variety of human tumors, overexpression of cyclin D is found, this shows that cyclin D may act as a positive growth regulator (Shackelford et al., 1999). However, in G1 progression, there is a point which is called restriction point, when the cell reach that point, there is no need for growth factors for the cell cycle progression (Pardee et al., 1978). At that point it is thought to coincide with pRb phosphorylation (Pardee, 1989; Hatakeyama and Weinberg, 1995). It is found that restriction point is lost in many human tumors (Levin et al., 1991)

E2F is a trancription factor, phoshorylated Rb protein become active and repress the function of the E2F. E2F is essential for the progression of G1-S phase transition, and it functions in S phase (Lin et al., 2007).

It is thought that cyclin D or CDK4/6 plays a significant role in tumor formation (Ortega et al., 2002). For instance, in breast cancer researchs, it is found that in about 50% of breast cancers exibits high levels of cyclin D (Steeg and Zhou, 1998).

CDK Inhibitors : INK4 and CIP/KIP Family

Cyclin dependent kinase inhibitors (CKIs) are tumor suppressors and able to bind to CDKs cause the inactivation of CDKs’ kinase activity, leading to a negative regulation of the cell cycle. ( Ekholm and Reed, 2000) There are two CKI families that play important role in regulation of cell division. These are INK4 family and the CIP/KIP family (Roussel, 1999). INK4 family inhibitors bind and inhibit the D-type cyclin-dependent kinases that are CDK4 and CDK6 while the CIP/KIP family inhibitors are universal inhibitors, they inhibit the activity of most of the CDK-cyclin complexes ( Sherr et al., 2004). The INK4 family consists of p16INK4A, p15INK4B, p18INK4C, and p19INK4D, all of which bind to CDK4 and CDK6 and cause the inhibition of their kinase activities by interfering with their association with D-type cyclins. The KIP/ CIP family consists of p2CIP1, p27KIP1, and p57 KIP2 ( Sherr and Roberts, 1995). This classification is based on their structure and CDK affinity.

Tumor Suppressor Genes and Rb Protein

Tumor suppressor genes and oncogenes are important regulatory genes that encode proteins which regulate transitions in and out of the cell cycle (Tripathy & Benz, 1992). Defects in tumor suppressor genes and oncogenes cause uncontrolled cell division, that leads to cancer (Tripathy & Benz, 1992). Tumor suppressor genes have many roles; they protect cells from undergoing malignant transformation, protect the genome from mutagenic events, prevent dysregulated progression through the cell cycle, induce apoptosis in cells that escape normal cell cycle controls, and inhibit cellular migration and metastasis (Hayslip and Montero, 2006) Tumor suppressor genes can be inactivated by either deletion of one allele and somatic mutation of the other allele, cause a loss of heterozygosity (Swellam et al., 2004). Alternatively, epigenetic events, such as hypermethylation of the gene inactivates the tumor suppressor genes, and cause promoter suppression so that genes can not be transcribed further (Herman et al., 1997). Tumor suppressor gene promoter hyper-methylation can be used as potential breast cancer biomarkers (Radpour et al., 2010). Many tumor suppressor genes have been identified untill today (Table).

Rb protein (pRb) is a first identified tumor suppressor protein (Sherr, 2004). It is known as a negative regulator of the cell cycle due to its ability to bind the transcription factor E2F and repress transcription of genes required for S phase (Du and Pogoriler, 2006). There are more around 100 proteins that interact with Rb. The retinoblastoma family includes three members, Rb/p105, p107 and Rb2/p130, they are called as ‘pocket proteins’. pRb represses transcription also by remodeling chromatin structure through interaction with proteins such as hBRM, BRG1, HDAC1 and SUV39H1, that are involved in nucleosome remodeling, histone acetylation/ deacetylation and methylation, respectively. Loss of pRb functions may induce cell cycle deregulation and so cause to a malignant phenotype (Giacinti and Giordano, 2006).

CHAPTER 2: p16 TUMOR SUPPRESSOR

Discovery of p16 Tumor Suppressor

P16 tumor suppressor is first discovered by Manuel Serrano and his colleagues in 1993. They isolated human p16 complementary DNA and demonstrated that p16 binds to CDK4 and inhibit the kinase activity of CDK4/cyclin D complex. They identified the p16 by using yeast two hybrid screen (Serrano et al., 1993) Yeast two hybrid screening is used to discover protein-protein interactions. (Young, 1998). A variety of extracellular interactions have been established by using two hybrid methods. Basically, the base components of yeast two hybrid are transcription activator proteins. The yeast GAL4 is a transcription activator protein that contains DNA binding and activation domain (Fields et al., 1989). The productive interaction between partner proteins ( X and Y) brings the separate activation domain (AD) into close proximity to the DNA binding domain (BD), thereby reconstituting the function of the GAL4 transcription activator protein and driving expression of a downstream reporter gene (Young, 1998). Serrano and his colleagues used yeast two hybrid screen to search for proteins that can associate with human CDK4. To test the specificity of the association between p16 and CDK4, yeast cells were co- transformed with a plasmid encoding the GAL4AD- p16 fusion and with plasmids encoding several different targets. Only the GAL4DB- CDK4 fusion interacted with GAL4AD- p16 (Serrano et al., 1993). By using CDK4 as bait in a yeast two hybrid screen, they cloned human CDKN2A, which they named p16INK4(inhibitor of CDK4) (Serrano et al., 1993)

In 1994, Kamb et al., made a research on melanoma patients and they identified a putative cell cycle regulator, p16INK4 (CDKN2) MTS1 (Mutltiple tumor suppressor-1). This gene is located on chromosome band 9p21, which in a region of less than 40 kb and it is homozygously deleted from many tumor cell lines, including melanomas. It encodes previously identified inhibitor (p16) of CDK4. It is found that this MTS1 gene is identical to that of p16 gene as identified by Serrano et al., 1993.

In 1998, it is found that the alpha transcript of CDKN2A encodes p16(INK4a), The beta transcript of CDKN2A encodes p14(ARF). P16 is a recognized tumor suppressor  which cause cell cycle arrest in G1 by binding to CDK4/6 and inhibits Rb phosphorylation. On the other hand, P14(ARF) is called p19(ARF) in mouse. It is found that p14(ARF) is shorten than p19(ARF) and these two proteins share only %50 identity (Stott et al., 1998). However both of them play role in the regulation of the MDM2-mediated degradation of p53. ARF binds to MDM2 and inhibits the ubiquitination of p53, thereby stabilizing the p53 (Pomerantz et al., 1998).

