Mitosis (M phase), obviously is an interesting phase of the cell cycle, in which cell division takes place. But Interphase, the interlude between two M phases has also caught eye of the researchers now and it is known that this phase is indeed playing many important roles.
Mitosis is further divided into prophase, metaphase, anaphase and telophase. Interphase includes G1, S and G2 phases. Replication of DNA occurs in a specific part of the interphase called S phase which is preceded by a gap called G1 phase during which the cell is preparing for DNA synthesis and is followed by a gap called G2 phase during which the cell prepares for mitosis. Cells in G1 can, before commitment to DNA replication, enter a resting state called G0 phase which accounts for the major part of the non-proliferating cells in the human body.
The transition to different phases during the cell cycle progression is ordered and tightly-regulated process involving many checkpoints for assessing extracellular growth signals, cell size and DNA integrity.
Cyclin-dependent kinases (CDKs), a family of serine / threonine protein kinases are key regulatory proteins. Out of the nine CDKs which have been identified, five are active during the cell cycle, i.e. during G1 (CDK4, CDK6 and CDK2), S (CDK2), G2 and M (CDK1). These proteins maintain stable levels during the cell cycle, in contrast to their activating proteins, the cyclins, which increase and decrease periodically activating CDK proteins. CDKs and their cyclin partners are positive regulators whereas, cyclin dependent kinase inhibitors (CKIs) which act as brakes to stop cell cycle progression in response to regulatory signals are important negative regulators. If there is abnormal expression in any of these regulators, it leads to alterations in fundamental genetic control of the cell division and leads to cancer. Therefore, it is very important to fully understand the molecular mechanisms of cell cycle and to use these as targets for the therapeutic intervention.
Cell cycle regulation:
Different cyclins are present in different phases of the cell cycle and they will periodically activate CDKs which will induce downstream processing by phosphorylating other proteins. CDK-cyclin D complexes are essential for entry in G1 phase. The three D type cyclins (cyclin D1, cyclin D2, cyclin D3) bind to CDK4 and to CDK6. Another G1 cyclin is cyclin E which associates with CDK2 to regulate progression from G1 into S phase. Cyclin A-CDK2 complex is present in G2 phase. Then during late G2 and early M, cyclin A complexes with CDK1 to promote entry into M phase. Mitosis is further regulated by cyclin B in complex with CDK1.
Activity of CDK is also regulated by phosphorylation of the T-loop threonine by the CDK activating kinase (CAK), a serine/threonine kinase that is also involved in transcription and DNA repair.
CDK activity can be counteracted by cell cycle inhibitory proteins, called CDK inhibitors (CKI) which are divided into INK4 family and Cip/Kip family. The INK4 family includes p15 (INK4b), p16 (INK4a), p18 (INK4c), p19 (INK4d), which specifically inactivate G1 CDK (CDK4 and CDK6). The second family of inhibitors, the Cip/Kip family, includes p21 (Waf1, Cip1), p27 (Cip2), p57 (Kip2). These inhibitors inactivate the G1 CDK cyclin complexes and also CDK1-cyclin B complexes.
The restriction point (R) is defined as a point of no return in G1, following which the cell is committed to enter the cell cycle. This control is mediated by the cyclin D and cyclin E-dependent kinases. The primary substrates of CDK4/6 and CDK2 in G1 progression are the members of the retinoblastoma protein family pRB, p107, and p130. During early G1 cell phase pRb becomes phosphorylated and this leads to disruption of the complex with the histone deacetylase protein (HDAC) and releases transcription factors E2F-1 and DP-1, which positively regulate the transcription of genes whose products are required for S phase progression, including cyclin A, cyclin E, Cdc25.
When cancer happens:
When cells become cancerous they develop some special properties as loss of differentiation, self sufficiency in growth signals, limitless replicative potential, increased invasiveness, and decreased drug sensitivity.
It has been observed that the cell cycle regulators are frequently mutated in human tumours.
• Alterations in CDK molecules have been seen in few cases. CDK4 and CDK2 over expression were seen in melanoma, sarcoma, focal carcinoma and glioma.
• Involvement of cyclins has been observed in many human cancers. Indeed, the increased expression of cyclin D1 is one of the most frequent, since it occurs in ~60% of breast cancers, 40% of colorectal cancers, 40% of squamous carcinoma of the head and neck, and 20% of prostate cancers. Cyclin D2, cyclin D3 and cyclin E have also been reported in some cancers.
• Enzymes which activate CDK such as members of cdc 25 phosphatase family are also associated with tumours. Cdc25A and Cdc25B are potential human oncogenes with Cdc25B overexpressed in 32% of primary breast cancers.
• The p16 gene is altered in a high percentage of human tumours. Deletions of p16 have been reported in approximately 50% of gliomas and mesotheliomas and in 40-60% of nasopharyngeal, pancreatic and bilary tract tumours.
• pRb is also frequently mutated in human retinoblastoma and lung cancer. Approximately 90% of human cancers have abnormalities in some component of the pRb pathway.
Knowing how cell cycle regulators are playing major role in causing genetic alterations in different types of cancers, many strategies have been developed for drug development. These generally target CDKs and are known as CDK inhibitors. They are potent anti- proliferative agents and also trigger apoptosis. They act by competitive inhibition of ATP binding to CDK.
Many purine analogues, plant cytokinin analogues and pyrimidine analogues are CDK inhibitors. Roscovitine, a C2-, C6-, N9-substituted purine has a potent inhibitory activity on CDK1. CVT-313 is another purine analogue which blocks proliferation of human lung fibroblasts. Olomoucine II is a specific CDK1 inhibitor with 10 times higher inhibitory activity than roscovitine. Butyrolactone inhibits CDK1, CDK2 and CDK4 and is also known to inhibit phosphorylation of pRb and of histone H1, G1/S and G2/M transition and DNA synthesis in human fibroblasts. Flavonoids are also known to inhibit protein kinases specially Protein Kinase C (PKC). Flavopiridol specifically inhibits CDK1 and CDK2 and is a potent growth inhibitor of several breast and lung cancer cell lines. Many other CDK inhibitors have been discovered till now and many are still in process of being identified. Some other examples include kenpaullone, indirubin, Staurosporine, suramin, toyocamycin, quinazolines and aminothiazoles.
Consequently, it is very important to know about the origins of cancer so that best therapeutics can be designed to control cancerous growths.
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