1 Course : PGPathshala-Biophysics
Paper 11 : Cellular and Molecular Biophysics Module 5 : Cell Cycle Regulation and Cancer
Content Writer: Dr. Sandeep Agrawal, AIIMS, NEW DELHI Objectives:
--- 1. Overview of the eukaryotic cell cycle
2. Cell cycle regulation machinery
3. Alteration in cell cycle regulation machinery in cancer cells
--- Introduction:
Cell cycle regulation machinery functions in all eukaryotic organisms to control the cell proliferation, while cancer is a disease of uncontrolled cellular proliferation. Proper functioning of the cell cycle regulation machinery plays the central role in preventing cells from becoming cancerous.
One or the other component of cell cycle regulation is defective or mutated in most of the known cancers. In this module we will discuss the mutations in cell cycle regulation and checkpoint pathways which lead to tumorigenesis.
1. Eukaryotic cell cycle
During the embryonic growth and later development, cell division takes place in virtually every tissue.
In adult organisms most of the cells are differentiated and become quiescent. Whether a cell will undergo division or not, is of crucial importance and depends on the complex system of growth signals and the cell cycle regulatory mechanisms.
The eukaryotic cell cycle occurs in four well-defined stages:
a) S (synthetic) phase- The DNA replication takes place and the amount of DNA doubles in the cell.
b) G2 (Gap) phase- Proteins essential for cell division are synthesized and the cell size approximately doubles.
c) M (mitosis) phase- Maternal nuclear envelope breaks down, paired chromosomes are pulled to opposite poles of the cell, and nuclear division takes place followed by cell division (cytokinesis), producing two daughter cells.
d) G1 (Gap) phase- This phase signifies a waiting period before the daughter cells can undergo cell division again which happens in embryonic and rapidly proliferating tissues. After passing through M phase and into G1, a cell can continue to divide and produce daughter cells or it ceases to divide and enter a quiescent (G0) phase, which may last hours, days, or even the lifetime of the cell. When a quiescent cell gets signals to divide again, it reenters the cell cycle through G1 phase.
2 2. Cell cycle regulation machinery
2.1 Cyclin and cyclin dependent kinases (CDKs)
A cascade of protein phosphorylations relay the progression of cells through one stage of cell cycle to the next. This system involves highly regulated cyclin dependent kinases (CDKs) which are activated only after their association with another subunit called cyclin. Different CDKs associate with their corresponding cyclin subunits, which are transiently expressed at the appropriate period of the cell cycle to form an active complex with unique substrate specificity. Activated CDKs in these complexes drive the cell cycle by phosphorylating key proteins like Rb that regulate the cell cycle transitions.
2.2 CDK inhibitors (CDKIs)
The activity of CDK-cyclin complexes is controlled by CDK inhibitors (CDKIs), which constitute cell cycle checkpoints. The surveillance mechanism senses the damage in DNA and chromosomes.
The checkpoints ensure that cells with defective DNA or chromosomes do not replicate. The G1-S checkpoint monitors the integrity of DNA before its replication, while G2-M checkpoint checks DNA after replication and ensures that cell is suitable to enter mitosis. When DNA damage is detected, the checkpoint activation halts the progression of cell cycle and provides time for DNA repair machinery to function. If an irreparable damage to DNA has occurred, p53 mediated responses lead to elimination of cell by apoptosis or it enters a nonreplicative state called senescence.
Non-specific CDKIs- p21, p27 and p57
Specific cyclin D-CDK4/6 inhibitors- p15, p16, p18 and p19 collectively called as INK4 (A to D) proteins.
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3. Alterations in cell cycle regulation machinery in cancer cells
The whole cell cycle control and checkpoint system function to prevent cells from becoming cancerous and alteration in any of the component predisposes cells for development of tumor. Almost all the cancers appear to have genetic defect that inhibits proper functioning of the G1-S checkpoint, causing cells to continually reenter the S phase.
3.1 Alterations in activity of cyclins, CDKs or CDKIs
Cyclin D genes are overexpressed in many cancers including breast, esophagus, liver and certain type of lymphomas and plasma cell tumors.