INK4/ARF Locus and p16INK4A

The INK4 gene family encodes p16INK4A, p15INK4B, p18INK4C, and p19INK4D. The four mammalian INK4 proteins have similar biochemical properties: binding of INK proteins to CDK4 and CDK6, change the conformation, and prevent the binding of these kinases to D type cyclines, that inhibits CDK4/6 mediated phosphorylation of retinoblastoma (Rb) proteins and progression into S phase. cause G1 phase arrest. INKA/ARF/INKB Locus is also called as CDKN2B/CDKN2A, it is located on chromosome 9p21 in human, on chromosome 11 in dogs, chromosome 4 in mause, chromosome 5 in rat. (Ruas and Peters, 1998; Sharpless, 2005)

The INK4/ARF locus encodes three tumor suppressors, p15INK4B, p16INK4A and p19ARF.

There are two overlapping genes in this locus. p16 and ARF have different first exons (1α and 1β) that are spliced to a common exon 2 and exon 3. The two transcripts share identical exons 2 and 3., but each regulated by its own promoter, so One promoter produce a transcript that is formed by exons 1α –2–3 and encodes p16 INK4a, while the other promoter produces a transcript that is formed by exons 1β–2–3 and encodes p19ARF (alternative reading frame). The two transcripts share identical second and third exon, but the reading frames are different and, therefore the amino acid sequences of p16INK4a and p19ARF are completely different, they are not isoforms so they have different functions. p16INK4A(also called CDKN2A) regulates the cell cycle by inhibiting the CDK4 or CDK6 cyclin dependent kinases. Loss of kinase activity inhibits the phosphorylation of pRb proteins and progression into the S phase. Additionally, in many sporadic primary tumors p16INK4a inactivation by deletion, point mutation and promoter methylation is demonstrated. These data represents p16INK4a as major tumor suppressor at 9p21. The other protein is p19ARF, the molecular weight of ARF is different in human and mice. In human it is 14kDa, 132 amino acid, so it called p14ARF, in mice it is 19kDa and169 amino acid so its called p19ARF. ARF regulates p53 pathway. knock-out data from the mouse suggesting ARF is a tumor suppressor at 9p21. ARF plays a role in the regulation of the MDM2-mediated degradation of p53, it binds to MDM2 and inhibits the ubiquitination of p53, thereby stabilizing this tumor suppressor protein.

P15 has its own readind frame and physically distict from the others.

P16INK4 Tumor Suppressor

P16 Tumor suppressor is a cyclin-dependent kinase inhibitor (CDKI) (Robertson et al., 1999). Human p16 is a 156-amino-acid protein and its molecular weight is 16 kDa (Marchetti et al., 1998). P16 tumor suppressor contains four ankyrin repeats in its structure for protein–protein interactions (Ruas and Peters, 1998). These ankyrin repeats bind to the non-catalytic side of CDK4 (Russo, 1998; Brotherton et al.,1998). P16 has biochemical ability to bind to both CDK4 and CDK6 and inhibit the catalytic activity of the CDK4 –6/cyclin D complex which is required for pRb phosphorylation and cell cycle progression (Serrano et al., 1993).

Disruption of p16/CDK4 binding inactivates the tumor suppressive ability of p16 (Russo, 1998). Mutations in the p16 binding site affect the capability of p16 binding to CDK4 and also interrupt the binding of cyclin D to CDK4, which can also lead to melanoma. (Coleman et al., 1997; Tsao et al., 1998). In addition to inhibiting the pRb/E2F pathway, the very recent researchs show that p16 has function to downregulate CDK1 expression by upregulating miR-410 and miR-650 (Chien et al., 2011). CDK1 is a very important protein kinase for cell cycle regulation during G2/M phase (Santamaria et al., 2007).

Structure of p16

P16 contain four ankyrin repeats (Tevelev et al., 1996; Byeon et al.,1998) (Figure.), each comprised of a helix–turn– helix (HTH) motif linked by β-hairpin-containing loops (Byeon et al.,1998). Ankyrin repeats are relatively conserved motifs of approximately 31-34 residues (Li et al., 2006) and they are exist in plant, prokaryotes, viruses, yeast, invertebrates and vertebrates proteins. Besides, they are involved in in numerous physiological processes through the mediation of protein_protein interactions (Li et al., 2006). The four AR motifs of P16 are arranged together in a linearly to form a helix bundle with a concave surface, clusters of charged groups are present for target binding (Byeon et al.,1998). The structure of P16 is basically does not change by binding to CDK6 (a close homologue of CDK4) (Li et al., 2006).

As it is discovered in the the crystal structures of P16_CDK6 (Russo et al., 1998) P19INK4D_CDK6 (Brotherton et al., 1998) and P18INK4C_CDK6_viral cyclin D complexes (Jeffrey et al., 2000), binding of CDK6 to the concave surface of P16 (or P18 or P19) reveals the catalytic cleft of CDK6 to P16, therefore an electrostatic interaction is formed between D84 of P16 and R31 of CDK6 (R24 in CDK4). Because R31 is present at the active site of CDK6 and its positively charged side chain could stabilize the transition state of CDK6 (Russo et al., 1998). D84 (P16)_R31 (CDK6) interaction could destabilize the transition state and thus decrease the kinase activity. This findings shows that cancer- related mutations at either R24 of CDK4 (R24C) or D84 of P16 (D84N) leads to uncontrollable cell proliferation (Sotillo et al., 2001). Furthermore, P16 inhibit the activity of CDK4/6 by interfering the binding of its activator cyclin D, however binding of p16 to CDK6 cahnges the binding surface for cyclin D, despite of the P16-binding and cyclin D-binding surfaces in CDK4/ 6 are opposite to each other (Jeffrey et al., 2000).

Most CDK4-interacting residues in P16, such as D84 are present in the second and third ankyrin repeats ( Figure) (Russo et al., 1998). Residues in the first and fourth ankyrin repeats (contains the flexible N- and C-termini) contribute little to P16_CDK4 association (Russo et al., 1998). Biochemical studies reveald that point mutations and removal of the N- and C-termini caused a decrease in the stability and solubility of P16 (Tevelev et al., 1996).

The first and fourth ankyrin repeats play important roles in binding to non-CDK proteins (Sun et al., 2010; Choi et al., 2005) as well as in posttranslational modification of P16 (Gump et al., 2003; Guo et al., 2010) First of all, according to protein fragmentation experiments it is revealed that residues 1-60 and residues in the fourth ankyrin repeats are responsible for binding to GRIM-1919 and JNKs respectively (Choi et al., 2005) Second, P16 has four phosphorylation sites (Ser7, Ser8, Ser140, and Ser152) on the N- and C-termini, phosphorylation at these four sites result different changes in the structure, function, and stability of P16 (Guo et al., 2010). Third, previous studies revealed that the first ankyrin repeats of P16 might be involved in inhibiting CDK7-CTD kinase, TFIIH (Sun et al., 2010; Serizawa, 1998). As a whole, for the structural integrity of P16, all four ankyrin repeats are required.

