FIGURE 1-3
Diagram demonstrating the basic phases of the cell cycle
Cellular division and death is controlled by a series of specific mechanisms which if aberrant can lead to the formation of abnormal pathology. Knowledge of the cell cycle is imperative to understanding the balance between cellular proliferation and death.
Interphase encompasses the G1, S and G2 phase (Figure 1-3). It is a growth phase where cellular metabolic activity occurs in preparation for mitosis. Cellular organelles double, DNA replication and protein synthesis occurs. Chromosomes are not clearly visible in the nucleus, the nucleolus usually is. G1 is the phase where the cell grows, organelles are synthesised and cytoplasmic volume increases, a cell which does not divide again remains in this phase. The S or synthesis phase is also known as the Swanson phase where cellular DNA duplication occurs. G2 phase is the phase in which the cell resumes its growth in preparation for mitosis.
Mitosis (M phase) is subdivided into further components: Prophase, Metaphase, Anaphase, Telophase and Cytokinesis. In prophase, the chromatin condenses becoming visible in the form of chromosomes, centrioles move to opposing ends of the cell, the nucleolus disappears and fibres in the cell form the mitotic spindle. Prometaphase is marked by dissolution of the nuclear membrane and proteins attaching to the centromeres forming kinetochores. Metaphase occurs when spindle fibres cause alignment of the chromosomes forming a metaphase plate. Anaphase is marked by the separation of the paired chromosomes at the kinetochores and their movement towards the opposite poles of the cell along the spindle microtubules. Telophase starts once the chromatids arrive at the cellular opposite poles and new membranes form around the new daughter nuclei. Chromatomes are no longer visible, spindle fibres dissipate and the commencement of cytokinesis occurs. Cytokinesis is the splitting of the original cell into two daughter cells.
1.14.1 Cell cycle regulators and checkpoints
Checkpoints within the cycle assess for DNA damage. Should this be identified, the cell cycle is stalled until repairs are made. Alternatively, should repair be impossible, an effector mechanism causing cellular destruction by apoptosis is initiated. The cell cycle is controlled by proteins called cyclin-dependent kinases (CDKs) which are activated by another group of proteins called cyclins. They enable the movement of the cell between phases, acting as major control mechanisms.
FIGURE 1-1
Schematic representation of cyclin alterations within the normal cell cycle phases319
The maturation promoting factor or Mitosis promoting factor (MPF) includes the cyclin B and Cdk1 which trigger movement in the cell cycle. In the G1 phase, the main implicated cyclins are the D cyclins together with Cdk4 and Cdk6 , followed by cyclins E and Cdk2 in the S-Phase, Cyclin A and Cdk2 in the G2 phase and B cyclins with Cdk1 (Cdc2) in the Mitotic phase (Figure 1-4).
FIGURE 1-4
Diagram demonstrating cell cycle regulators and checkpoints
The replication of DNA in the S phase of the cycle is dependent on the expression of required proteins activated by the E2F transcription factor. This transcription factor is regulated by the protein product of the retinoblastoma gene pRB. Between the G0 or early G1 phase, the E2F is inactivated by pRB. In the late G1 phase the CyclnD/Cdk4 complex phosphorylates sites on the pRB releasing the E2F. This then produces proteins for DNA replication to occur. The activated E2F, stimulates in turn the production of further E2F, and Cyclins A and E enabling the formation of the Cyclin E/Cdk2compex. This further phosphorylates pRB moving the cell cycle into the S phase. The formed Cyclin A/Cdk2 complex acts on terminating the DNA replication by phosphorylating the E2F.
1.14.2 Apoptosis
FIGURE 1-5
Cellular signal transduction pathways320
The caspase class of proteins regulates apoptosis. They control its initiation (e.g. caspase 8 and 10) and execution (caspase 3, 6 and 7) by destroying cellular proteins. Initiating caspases cleave to inactive caspases at specific sites, resulting in the executing caspase degradation of cellular proteins by their proteases. Inducers of apoptosis vary. Some of these include the Fas ligand (FasL/CD95L), a type-II transmembrane protein belonging to the tumour necrosis factor family, TNF-related apoptosis-inducing ligand (TRAIL), the Apo 2 or 3 Ligands (APO-2L, APO-3L) and the tumour necrosis factor (TNF). These bind to apoptosis inducing receptors such as Fas/CD95, TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 (DR4/DR5), DR3, and the tumour necrosis factor receptor (TNFR). Once the ligands bind to receptors, recruitment of cellular adaptors such as FADD (Fas-associated death domain protein) and TRADD (TNFR-associated death domain protein) occurs and caspases are activated (Figure 1-6).
FIGURE 1-6
The role of Caspases in cellular signal transduction321
1.14.3 P21
P21or WAF1 is also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1. It is found on chromosome 6 encoded by the CDKN1A gene and binds to and inhibits the activity of CDK1 or CDK2 complexes regulating the cell cycle at G1. It is tightly regulated by p53 and in response to stress stimuli meditates the cell cycle arrest at the G1 phase. It also interacts with a DNA polymerase known as proliferating cell nuclear antigen (PCNA) regulating DNA replication and repair in the S phase of the cell cycle. Unlike p53, mutations in P21 are not associated with increased incidence of malignancy.
1.14.4 P27
P27 is a member of the gene family of Kip/Cip CDK inhibitors (CDKIs) which acts on cyclin-dependent kinases (CDKs) to exert positive and negative regulatory functions at the G1/S phase of the cell cycle95. Mutations of the Kip/Cip genes are rare but knock-out mice models of P27 have increased predisposition to tumours. Numerous studies link reduced levels of P27 with worse prognostic tumour outcomes322,323.
1.14.5 P53
p53 is a tumour suppressor protein encoded by the TP53 gene located on the short arm of chromosome 17 (17p13.1) with an important role in the cell cycle and apoptosis. It is found at low levels in normal cells.
Stress signals within the cell or damage to the DNA elevate p53 protein levels. A defect in the protein is linked to abnormal cellular proliferation and malignancy with around 50% of tumours containing p53 mutations. Its role is to arrest the cell cycle in the event of minor DNA damage and to induce apoptosis if the damage is severe. The presence of a genetic defect in p53, also known as Li Fraumeni’s syndrome, leads to a high frequency of cancer in affected individuals. Whereas increased levels of p53 induces apoptosis suppressing tumour growth, excess elevation of p53 causes excess apoptosis increasing the
ageing process. The gene Mdm2 via the Ubiquitin system is the major regulator of p53. DNA which is damaged activates protein kinases such as ATM, DNA-PK, or CHK2. Phosphorylated p53 secondary to DNA damage cannot bind to Mdm2 increasing its levels. Once DNA damage is repaired, the kinases are no longer active and p53 dephosphorylation occurs and can be destroyed by the Mdm2. p53 is also known to induce apoptosis via the mitochondrial release of Cytochrome c when it binds to Caspase9. It activates the expression of the Bax gene and Apoptotic protease activating factor 1 (Apaf1). The Bcl-2-associated X protein (Bax) gene stimulates the Cytochrome c release, which on binding to Caspase 9, induces apoptosis. A few studies have looked at the relation between p53 and CDKN1A genotypes in endometriosis. p53 is responsible for the transcriptional induction of the p21 gene (CDKN1A/WAF1/CIP1). These two molecules are known in literature to be associated with pathology sensitivity to cancer. In this study324, it has been suggested that there are significant differences in the p53 but not the CDKN1A genotype in women with endometriosis. It is postulated that the C (prp) allele of the p53 codon can be associated with development of endometriosis and potentially serve as a marker for this disease.