CDK4 gene overexpression is reported in melanomas, sarcomas and glioblastomas.
Inhibition of CDKI (p16) activity is seen in many malignancies including some subsets of melanomas, pancreatic cancers, glioblastomas, esophageal cancer, lung cancers, soft tissue sarcomas and bladder cancers. MYC, a transcription factor, overexpressed in many cancers, is implicated in repressing the activity of CDKIs.
3.2 RB gene: governor of the cell cycle
The product of RB gene is a DNA binding protein which regulates G1-S transition in cell cycle. Inside the cells Rb is present in an active hypophosphorylated form and an inactive hyperphosphorylated form. In its active hypophosphorylated form, Rb binds and inactivates E2F family of transcription factors; preventing transcription of cyclin E. Active Rb blocks E2F mediated transcription in following two ways:
a) Rb sequesters E2F, preventing it from interacting with other transcriptional activators.
b) Rb recruits chromatin remodeling proteins like histone deacetylases and histone methyltransferases, which inhibit transcription of E2F responsive genes like cyclin E.
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When cells receive mitogenic stimuli, cyclin D is expressed and activated cyclin D-CDK-4/6 complex is formed which inactivates Rb by phosphorylation and E2F is released leading to cyclin E expression. Cyclin E expression stimulates DNA replication and progression through the cell cycle.
The loss of RB gene was first discovered in retinoblastomas but now it is evident that homozygous loss of RB gene is a common feature of several tumors including breast, bladder and small cell lung cancer. Patients of familial retinoblastoma are also at increased risk of developing osteosarcoma and certain soft tissue sarcomas. A cell heterozygous at the RB locus is not neoplastic. Tumors develop when the cell loses its normal RB gene copy and thus becomes homozygous for the mutant allele.
In cervical cancers Rb function is eliminated by the binding of an inhibitory protein, designated E7, which is encoded by human papillomavirus (HPV).
3.3 Mutation at INK4b-ARF-INK4a locus
The locus encoding p16 is most highly vulnerable genetic locus in human genome. This locus encodes for three tumor suppressor genes. P16 is encoded by gene INK4a, immediately upstream of which is INK4b locus coding for p15, another cyclin D-CDK4/6 inhibitor. P14ARF is encoded by an exon upstream of the first INK4a exon and shares exon 2 and exon 3 with INK4a. p14ARF controls the stability of p53 and acts as a positive regulator of p53. The p14ARF gene is induced by high levels of mitogenic signaling. p14ARF binds and sequesters Mdm2 in nucleolus, where it cannot access p53 thereby playing an important role in stabilization of p53 by controlling levels of Mdm2. Thus mutations in this locus would simultaneously affect two major tumor suppressor pathways in the cell, the Rb and p53 pathways, resulting in carcinogenesis.
5 3.4. TP53 gene: guardian of the genome
p53 is the most important molecule implicated in tumorigenesis. It is thought that most of the human cancers involve either mutations in p53 itself or in proteins regulating its activity. p53 response pathways are triggered by under stresses like anoxia, inappropriate oncoprotein activity (e.g., MYC or RAS) and damage to integrity of DNA. p53 carries out its tumor suppressor activity by following mechanisms:
a) Activation of temporary cell cycle arrest, termed quiescence.
b) Induction of permanent cell cycle arrest, termed senescence.
c) Induction of programmed cell death, termed apoptosis.
In a healthy cell, the activity of p53 is normally kept low by a protein called Mdm2. The activated Mdm2 protein forms a complex with p53, inhibiting the transcription factor and ubiquitinating it for proteasomal degradation. The protein kinase activity of ATM and/or ATR is activated following DNA damage is sensed. ATM inactivates Mdm2 by phosphorylating it thereby increasing the half-life of p53 and activating its ability to drive transcription of various target genes.
p53 mediated G1 cell cycle arrest is result of p53 dependent transcription of CDKI gene CDKN1A (p21). p21 inhibits cyclin-CDK complex and prevents phosphorylation of Rb, thereby arresting cells in G1 phase. The p53 protein also induces expression of DNA damage repair genes.