Function of p16

P16 acts as a negative regulator pRb/E2F pathway (Sherr and Roberts, 1999) During early G1 phase, CDK4 and CDK6 form a complex with cyclin D, this cyclin D- CDK4/6 complex phosphorylate the Rb protein family (Kim and Sharpless, 2006) E2F is a trancsription factor that initiates transcription of S phase genes. E2F is a complex with Rb protein in hypo phosphorylated status so Rb inhibits EF2 transcription factor to do its fuction (Weinberg, 1995). These inhibitory phosphorylation of Rb cause release of E2F transcription factor from Rb/EF2 complex. Therefore EF2 has access to entry into S phase (Harbour and Dean, 2000). Binding of p16 inhibits kinase activity of CDK4 and CDK6 with cyclin D that leaves Rb in un-phosphorylated form with EF2 and inhibits EF2 transcription factor (Walkley and Orkin, 2006). S phase genes can not be transcribed so cell enter G1 arrest. Binding of p16 cause a stractural changes on cyclin D binding sites of CDK so they become unable to bind to cyclin D no longer ( Russo et al., 1998). Furthermore, P27Kip1 is an inhibitor of CDK2, there is an association between p16 and P27Kip1, p16 mediated growth inhibition may occur only when cyclin E/Cdk2 complexes are inactivated by p27Kip1 (Jiang et al., 1998).

Recently it is reported that, there is a physical association between P16 and GRIM- 19. GRIM- 19 (gene-associated retinoid-IFN-induced mortality-19) is a tumor suppressor and proapoptotic protein. P16 and GRIM- 19 inhibit the cell cycle progression bia E2F pathway (Sun et al., 2010)

In addition to the pRb_E2F pathway, there are alternate and independent regulatory pathways for p16 and controlling cell cycle progression (Nishiwaki et al., 2000). TFIIH is an essential factor for transcription by RNA polymerase II (RNA pol II) (Zawel et al., 1995). CDK7 is a kinase subunit of TFIIH which phosphorylates the carboxyl-terminal domain (CTD) of the largest subunit of RNA pol II (Roy et al., 1994) First, phosphorylation of CTD of the large subunit of RNA polymerase II by the CDK7 subunit of general transcription factor TFIIH is a necessary regulatory event in transcription (Serizawa, 1998; Nishiwaki et al., 2000). P16 can interact with TFIIH in the preinitiation complex and inhibit CTD to to induce cell cycle arrest. Second, it has been demonstrated that p16INK4a inhibits the activity of c-Jun N-terminal kinases (JNKs) and it connects to the glycine-rich loop of the N-terminal domain of JNK3(Choi et al., 2005). Interaction of p16INK4a with JNK inhibits c-Jun phosphorylation which is induced by UV exposure (Choi et al., 2005). In addition, it is explicitly elucidated that p16 play role in cellular senescence and aging (Yang et al., 2008) In human and rodent tissues, it is found that level of expression of P16 is directly propotional with aging in case of both health and disease (Kim and Sharpless, 2006)

This high level of p16 expression trigger cellular senescence and aging in various progenitor cells and premalignant tumor cells such as (Collado et al., 2007) neural progenitor cells (Krishnamurthy et al., 2006), pancreas islet progenitor cells (Molofsky et al., 2006) and hematopoietic stem cells (Janzen et al., 2006).

Recent studies show that P16 could be involved in the cellular response to genotoxic agents (Serrano et al., 1993). P16- deficient cells indicated sensitivity to ultraviolet (UV) light induced apoptosis so the absence of functional P16 leads propagation of proapoptotic signaling (Al-Mohanna et al., 2004) Oppositely, high level of P16 expression in tumor cells caused cell cycle arrest and inhibition of apoptotic events, including cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These evidences show that P16 is capable of mitigating the sensitivity of mitochondria to proapoptotic signals in cancer cells with damaged DNA (Le et al., 2005)

In humans, deletion of the 9p21 region that encodes p16INK4A/p14ARF/p15 tumor suppressor loci cause tumor formation in a wide range of tissues (Kamb et al., 1994; Kleihues et al., 1994; Packenham et al., 1995).

In case of loss of p16 expression, loss of heterozygosity (Swellam et al., 2004), loss of homozygosity (Ranade et al., 1995; Quelle et al., 1997), and hypermethylation of the promoter (Herman et al., 1997) in the 9p21 region have been observed observed. Besides those mechanism also promote p16-related neoplasms. Before development of tumorigenicity in rat respiratory epithelium, hyper-methylation of the p16 promoter region appears to occur early in neoplastic transformation (Yamada et al., 2010). Loss of p16 activity as an early event in cancer progression, frequency of loss of p16 is high in pre-malignant lesions is the best prove of it (Liggett & Sidransky, 1998). Expression of p16 is an early prognostic indicator for predicting cancer recurrence (Bartoletti et al., 2007). In human colorectal cancer, hypermethylation of CpG islands in the p16 promoter causes enhanced cell proliferation, and can induce DNA demethylation in the invasive region (Jie et al., 2007). In addition, INK4A/ARF hypermethylation is seen in in mammary epithelial cells in high risk women with sporadic breast cancer (Bean et al., 2007; Jing et al., 2007; Sharma et al., 2007).

K-cyclin (ORF72) is a human homolog of cyclinD1 in Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) that is oncogenic in immune suppressed individuals. Balance of expression of p16 and CDK6 is associated with inhibition of unphophorylated CDK6-K-cyclin complex and functional availaibility of K-cyclin for tumorogenesis by p16. (Yoshioka et al., 2010). p16 forms a complex with HIF-1α, the transcription factor for the VEGF gene promoter, therefore represses the transactivation of VEGF. p16 inhibits VEGF gene expression and inhibits cancer cell induced angiogenesis in breast cancer cells and the loss of p16 is a significant transition in neoplastic development (Zhang et al., 2010).

