Figure The structure of the cell cycle and position of checkpoints within it (ftom Genetics, Klug et al.)The cell cycle is a closely regulated mechanism, which, when the regulatory steps fail, can have huge consequences. Interphase and mitosis are the two stages of the cell cycle, and they alternate. Interphase is split into 3 distinct phases known as G1, S, and G2. During the S phase of interphase the DNA of the cell (nuclear DNA doubles) in preparation for formation of two new daughter cells. In mammalian cells the entire cell cycle can take between eighteen and twenty-four hours. The length of the G1 phase can vary hugely between cells, because this is the stage at which the cell makes the decision whether or not it will continue to replicate, and when. This pause in the cell cycle, when the cells are suspended in the G1 phase is called the G0 phase, and some cells leave the cell cycle here, and they will never replicate again, as is the case with nerve cells. This process is known as terminal differentiation, and if cells are too badly damaged, apoptosis may occur, if the damage cannot be repaired. Regulatory steps occur between the G1 and S phases, G2 and mitotic (M) phases, as well as between metaphase and anaphase of mitosis. S phase cells contain factors which induce them to enter the S phase, as well as a factor that will not allow M phase to start until DNA replication has occurred, and mitotic cells contain a factor which triggers the start of mitosis. cell cycle.jpg
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During the G1 phase a cyclin dependent kinase (cdk4), and cyclin D complex forms. This complex activates proteins such as pRB, which in turn activate the transcription of genes to produce products which are required for DNA synthesis in the S phase of interphase. If levels of pRB are reduced the cell will leave the G1 phase to enter the G0 phase. The G1 checkpoint is where the cell assesses any DNA damage, and whether the cell is the correct size. G0 phase is entered until any errors are corrected. When these corrections occur, the cell can enter the S phase, and DNA can replicate.
In normal cells a signal which informs it not to replicate is present in the G2 phase. This is true until the maturation promoting factor (MPF) is present, and active. MPF is a kinase, which means that it transfers a phosphate group to the protein, thus changing the properties of the protein. MPF is made up of two different molecules: mitotic cyclin and cyclin dependent kinase (cdk), with the cdk being the kinase part of the molecule. The mechanism by which MPF is activated involves the union of mitotic cyclin and cdk, to form inactive MPF. Cdk-activating kinase (CAK) is used to phosphorylate MPF and activate it, the CAK is followed radiply by the wee1 kinase which phosphorylates the MPF another two times as thus activates it again. Re-activation requires the presence of a phosphotase called cdc25 in its activated form. In the presence of DNA damage, such as single stranded DNA or misplaced base pairs, a kinase (chk1) is activated, and phosphorylates cdc25 and deactivates it. As long as there is no damage to DNA or mutations within genes coding for kinases, cdc25 is active, and removes two of the phosphate groups bound to the MPF, activating it again. This process is necessary to prevent the cell progressing between phases in the presence of damaged DNA.
The MPF has its effect in normal cells, on the regulatory stage between G2 and M phases. The effects of the MPF are the phosphorylation of both condensins, and H1 histones, causing chromosomal condensation. Phosphorylation of microtubule associated proteins (MAPs) increases the polymerisation and depolymerisation of microtubules and the assembly of spindles for use during mitosis, and phosphorylation of nuclear lamins, causes them to depolymerise, and cause the nuclear membrane to disintegrate. The condensation of chromosomes caused by phosphorylation of condensins and histones can be seen during the prophase stage of mitosis, and spindle formation can be seen here also. The degradation of the nuclear membrane also can be seen during prophase, and so all of these factors can be seen to initiate mitosis. The MPF also regulates itself, as it activates cyclin degradation, so switches itself of by negative feedback. Another gene which controls the cell cycle is called p53, and is normally very unstable, but when DNA is damaged p53 can become phosphorylated, and cause a series of events which lead to the blocking of DNA replication and death of the cell. When this mechanism fails cancer can develop within the body.