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p53 acts as a “guardian of genome” and guards the cell from genetic changes. When p53 mediated G1 checkpoint does not function properly, defective DNA can replicate, perpetuating mutations and DNA rearrangements that are passed on to daughter cells. This can result in transformation into cancerous and metastatic cells. p53 loss of function mutations can also result in inhibition of apoptosis, contributing to transformed cells. More than 70% of human cancers have a defect in TP53 gene including breast, colon and lung cancers.
The active form of p53 is a tetramer of four identical subunits. A missense mutation in one of the two p53 alleles leads to a loss of function because virtually all the oligomers will contain at least one defective subunit, and such oligomers have reduced ability to activate transcription. Such a mutation is an example of dominant negative mutation. Less commonly, some patients inherit a mutant copy of TP53; resulting in Li-Fraumeni syndrome. Patients of this syndrome are several folds more susceptible for developing malignant tumors and also at a younger age as compared to general population.
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In cervical cancers, HPV proteins E6 and E7- bind to and inhibit the p53 and Rb tumor suppressors respectively. HPV E5 protein is responsible for sustained activation of the PDGF receptor leading to continuous proliferation of transformed cells.
3.5 Transforming growth factor–β (TGF-β) pathway
TGF-β is a member of a family of dimeric growth factors which includes bone morphogenetic proteins and activins. In most of the normal cells, TGF-β is a potent inhibitor of proliferation. This growth factor acts by binding to a complex formed by TGF-β receptors I and II. Dimerization of receptor following ligand binding leads to a cascade of downstream signaling which results in transcriptional activation of growth inhibiting CDKIs and repression of growth promoting genes like MYC, CDK2, CDK4 and genes encoding cyclins A and E.
Mutations in TGF-β signaling pathway is common in pancreatic, colon, stomach and endometrial cancers. It is thought that TGF-β signaling pathway is involved in activation of epithelial to mesenchymal transition (EMT) in certain late stage malignant tumors.
3.6 Mutations affecting apoptosis
Abnormalities such as errors in mitosis and DNA damage can trigger apoptosis or programmed cell death. Apoptosis plays a very significant role in getting rid of excess and abnormal cells. If cells do not die when they should and instead keep proliferating, a tumor may form. For example:
In chronic lymphoblastic leukemia (CLL), cells have chromosomal translocation that activate bcl-2 gene which is known to be a critical blocker of apoptosis. This inhibits apoptosis and allows survival of tumor cells.
The most common tumor suppressor gene implicated in human cancers is p53. P53 induced expression of pro-apoptotic protein like Bax lead to programmed death of cells having extensive DNA damage. Cancers having mutated p53 gene lack proper apoptotic response and accumulation of abnormal and tumor cells.
PTEN is another tumor suppressor gene implicated in several cancers. PTEN normally acts as a pro-apoptotic protein by dephosphorylating phosphatidylinositol 3,4,5-triphosphate, a secondary messenger that functions in activation of Akt pathway which promotes cell survival, growth and proliferation and prevents apoptosis by several pathways.
8 SUMMARY:
1. The eukaryotic cell cycle has four defined phases: S, G2, M and G1. DNA synthesis takes place in S phase.
2. CDKs when associated with their corresponding cyclin subunit phosphorylate key proteins responsible for smooth transition from one stage of cell cycle to the next.
3. CDK inhibitors constitute G1-S and G2-M checkpoints which check the suitability of the cell for entering next phase of the cell cycle.
4. Almost all the malignancies have defect in cell cycle regulation and checkpoint control system leading to accumulation of damaged DNA and proliferation of cells with this abnormal DNAs.
5. In its active hypophosphorylated form Rb binds to E2F transcription factors and inhibits the transcription of genes needed for DNA replication.
6. More than 70% of human cancers have defect in p53 pathway.
7. p53 carries out its tumor suppressor activity by inducing cell cycle arrest in cells with DNA damage or inducing apoptosis if an irreparable damage has occurred to the DNA.
8. Levels of p53 are normally kept low in healthy cells by Mdm2.
9. Defects of TGF-β pathway are common in pancreatic, colon and stomach cancers.
10. PTEN is a proapoptotic tumor suppressor often mutated in certain types of cancers.
--- End of Module 5
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