Role of p16 in Senescence

Cell senescence is a phase which cell resting permanently, its is associated with cell aging (Smith & Pereira-Smith, 1996). There are some factors which induced the senescence. It can be induced by DNA replication stress or by oncogene expression however is most often the result of replicative senescence. Presence of p16 is related to replicative senescence. Replicative senescence is a fundamental feature of somatic cells, with the exception of most tumour cells and possibly certain stem cells (Campisi, 1997). In lymphocytes increased level of p16 expression is found before replicative senescence (Chebel et al., 2007). Senescence, induced by oncogene is connected with high expression of p16 and p14ARF(Serrano, 1997; Markowski et al., 2010). In K-ras constitutively expressing mice, deletion of the INK4/ARF gene locus causes loss of senescence and invasive, metastasizing tumors (Bennecke et al., 2010). Level of p16 expression increases as mouse embryonic fibroblasts reaches senescence (Zindy et al., 1997). It has been reported that T box proteins (Tbx2) and polycomb proteins (BMI1, Cbx7, Mel18) are repressor of INK4 locus. (Jacobs et al., 1999; Gil et al., 2004). Reppression of the INK4 locus controlled by methylation of histone 3 at lysine 27, by binding of chromobox 7(CBX7) within the polycomb repressive complex 1 to ANRIL (antisense non-coding RNA of INK4B/INK4A/ARF locus) (Yap et al., 2010). Bmi-1 encodes the polycomb protein, that represses both p16 and p14ARF and is connected with regulation of the replicative life span of human fibroblasts (Itahana et al., 2003).

The Activators INK4/ARF Locus

RAF–MEK–ERK kinase cascade, is a signaling pathway that respond to activated RAS, affects INK4a, and the mutant form of B-RAF which is prevalent in human melanomas strongly activates INK4a (Lin et al., 1998; Zhu et al., 1998; Ohtani et al., 20001) It is assumed that signalling by the RAF– MEK–ERK pathway will culminate in phosphorylation of a key transcription factor that regulates INK4a expression.

The p38 family which are also known as stress-activated protein kinases (SAPKs). The p38 family can influence cell-cycle progression in several ways and, in vivo, the accumulation of INK4a and other markers of senescence can be observed with single alleles of mutant B-RAF or RAS (Michaloglou et al., 2005; Collado et al., 2005). p15INK4b is one of the senescence markers which is identified in these studies (Collado et al., 2005). This findings suggest that Ink4b expression is increased in response to RAS–RAF–MEK signalling (Latres et al., 2000; Palmero et al., 1998 ) For ARF, RAS signalling is more complicated. Ras induces ARF expression MEFs (mouse embryo fibroblasts) (Palmero et al., 1998; Lin et al., 2001) but it has not same effect in human cells (Ferbeyre et al., 2000; Berkovich et al., 2003). RAS causes increased binding of transcription factor DMP1(cyclin D-binding Myb-like protein) to the promoter, and is unable to activate Arf in DMP1-null MEFs (Sreeramaneni et al., 2005)

2.7 Regulation of p16 Tumor Suppressor

Regulation of p16INK4a Tumor Suppressor

Since, discovery of roles of INK4A/ARF locus, it is revealed that INK proteins play role in tumor suppression and senescence, thus many scientist’s entire attention centered on the regulation of INK4A/ARF locus studies. Especially transcriptional regulation of p16INK4 has been an area of intense study in the past decade. In 1996 scientist Rodney S. Nairn and his colleagues made a research on Xiphophorus (swordtail fish), the finding of polymorphisms of the p15INK4b/p16INK4a homolog of Xiphophorus segregate with melanoma susceptibility is the prove that INK4 proteins play role in tumor suppression for more than 350 million years (Nairn et al., 1996). Regulation of p16INK4a protein may be in different stages: regulation in transcriptional level, post transcriptional level, post translational level.

Many evidence shows that INK4/ARF expression is seen in the early stages of cancer formation (Sherr, 2000) but the accurate stimuli relevant to cancer which trigger the activation of expression of the locus is still unclear.

p16INK4A and p19ARF proteins are co-regulated in rodents. There are some stimuli which particularly regulate only p16 INK4A or contrariwise p19ARF (Krishnamurthy et al., 2004; Zindy et al., 1997). Co-regulation of both p16INK4A and p19ARF proteins in human is not deeply rooted (Kim and Sharpless, 2006) For instance, senescence is related to increased expression of p16INK4A, but not ARF, in addition compelled RAS-RAF activation is just for the induction of p16INK4a in human cells (Huot et al., 2002; Michaloglou et al., 2005; Munro et al., 1999). Similarly in human aging, only p16 expression has been detected (Melk et al., 2004)

There are many noxious stimuli which induce p16INK4A and/or ARF expression in vitro and in vivo. Expression of p16INK4 increases by DNA damaging stimuli, for example, Oxygen radicals, ionizing radiation, chemotherapeutic agents, telomere dysfunction and UV light. There is an association between induction of p16INK4 expression, which is caused by these stressors, and MAPK activation (Bulavin et al., 2004; Ito et al., 2006; Iwasa et al., 2003). There are many activators which lead to induction of INK4/ARF expression, Ras activation is one of them (Ohtani et al., 2001) and p16INK4A expression can be triggered by different stimuli which are associated with cellular signaling pathways.

Regulation p16 at DNA Level

The INK4b/ARF/INK4a locus is located on human chromosome 9p21(Hannon and Beach, 1994). There is a high incidence of genetic deletion of this locus in a variety of human malignancies, such as melanoma, pancreatic adenocarcinoma, bladder carcinoma, and leukemia (Gill and Peters, 1994). There are five possible candidate tumor suppressors, P16 (Kamb et al., 1994), P15INK4B (Hannon and Beach, 1994), P14ARF (Mao et al., 1995), P16γ (Lin et al., 2007) and P12 (Roberts and Jones, 1999). The complexity of this locus and its susceptibility to genetic alterations have a bearing on P16 functions.

By using knockout mice, researchers have showed that p16, p15, and p14ARF genes are defficient in mice and more prone to spontaneous cancers than wild-type mice however pretend to be less less tumor prone than animals deficient for both p16 and p14ARF. (Latres et al., 2000; Bardeesy et al., 2006)

Overexpression of each (p16, p15, and p14ARF) leads to cell cycle arrest at the G1-G0 boundary, this shows that P16, P15, and P14ARF intensly suppress tumorigenesis synergistically. P16, P15, and P14ARF together set up one of the primary antitumor defenses in humans through the regulation of both pRb and P53 pathways. The INK4b/ARF/INK4a locus exceedingly capable of damaged because of its complexity. A single genetic alteration, such as homozygous deletions, may effect the multiple tumor suppressors (Kim and Sharpless, 2006). Besides to the homozygous deletion of the INK4b/ARF/INK4a locus which prevent the expression of p16, p15, and p14ARF, some point mutaion which is related to cancer or small deletions in exon 2 damage the both p16 and p14ARF (Kresty et al., 2008). It has also has been found that multiple genetic events, such as methylation of the p15 promoter and point mutations of p16, which accurs at the same time may effect p16, p15, and p14ARF differently (Esteller et al., 2001). Co-inactivation of two or all three p16, p15, and p14ARF genes may be more oncogenic in certain tissues than loss of just one. Likewise P15, P16, and P14ARF are all potent tumor suppressors, point mutations and intragenic alterations in the INK4b/ARF/INK4a locus impair p16 separately or together with p14ARF (Ruas and Peters, 1998). After p53, the p16 gene is the most frequently mutated gene in human cancers (Sherr and Roberts, 2004).

