The various facets of the cell cycle have been studied in detail using biochemical and genetic techniques. Recombinant DNA technology has also been used to study these aspects of the cell cycle. An array of several incidences in the cell cycle is responsible for cell division. This array of events gives rise to the production of two daughter cells, each of which contains chromosomes that are identical to the parental cell. The cell cycle consists of four separate stages. The first gap phase also known as G1 phase :- is characterized by the production of proteins and RNA's by the cells. S phase:- In the S phase, the proteins and RNAs that were synthesised in the G1 phase are required for DNA synthesis and chromosome replication which occurs during the S phase. G2 phase :- The cell checks to confirm that the DNA is intact and that the components of the cell have been replicated accurately. During the G2 phase, the cell grows further before it can divide which occurs during the mitotic phase. The phases, G1, G2 and S are together known as interphase. M phase :- eventually, the cells enter the mitotic phase, after it has proceeded through the G2 phase. During certain circumstances when mitogens or nutrients are absent, the cells go into a resting phase known as Go. (Lodish, H. et al., 2007). The progression from one phase to another is controlled by numerous checkpoints. These particular checkpoints restrict the untimely progression of the cell into the subsequent stages of the cell cycle. In the beginning, mammalian cells react to certain triggers such as growth factors when it enters the G1 phase. Towards the latter stage of G1, these cells no longer respond to these factors such as signals produced by growth factors or to growth factor regulators like TGFß. Instead, certain cell cycle proteins control the advancement of the cell through the phases of the cell cycle. The generation, stimulation and disintegration of a group of cyclins and their associates, the cyclin dependent kinases are responsible for the advancement of the cell from one stage to another. There is an additional class of proteins known as the cyclin dependent kinase inhibitors (CDKIs) which play an important role in coordinating the signals in each phase of the cycle.
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Checkpoints are present which play an important role in confirming that the DNA was copied accurately. In case there are errors in the DNA, defective DNA may be transmitted. START or restriction point (R) is the initial checkpoint present in mammalian cells. It is present in the late G1 phase. The cells undergo DNA duplication at this point and if defects are detected, the cell cycle is paused and the defect is corrected. If such a checkpoint failed to exist, the genome would be unreliable and defective cells would continue to proliferate. The final checkpoint which occurs during the S phase and at G2, confirms that a complete set of chromosomes are present. The cell enters subsequent phases when the cyclins disintegrate at each checkpoint and when they collaborate with the CDKIs. The normal development of multicellular organisms relies on the effective regulation of the cell cycle. Mismanagement of the cell cycle would eventually progress into the development of cancer.
Control of the cell cycle in Mammalian cells: Cyclins
There are thirteen mammalian cyclins that have been determined. Depending upon the phase of the cell cycle during which these cyclins are generated, cyclins are categorized into the G1 and mitotic cyclins. A section of the cyclin which consists of 100 amino acids called the cyclin box adheres to the cyclin dependent kinase or the CDK. The PEST sequences which are located on the C terminal of the cyclin box are responsible for the degeneration of the G1 cyclins. Preceding mitosis, the longer lived mitotic cyclins undergo degeneration by certain proteinases.
Cyclins adhere to the CDKs resulting in their activation. The activation of the CDKs is also dependent on a system of phosphorylation and dephosphorylation at certain residues on the kinases. p40mol5 is a protein that causes the phosphorylation of CDC2, CDK2 and CDK4. Cyclin H activates these cyclin dependent kinases. ( MacDonald F. et al., 1997)
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The cyclin dependent kinases and cyclins are regulated by certain proteins known as cyclin-dependent kinase inhibitors (CDKIs). CDKIs are classified into two categories. p21Cip1, p27 Kip1 and p57 Kip2 constitute the first class of inhibitors which restrict the functioning of all cyclin-CDK complexes except CDK4 and CDK6 complexes. CDK4I also known as 'inhibitor of CDK4' or the INK4 proteins comprise of p15 INK4B, p16 INK4A, p18 INK4C and p19 INK4D. These inhibitors affect CDK4 and CDK6 which are bound to cyclin D. (Weinberg RA., 2007)
Once DNA damage is detected, it is prohibited from progressing through the stages of the cell cycle due to the functions carried out by each of the cyclin-CDK complexes and the CDKIs. There are certain cyclin-CDKs which are involved in the control of the restriction point and they are known as the D-type cyclins. There are 4 types and they are D1, D2, D3 and D4. When CDK4 or CDK6 associate with D1 they are collectively called D cyclins which are generated in the presence of growth factors called mitogenic factors. Mitogenic signals are transmitted via the ras protein which in turn triggers the transcription of genes that code for the D-type cyclins. The Fos and Jun proteins bind together to form the AP-1 transciption factor which in turn act on cyclin D1 promoters inducing the transcription of cyclin D1. Cyclin D2 and CDK4 drive the cell through the G1 phase. A protein called Myc along with its associate, Max form a complex which promotes the transcription of the cyclins D2 and CDK4. Myc also amplifies the transcription of the E2Fs 1, 2 and 3and the protein Cul1 which causes the degeneration of p27Kip1. By increasing their concentrations, Myc drives the cell through the G1 phase into the S phase. However, when Myc affiliates with another protein, Miz-1, the transcription of p15INK4B, p21Cip1 and p27Kip1 inhibitors is repressed before the R point. (Macdonald F. et al., 1997) E-type cyclins, E1and E2, which are produced during the latter stage of G1, past the R point, affiliate with CDK2. This results in the phosporylation of several substrates that are essential for the progression of the cell cycle into the S phase. Once the cell enters the S phase, CDK2 substitutes the E-type cyclins for the A-type cyclins allowing the advancement of the cycle. While the S phase proceeds, the A-type cyclins detach from CDK2 and combine with a new CDK (CDK1 or CDC2). During the G2 phase, the A-type cyclins detach from CDC2. CDC2 then binds to the B-type cyclins (B1 and B2). The activation of several incidences which lead to mitosis is performed by CDC2, which forms a complex with the B cyclins.