The estimation of p16 inactivation frequencies in different types of human tumors are (Li et al., 2000): breast cancer, 20%; non-small cell lung carcinoma (NSCLC), 65%; colorectal cancer, 30%; bladder cancer, 60%; squamous cell carcinoma of the head and neck (SCCHN), 50_70%; melanoma, 60%; leukemia, 60%; esophagus cancer, 70%; multiple myeloma, 60%; pancreatic carcinoma, ≥ 85%.

Inactivation of p16 includes four types of genetic alterations, those are: homozygous deletion, promoter hypermethylation, loss of heterozygosity (LOH), and point mutation (Li et al., 2000; Ortega et al., 2002; Forbes et al., 2006) Majority of them in p16 alterations are homozygous deletion and promoter hypermethylation (Ortega et al., 2002). The incidence and the mechanisms of p16 inactivation vary with tumor type and tumor developmental stage. Whereas homozygous deletions and abnormal methylation-mediated silencing may cause complete loss of P16 function in cells, point mutations, especially missense mutations and in-frame small deletions, may only partially impair the structure and function of P16 (Byeon et al., 1998; Guo et al., 2009) Scientist have been found that cancer-related missense mutations in at least 76 residues of P16 (Sharpless, 2005). There are four groups of residues with cancer-related missense mutations according to their mutagenic effect on the CDK4-inhibitory ability, conformational stability, and structural integrity (Guo et al., 2009).

The first group includes residues which directly involved in the association with CDK4/6, for instance; E26, D74, D84, and D92. Location of these residues are on the concave face for CDK4 binding.(Figure) Missense substitutions at these residues cause to unaltered structures, comparable conformational stability, however significantly decreased CDK4-inhibitory activities. For instance, D84H is a mutation which is commanly found in cancers and does not shows any detectable CDK4-inhibitory activity. In the second group there are most of the residues with cancer-related missense mutations as W15, E69, N71, F90, W110, and L121. These residues does not directly bind to CDK4, by stabilizing the global structure of P16, they contribute to P16-CDK4 association. The third group includes residues which are important for the formation of the core structure of P16, for instance L63, L78, P81, A100, G101, and P114. Any mutaions in these residues alarm the the global structure of P16 and therefore eliminate its CDK4-inhibitory activity. The last one consist of residues whose missense substitutions do not lead any detectable changes in the CDK4- inhibitory activity, stability, or structure. Location of residues of this group are in the first and fourth ARs and the N- and C-termini.

Regulation at Transcriptional Level

Regulation of p16 is a complex process, the reason is that the unique nature of INK4b/ARF/INK4a locus. p16, p15, and p14ARF have different independent promoters, so the product proteins have function in different pathways. They independently activated or repressed under different circumstances (Gill and Peters, 2006). Grouping genes in a single chromosomal domain have advantages, they can be regulated as a whole by the same remodeling event(s) (Kim and Sharpless, 2006), so INK4b/ARF/INK4a locus is coordinately regulated. Recently scientist proved that there is two type regulatory mechanism for p16. These are independent and coordinated regulation of p16.

Independent Regulation of p16

In the past decade transcriptional regulation of p16 has been one of the intense study area because of its role in tumor suppression, senescence, and aging. It is found that transcription of p16 is related to various regulatory elements which present in the p16 promoter (Li et al., 2011).

Ets-Binding Site-Mediated Regulation

There is a conserved Ets-binding site in the p16 promoter in position _124 to _85 interval. Ets1 and Ets2 transcription factors that are able to bind to Ets binding site which activate the p16 promoter and trigger the expression level of p16 in human fibroblasts. Ets1 and Ets2 transcription factors can be activated by MAPK-mediated phosphorylation (Ohtani et al., 2001). Id1 is a a helix_loop_ helix (HLH) protein which is a repressor of p16 expression, physical association of Ets2 with Id1 makes incapable the Ets2-mediated transactivation, while in human diploid fibroblasts abnormal expression of oncogenic Ras increase the binding of Ets1 and Ets2 to the p16 promoter (Zheng et al., 2004). The reason is that Ets2 controls the transactivation of p16, Id1 counterpoise the activation of the p16 promoter which is mediated by Ras-Raf-Mek signaling (Ohtani et al., 2001). In young fibroblasts, p16 is expressed at low levels because of a balance between Ets2 and Id1. Abnormal phosphorylation of Ets2 by oncogenic Ras may override this steady state, thus activate the 16 promoter and transcription. During senescence, the Ras-Raf-Mek signaling is reduced and the Ets2 level is low; consequently the increased level of expression of Ets1 and concomitant downregulation of Id1 result in upregulation of p16. The balance between Ets1/2 and Id1 is like a sensor which detects aberrant growth signals (mitogenic stress and oncogenic stress). Current studies demonstrate that ROS (reactive oxygen species) is a contributor to senescence which is attributed to its transactivation of p16 expression by the Ras-Raf-Erk-Ets signaling pathway (Schmid et al., 2007).

E-Box-Mediated Regulation

Id1 may also effect the transactivation of E47. E47 has an HLH domain, like other class I basic helix_loop_helix (bHLH) proteins (also known as E proteins) (Pagliuca et al., 2000). E47 mediates homo- and heterodimerization with other HLH proteins to regulate gene expression. promoter sequence of p16 contains E-box sequences. There are two E-box elements in the p16 promoter, located at positions _349 (CAGGTG) and _615 (CAGGTG) (Zheng et al., 2004). In case of homodimerization or heterodimerization with other E proteins E47 binds to these two E-boxes and activate the transcription of p16 in senescent cells. Id1 can form only a heterodimer with E47, Id1 has a highly conserved HLH domain but lacks the basic DNA binding domain. Id1 binds to E-box elements and inhibits E47-mediated activation of the p16 promoter (Li et al., 2011). In addition, Myc, is an E-box-binding transcription factor, which binds to the promoter and the first intron of p16 and upregulate its expression in human cells (Gill and Peters, 2006).

Sp1-Binding Site-Mediated Regulation.

There is a GC-rich region within the p16 promoter, which contains at least five assumed GC boxes (positions _474 to _447, _462 to _435, _380 to _355, _76 to _49, and _26 to _1), that are a sign of assumed binding target sites for Sp transcription factors which are Sp1, Sp3, and Sp4 (Wu et al., 2007). In the p16 promoter, there exist a positive transcription regulatory element ( in positions _466 to _451) and also called GC box for for Sp1 binding (Wu et al., 2007). The amount of Sp1 protein does not influence the binding of Sp1 to GC box, Sp1 binding is enhanced by increase in Sp1 affinity during cellular senescence. Furthermore, abnormal expression of Sp1 triggers the transcription of p16 in human fibroblasts (Wang et al., 2008). It is suggested that binding of Sp1 to GC box has a possitive influence on p16 transcription. Moreover, there is a coavtivator with histone acetyltransferase activity which cooperates with Sp1, it is called P300/ CBP. They both activate the p16 promoter activity and p16 mRNA expression. The histone acetyltransferase domain of P300 is able to contribute to p16 transcriptional activation by inducing hyperacetylation of histone H4 at the p16 gene (Xue et al., 2004). P300 is Sp1-dependent in p16 transcription, whereas abnormal expression of P300 is able to induce cell cycle arrest by upregulating p16 expression.

HBP1-Binding Site-Mediated Regulation

There is a binding site for the HMG box-containing protein 1 (HBP1) transcription factor at possition _426 to _433 in the p16 promoter. It is a downstream effector in the Ras-Raf-Mek signaling pathway (Li et al., 2010). Binding of HBP1 to the p16 promoter positively regulates the expression of p16 and induce premature senescence in primary cells.

ITSE-Mediated Regulation.

There is a a negative regulatory element in the p16 promoter which is called ITSE (the INK4a transcription silence element) in location _491 to _485 (Wang et al., 2001). In case of deletion of ITSE in young 2BS cells, the p16 promoter activity increase. In addition, ITSE has a binding site for Myb-related protein B (B-MYB), which is a transcription factor play role in the regulation of cell survival, proliferation, and differentiation (Huang et al., 2011)

Ap1 Site-Mediated Regulation

Ap1 is a transcription factor which is a heterpdimeric and homodimeric protein, Ap1 proteins binds to leucine zipper (bZIP) region in DNA sequence. Ap1 composed of Jun proteins (c-Jun, JunB, and JunD), Fos proteins, Jun dimerization partners (JDP1 and JDP2), and related activating transcription factors (ATF2, LRF1/ATF3, and B-ATF) (Li et al., 2011). There are three AP1-like sites in the mouse p16 promoter, which includes TGACTGA at position _1189, TGACTTCA at position _783, and TGACACA at position _484 (Huang et al., 2011). Ap1 transcription factors has different effects on cell proliferation, arrest and apoptosis (Gill and Peters, 2006). For instance ectopic expression of JunB induces high levels of P16 and induces growth arrest in mouse fibroblasts and reduce the level of proliferation in T3 cells (Shaulian and Karin, 2001). Induction of p16 expression is related to transactivation by binding of JunB to the Ap1 sites in the p16 promoter. Additionally JunB plays role as a downregulator of the expression of cyclin D1. Overexpression of JunB in 3T3 cells prevents the kinase activity of CDK4, thus pRb hyperphosphorylation reduces and G1 phase extends.

In an opposite manner, Conversely, c-Jun upregulates cyclin D1 but downregulates P16, so it promotes cell proliferation (Li et al., 2011).

PPRE-Mediated Regulation

PPRE is a peroxisome proliferator response element which is located at position _1023 in the p16 promoter (Gan et al., 2008). Peroxisome proliferator-activated receptor R (PPARR) negatively regulates the cell cycle progression at the G1 to S phase transition by triggering p16 expression in vascular smooth muscle cells (SMC). There is an association between PPARR and Sp1, PPARR binds to the canonical PPRE region and interacts with Sp1 in the proximal Sp1-binding sites of the p16 promoter, therefore enhance the p16 expression (Li et al., 2011).

Regulation Mediated by Unspecified Elements

There are some regulatory elements which are unsipesifc element but they regulate the p16 expression. SWI-SINF (SWItch/Sucrose NonFermentable) (Stern et al., 1984) is a chromatin remodeling complex,which destabilise histone-DNA interactions to regulate access of binding domains to transcription machinery (Betz et al., 2002) and capable of altering the position of nucleosomes along DNA (Whitehouse et al., 2009) SNF5 is a component of the SWI_SNF chromatin remodeling complex. In malignant rhabdoid tumor cells (MRT) re- expression of hSNF5 cause to G1 arrest by triggering the p16 expression and transcriptional repression of cyclins A, D1, and E (Oruetxebarria et al., 2004). The reason is that there is BAF60a subunit in the mammalian SWI_SNF complex which interacts with JunB, and SWI_SNF complex may initiate the p16 promoter, thus hSNF5 may activate p16 transcription.

Additionally there are a few repressors of INK4a/ARF/INK4b expression. For example Polycomb group (PcG) genes (BMI-1, Cbx7, Mel18) which are first identified in Drosophila, have been reported to repress all three genes (p16 INK4a, p15INK4b, and ARF) (Gill et al., 2004; Jacobs et al., 1999). These Polycomb group of silencers (PcG) maintain patterns of developmental gene expression (Orlando et al., 1998; Francis et al., 2001)

In recent studies, there is a newly identified transcription activator which is called BRG1 is a catalytic component of the SWI_SNF complex (Medina et al., 2008; Becker et al., 2009). Whereas BRG1 I not necessary for cell cycle inhibition which is induced by p16, P16 and BRG1 interaction negatively regulates the chromatin remodeling activity of BRG1 (Li et al., 2011) Recently, lymphoidspecific helicase (Lsh) is demonstrated as a a member of the SNF2/helicase familywhich has a role in p16 regulation (Zhou et al., 2009). Overexpression of Lsh in human diploid fibroblasts postpone the cell senescence by silencing p16. This repression cause to Lsh-related deacetylation of histone H3 at the p16 promoter.

Coordinated Regulation of p16, p14ARF, and p15

Despite the fact that through regulation of p16, p14ARF and p15, there are some stimuli which regulate p16 but not p14ARF or p15, these three genes are expressed both in normal tissues and insignificant number of tumor specimens (with whole INK4b/ARF/INK4a locus) (Ortega et al., 2002; Kim and Sharpless, 2006; Collado et al., 2007). In spite of 50 different regulatory domains p16 and p14ARF mRNAs are extremely stable, which is  hypothetically attributed to their shared 30 sequences (Guo et al., 2010). According to these common regulatory features, there might be a mechanisms controlling p16, p14ARF, and p15 simultaneously. This assumption supports that, p14ARF, and p15 are simultaneously downregulated by expression of PcG proteins, such as Bmi1, Cbx7, Ring1b, or Phc2 (Jacobs et al., 1999). PcG proteins are

transcriptional repressors, they recognize histone modifications so transcriptionally silencing chromatin (Gonzalez et al., 2006).