The concentrations of p15INK48 and p21Cip1 increase due to the activity of TGF-ß. D-CDK4/6 complexes are prevented from phosphorylating the appropriate substrates due to the activity of p15INK48. p21Cip prevents other cyclin-CDK complexes from functioning throughout the rest of the cycle.
Role of pRB and E2Fs in the cell cycle
pRB is a protein that prohibits the cell from passing through the various stages of the cell cycle in order to monitor the transcription of genes accurately. It has certain related proteins (p107 and p130) and they are together known as the pocket proteins. The function of the pocket proteins have a direct effect on certain transcription factors known as E2Fs. E2Fs promote the transcription of genes that are involved in the cell cycle. During the G1 phase, hypophosphorylation of pRB occurs due to the action of the cyclin D-CDK4 or 6 complex. This allows pRB to function as an inhibitor to a large extent by adhering to the E2Fs. This binding prevents the E2Fs from transcribing certain genes, the products of which are necessary for the transition from G1 to the S phase and the subsequent phases of the cell cycle. After the R point, the hyperphosphorylation of the pocket proteins result in the release of the E2Fs from RB. The E2Fs then transcribe genes that give rise to proteins that are required for the transition from G1 to the S phase and the remaining phases.(Weinberg RA., 2007)
The Cell cycle and Cancer Therapies:
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The cyclins along with their associates, the cyclin dependent kinases play an integral role in the progression of the cell through its various stages. It has been found that the normal functioning of the CDKs and CDKIs has been disturbed in cancerous cells. Checkpoints, such as the R point, which are present at each stage of the cell cycle are responsible for scrutinizing the DNA that was duplicated during G1 and rectifying defects if they are detected. A reason for the development of cancer is a failure on the part of the checkpoints to detect defects in the DNA and to rectify them due to the inactivation of the CDKIs or the excessive expression of the cyclins. There are mutations in the INK4 inhibitors which which have been linked to ovarian, lung, pancreatic and esophageal carcinomas. The activity of Rb has also been repressed in many types of tumours. DNA damaging drugs or irradiation is one option which can restrict cell cycle checkpoints. (Ruddon RW., 2007). The Ras protein and its associated pathways, which are involved in receiving signals for the production of cyclin D1 have been used to develop anti-cancer drugs. CDK inhibitors such as Raf and PI3K are being studied for drug development.( Knowles and Selby., 2005) In several types of cancer, an overexpression of the cyclins is implicated. For instance, excessive expression of cyclin D, is implicated in breast cancer. P16 which is a CDKI is inactive in lung and colorectal tumours. If the expression of the CDKs is regulated, the checkpoints would rectify defects which would result in reduced proliferation or apoptosis.
Transcription is prevented by certain CDKIs. RNA polymerase II is an enzyme which is essential for the transcription of certain genes. This enzyme is phosphorylated by CDK9/cyclin T allowing the cell cycle to progress through the phases of the cell cycle leading to mitosis and proliferation of the cell. If CDK9/cyclin T is inactivated by flavopiridol which is a CDK inhibitor, RNA polymerase will be incapable of transcribing genes required for proliferation. This results in a decline in the anti-apoptotic protein Mcl-1 leading to apoptosis. Flavopiridol is a drug which restricts the activities of CDK2, CDK4 and CDK6. It is responsible for the suppression of the cell cycle in the late G1 stage preventing the cell from progressing into the S phase. It also prevents the cell from advancing through the G2 phase into the M phase. Flavopiridol has been found to augment the chemotherapeutic effects on cancer.