Bmi1 is a potent repressor of of INK4a. Bmi1 was first identified by its ability to cooperate with Myc in the induction of mouse lymphomas (Haupt et al., 1991; Van Lohuizen et al., 1991) In Bmi1 knockouts it is found that Bmi1-deficient mouse embryonic fibroblasts encounter premature senescence and accelerated accumulation of p16, p19ARF, and p15. Besides, abnormal expression of Bmi1 enlarge the age of death of both mouse and human fibroblasts. Presence of the multiprotein complex which contains EZH2 (a histone methytransferase) and (PRC 2) (Polycomb-repressive complex 2) cooperate with the Bmi1-mediated repression of the INK4b/ARF/INK4a locus (Canepa et al., 2007)

Additionally, Cbx7 is one of the Polycomb protein, , localized to nuclear Polycomb bodies and interacts with Ring1 (Bezsonova et al., 2009; Maertens et al., 2009). Cbx7 transcriptionally repress the INK4b/ ARF/INK4a locus independently from Bmi1 and extends the life span of normal human cells and immortalizes mouse fibroblasts (Gill et al., 2004) Recent studies revealed that CDK6 has a physical interaction with Bmi1 in young MEF cells, thus, recruiting PRC 1 and PRC 2 to the INK4b/ARF/INK4a locus and transcriptionally silence the locus as well as its replication during the late S phase (Agherbi et al., 2009).

There is another transcriptional corepressor which is called CtBP (COOHterminal binding protein), it is capable of access the p16 promoter enhance the PcG-based epigenetic histone mark, therefore help p16 silencing by DNA methylation (Mroz et al., 2008). CtBP-mediated repression of p16 can be reduced by increased level of ROS. CtBP has very little influence on the expression of p14ARF, its influence on the expression of p16 or p14ARF is different.

Regulation of p16 at Post Transcriptional Level

There are many stimuli or genetic alternations that regulate the mRNA expression of INK4A/ARF locus, there is less knowledge about post-transcriptional regulation of the p16INK4A. Posttranscriptionally, p16 mRNA splicing is influenced by ASF/ SF2 and the p16 mRNA stability which is reduced by RNA-binding proteins (RBPs) hnRNP A1, hnRNP A2, and AUF1 (Zhu et al., 2002; Wang et al., 2005). RNA binding proteins hnRNPA1, A2 and microRNAs: miR-24 play roles in p16INK4A regulation on mRNA level.

RBPs (RNA-binding proteins) are trans-acting factors which regulate gene expression after gene transcription. The regulation may be on different levels, for instance, pre-mRNA splicing and maturation, mRNA transport, storage, stability, and translation (Moore, 2005) HnRNP (Heterogeneous nuclear ribonucleoproteins) A1 and A2 are RNA binding proteins, may play important roles in the biogenesis of mRNA in vitro -in vivo and act as trans-factors in regulating gene expression (Chaudhury et al., 2010) HnRNP proteins are multifunctional: they participate in pre-mRNA processing such as splicing and are important factor of mRNA export, localization, translation, and stability (Dreyfuss et al., 2002) HnRNP A1 and A2 may affect gene expression of eukaryotes in a tissue-specific manner (Kamma et al., 1995; Hanamura et al., 1998; Kamma et al., 1995) HnRNP A1, hnRNPA2 and SF2/ASF (human premRNA splicing factor) are capable of modulating 5’splice site selection in a concentration dependent manner (Mayeda et al., 1992; Mayeda et al., 1994)

The alternations in the ratios of hnRNP A1 and A2 to human pre-mRNA splicing factor SF2/ASF may effect exon skipping (Mayeda et al., 1993) They play role in shuttle between the nucleus and cytoplasm to regulate the mRNA transport (Pinol-Roma and  Dreyfuss, 1992).

As a summary, hnRNP A1 and A2 participate in a variety of cellular functions, consist of exon skipping, mRNA splicing, trafficking, and turnover. HnRNP A1 and A2 RNA binding proteins are less active in senescent human fibroblasts. It is thought to be that changes in the mRNA ratios of ARF and p16INK4A influenced by the changes in the expression of hnRNP A1 or A2 proteins (Sherr, 1998)

In senescent human fibroblasts, when hnRNP A1 and A2 ration decrease versus to SF2/ASF ratio in the nucleus, this cause to a shift to the more proximal splice site which leads the predominance of the p16INK4A than p14ARF. Alternations in the ratios of hnRNP A1 and A2 to SF2/ASF may affect the exon skipping (Mayeda et al., 1993). However, increase in the levels of A1 or A2 related to SF2/ASF may cause to preferential generation of the ARF isoform (Zhu et al., 2002). If expression of hnRNP A1 and A2 reduce in senescent fibroblasts than in young cells, the ratio of ARF should be less than p16INK4A (Hubbard et al., 1995). This reduced rate of ARF mRNA in senescent cells, shows that p16INK4A protein levels may be higher than that of p14ARF. The ratio between hnRNP A1 or A2 proteins and SF2/ASF splicing factors has function in alternative splicing of INK/ARF locus. The increase in hnRNP A1 or A2 to SF2/ASF ratio will help the production of ARF mRNA whereas the decrease will help the production of p16INK4A mRNA. Overexpression of hnRNP A1 and A2 raise the steady state of p16INK4A mRNA and ARF mRNA, suggesting that hnRNPA1 and A2 may influence the mRNA stability (Zhu et al., 2002). Presence of hnRNP A1 or A2 protein in the cytoplasm may have interaction in direct manner with the mRNAs of ARF and p16INK4A conferring mRNA stability or they can bind mRNAs of transcription factors which regulate the transcription rates of ARF and p16INK4A mRNA.

For p16INK4A expression, the mRNA stability of p16INK4A by increase of hnRNP A1/A2 or the mRNA stability of transcription factors are responsible. HnRNP A1/A2 increase the steady state levels of ARF and p16INK4A (Zhu et al., 2002).

p16INK4A translation suppressed by miR-24

MicroRNAs , also called miRNAs are small, non-coding RNA molecules that are found in animals and plants, they play role in transcriptional and post-transcriptional regulation of gene expression (Chen and Rajewsky, 2007). miR-24-2 (hereafter miR-24) is described as a microRNA which suppresses p16INK4A translation in cultured human cells (Lal et al., 2008)

Hela cells are used to investigate the role of miR-24 on p16INK4A expression. By using Hela cells polysome fractionation followed by RT-qPCR analysis. As a result, it is found that miR-24 is localized predominantly in fractions 1 and 2, dissociated from the translational apparatus (Lal et al., 2008).

And by using RT-qPCR, it is monitored the overexpression of miR-24 in HeLa cells by transfecting premiR-24, consequently, it is revealed that overexpression of miR-24 in HeLa cells do not significantly alter the relative distribution of miR-24 on polysome gradients or influence the p16INK4A mRNA levels.