Indisulam is a drug that is responsible for lowering the expression of cyclin E. It is also involved in elevating p53 and p21 levels. p53 is a protein that is responsible for restricting the cell cycle when DNA damage is detected. If the defects in the DNA are large, p53 induces apoptosis.
This drug prevents CDK activity. The activity of pRB increases and as a result it prevents the E2F transciption factors from transcribing genes which are required for the progression of the cell cycle. (Dickson MA and Schwartz GK., 2009)
Figure 4: The figure above shows drugs that act on cyclin dependent kinases in order to regulate the cell cycle in cancerous cells. SNS-032 acts in the S phase of the cell cycle. It is involved in inhibiting CDK2, 7 and 9. Byrostatin-1 also functions in the S phase and is responsible for repressing CDK2 and activating p21. PD0332991 acts in the G1 phase and is involved in repressing the activities of CDK4 and CDK6. SCH727965, which acts in the M phase inhibits CDK1, 2, 5 and 9. Seliciclib suppresses the activities of CDK2/cyclin E, CDK1/cyclinB and CDK9/cyclinT. It acts in the S phase of the cell cycle. (Dickson MA et al., 2009)
UCN-01 is a CDK inhibitor. It is drug which is responsible for the restriction of the cell cycle at the G1/S transition. It is responsible for the dephosphorylation of pRB. pRB in its hypophosphorylated state adheres to the E2F transcription factor inhibiting the advancement of the cell cycle into the S phase. UCN-01 also induces the degeneration of the transcription factor E2F by ubiquitinization which takes place by the action of proteosomes. UCN-01 destroys the G2 checkpoint and amplifies the effect of cyclin B/CDK2. It also suppresses the activity of Chk1 kinase.
Cyclin dependent kinases undergoing clinical development
There are several drugs that are undergoing clinical trials. Numerous side effects have been documented.
The drug PD0332991, inhibits the activity of CDK4. It is involved in the suppression of the cell cycle in the G1 phase. Clinical trials have been performed with the drug for the treatment of human colon cancer consisting of the Colo-205 cell line. Regular administration of the drug resulted in a considerable reduction in the tumours.
CYC202 or R-roscovitine
It is a drug responsible for repressing the activity of CDK2/cyclin E. CYC202 induces the apoptosis of cells. It has been used against colon and uterine cancer. During clinical trials, the drug has been used for the treatment of HT29 and KM12 colon cancer cell lines. It has been found that CYC202 causes a reduction in the phosphorylation of pRB. Therefore pRB remains active preventing the E2F transcription factors from inducing protein synthesis. When the concentrations of the drug CYC202 were increased, phosphorylation of pRB at the SER-780 site reduced. At a concentration of 20µM, the phosphorylation at Ser-780 was reduced by 81%, a concentration of 50 µM resulted in 96% decrease in the phosphorylation of pRB and at 100µM, the decrease in phosphorylation at Ser-780 was found to be 97%. (Stephen RW et al., 2004)
SNS-032 is a drug which is responsible for the inhibition of CDK2, CDK7 and CDK9. CDK 7 and CDK 9 regulate transcription. SNS-032 prevents transcription resulting in a reduction in the proteins required for cell cycle progression. This process would eventually lead to apoptosis. Clinical trials conducted on chronic lymphocytic leukaemia cells using this drug caused apoptosis of the cells. The levels of certain anti-apoptotic proteins such as Mcl-1 and XIAP significantly depreciated due to the activities of SNS-032. However, SNS-032 had no influence on the levels of the protein, Bcl-1.(Chen R et al., 2009)
N-(3-chloro-7-indoyl)-1,4-benzenedisulphonamide functions in the G1 phase of the cell cycle and reduces the levels of cyclin E. It also increases the levels of p21 CDK inhibitors. During clinical trials, P388 murine leukaemia cells were treated with E7070 resulting in the inhibition of the cell cycle in the G1 phase. Clinical trials on mice have revealed that advanced tumours were completely reversed when treated with E7070 in 80% of the subjects. (Ozawa Y. et al., 2002)
Inhibition of the M phase of the cell cycle (Mitosis)
There are two classes of proteins known as Polo-kinase 1 and Aurora kinases that are involved in the G2-M transition. They are responsible for the binding of the tubulin fibres to the kinetochores. A complex known as the chromosome passenger complex (CPC) is present in the kinetochores. This complex contains Aurora kinase B and survivin which play an integral role in chromosome alignment and separation. If the activity of Aurora kinase B is repressed, the separation of the chromosomes would be aberrant leading to polyploidy and apoptosis. There are numerous classes of Aurora kinase B inhibitors that are undergoing clinical trials. (Schwartz GA et al., 2005)
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