However, when it is compared to the control group, a decrease is observed in p16INK4A protein level. This decrease in protein levels is not because of the lower p16INK4A translational initiation, it is because, overexpression of miR-24 reduces the elongation p16INK4A during translation (Lal et al., 2008).

In addition to regulation of miR-24 levels by transfection of premiR-24, a transcript antisense (AS) to miR-24 is introduced to get more insight about the influence of miR-24 to the translation of p16INK4A. After 48 h after transfection of AS-miR-24, the levels of miR-24 were reduced. In overexpressing AS-miR24 Hela cells a small increase in p16INK4A mRNA level is observed , when it is compared with control group (Lal et al., 2008).

The translational initiation is initiated after levels of miR-24 is decreased. Changes of miR-24 immediately influence the level of p16INK4A expression. It is predicted that miR-24 binds to the coding region and the 3’–untranslated region of p16INK4A mRNA. The results suggest that miR-24 does not influence the p16INK4A mRNA levels however suppresses p16INK4A translation

Regulation of p16 at the Posttranslational Level

Phosphorylation

It has been reported that senescence in human prostatic epithelial cells (HPEC) promotes phosphorylation of p16 other than inducing high level of expression of p16 protein (Sandhu et al. 2000). Scientific studies revealed that p16 is phosphorylated at four specific serine sites, Ser7, Ser8, Ser140, and Ser152, in human fibroblast cells (Gump et al., 2003). These four Ser residues do not directly bind to CDK4/6 as it is demonstrated in the crystal structure of the P16/CDK6 complex (Russo et al., 1998) but in familial and sporadic melanomas, in these residues mutations have been found. This shows the the importance of p16 phosphorylation in cancer.

Notably, in WI38 cells, just Ser152 is phosphorylated in CDK4/6-bound P16 (Gump et al., 2003), suggest that physiological effects of phosphorylation at these four residues may be different from each other. Moreover, It has been shown that p16 specifically binds to IKKβ, IKKβ is Serine kinase that plays an essential role in the NF-Κ-B signaling pathway, it is a primary kinase for phosphorylating IkBR, in human fibroblast cells (Gua et al., 2010). This binding leads the phosphorylation of p16 at Ser8, as a result inhibits its CDK4-inhibitory activity. These findings shows that phosphorylation of P16 represents an important mechanism of p16 regulation. Furthermore, protein phosphorylation is associated with the level of intracellular oxidative stress (Yang et al., 2008; Sandhu et al., 2000).

Oxidative stress may cause P16 phosphorylation, that allow tumor cells to enter cell division arrest and premature senescence, therefore keeping them from progressing into malignant ones.

Degradation

It is revealed that p16 is degraded in a proteasome-dependent manner in vivo (Chen et al., 2007; Ben-Saadon et al., 2004), it is assumed that as p53, p16 is led to ubiquitination-mediated proteasomal degradation by phosphorylation. However whereas conjugation of ubiquitin to an internal lysine is essential for the ubiquitination- mediated proteasomal degradation of most proteins, p16 is lysine-free (Ben-Saadon et al., 2004). Furthermore, N-terminus of endogenous p16 is totally acetylated, therefore this make p16 unsuitable for nonlysine polyubiquitination at the N-terminal residue (Chen et al., 2007). Additionally, The 26S proteasome consists of the 20S and 19S subunits. The degradation of p16 is independent from ubiquitination and it only requires the 20S catalytic core, not the entire 26S proteasome.

Coordination with Other Proteins

p16 has many interaction with other proteins (Sherr and Roberts, 1999; Li et al., 2006). The proteins which influence the interactions between p16 and its targets, contribute to the regulation of p16. Some proteins are found that positively and negatively modulate p16/CDK4 association. Some of these proteins are Cyclin Ds, other INK4 proteins, KIP inhibitors, GRIM-19, NF-kB, Gankyrin, SEI-1/TRIP-Br1, Tax.

GRIM-19 is capable of suppress STAT3-dependent transcription and oncogenic transformation in HeLa cells by IFN-β/RA induction (Sun et al., 2010). Ectopic expression of GRIM-19 suppress the expression of genes controlled by E2F1. Ectopic expression of GRIM-19 also leads formation of a ternary complex (CDK4, p16, and GRIM-19), and the presence of GRIM-19 enhances the binding of P16 to CDK4 (Sun et al., 2010). Conversely, high level of D1 cause to loss of the CDK4/P16/GRIM-19 complex. Binding of cyclin D1 prevents interactions of CDK4 with the P16/GRIM- 19 complex.

Gankyrin is a newly defined regulatory subunit associated with the 26S proteasome (Higashitsuji, et al., 2005; Li and Guo, 2010). There is a relation between overexpression of gankyrin and human cancers such as colorectal (Tang et al., 2010), pancreatic (Meng et al., 2010) and lung cancers (Man et al., 2010). Gankyrin plays role in two pathways INK4_CDK4_pRb and ARF_MDM2_p53 as a negative regulator. On one hand gankyrin binds to the ubiquitin ligase MDM2 and promotes p53 ubiquitination and subsequent proteasomal degradation (Higashitsuji et al., 2005). On the other hand, gankyrin physically interacts with pRb and facilitates the latter’s phosphorylation and degradation (Higashitsuji et al., 2000)

SEI-1 gene is a candidate oncogene, its location is 19q13.1 in human chromosome, it is a region frequently amplified in human breast, esophagus, ovarian, lung, and pancreatic cancers (Tang et al., 2002). The product of SEI-1 is P34SEI-1 (also named SERTAD1 and TRIP-Br1) plays role in multiple physiological processes (Hsu et al., 2001). P34SEI-1 inhibits apoptosis by stabilizing the X-linked inhibitor of apoptosis protein (Hong et al., 2009) and it binds to CDK4 (but not CDK6), this binding appears to antagonize the function of p16, therefore rendering CDK4-mediated phosphorylation of pRb resistant to the inhibitory effect of p16 during the late G1 (Li et al., 2004).

Tax is encoded by human T-lymphotropic virus 1 (HTLV-1) genome DNA exon 2, it is a transcription activator, very important for both HTLV-1 viral gene expression and transcription regulation in HTLV-1-infected cells (Higuchi and Fujii, 2009; Matsuoka and Jeang, 2011). There is a corrleation between the expression of Tax protein in HTLV-1-infected cells and increase in CDK4 activity (Higuchi and Fujii, 2009; Matsuoka and Jeang, 2011). Tax is capable of form a a binary complex with p16 in vitro and in vivo, so counteract the the CDK4/6-inhibitory activity of p16 , resulting in cell cycle progression (Li et al., 2003) and tax protein directly binds to CDK4 and stimulates the latter’s activity in phosphorylating pRb (Li et al., 2003).

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