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MPF is also important in the metaphase to anaphase regulatory step of mitosis, as it phosphorylates anaphase promoting complex (APC). However, phosphorylation alone will not activate APC, it must also bind to cdc20, which is bound by MAD/BUB complex to prevent it binding to APC. MAD and BUB are proteins which form the MAD/BUB complex on free kinetochores, found on the chromosomes. The kinetochore is where the microtubules attach to the chromosomes of a dividing mother cell. This means that if there are any chromosomes which havenâ€™t had the spindles attached to the kinetochore properly, or if spindles havenâ€™t formed properly, then APC cannot be activated so the cell remains in prophase. Even if there is only one free kinetochore, then MAD/BUB will assemble, bind cdc20 and inhibit APC. When there are no free kinetochores, MAD/BUB complex cannot form, leaving cdc20 free to bind APC, which is then activated. When activated, APC activates a pathway which degrades proteins including mitotic cyclin and securing. Securin is a protein which binds to and inhibits separin, a protein which degrades cohesions. Cohesins hold duplicated chromatids together during metaphase, and so anaphase occurs as the chromatids separate.
As explained earlier, the p53 gene codes for the p53 protein, and is commonly referred to as a tumour supressor gene. When the cell is exposed to carcinogens (such as UV and ionising radiation) and a lot of DNA damage occurs, the damage leads to the phosphorylation of p53 and the stimulation of the p53 pathway, which then leads to the stopping of the cell cycle and apoptosis (programmed cell death). This occurs because the p53 is phosphorylated, and a build up of this occurs within the cell. This activates the expression of certain genes within the cell, including the p21 gene which encodes the p21 protein. This protein inhibits both the G1 to S and the G2 to M transitions, as well as blocking DNA replication and inhibiting mitotic cdk. In many cancer cells, mutations within the p53 gene mean that the p53 pathway is inactive in the presence of DNA damage, and the damaged or mutated DNA are able to survive and replicate. Mutations within the p53 can occur naturally, but can also be inherited, and in some cases just one copy of mutated p53 gene is enough to inactivate the pathway, even when the other gene is wild type.
Another important gene in the control of cell replication, and another commonly mutated gene in cancerous cells is the Ras proto-oncogene. This gene encodes Ras proteins, and these allow the cell to respond to external growth factors by communicating signals between the cell membrane and the nucleus, and causing the cell to divide. The protein exists in both active form (when bound to GTP) and inactive (when bound to GDP). The mechanism for cell cycle regulation by this gene is as follows; a growth factor binds to a growth factor receptor on the plasma membrane of the cell. This causes the phosphorylation of the cytoplasmic part of the growth factor receptor by autophosphorylation. This phosphorylation in turn attracts the presence of nucleotide exchange factors which cause Ras to release GDP and bind to GTP (and cause activation). This leads to a series of protein phosphorylations (Raf, Mek, MAP kinase) ending with the phosphorylation of transcription factors within the nucleus. Once this has occurred, GTP is hydrolysed to GTP, inactivating Ras. Mutations can turn Ras into an oncogene, and prevent it hydrolysing GTP, so the cell is under constant instructions to divide. Oncogenes allow cell to undergo mitosis without the usual growth factors that are needed to stimulate cell replication such as S-cyclin (which combines with S-kinase to allow DNA synthesis to occur), and MPF. This is achieved by producing excessive quantities or mutant proteins that are involved in the process of growth stimulation. Another reason for the rapid proliferation of cancerous cells is the fact that they are often insensitive to the antigrowth signals that are present in the G1 phase to prevent the cell cycle transition from G1 to S.
pRB is another tumour suppressor protein, and it controls the G1 to S checkpoint within the cell cycle. It is not phosphorylated when cells are in the G0 phase, and binds to and inactivates transcription factors, but when the cell is in the presence of growth factors and moving towards the S phase, pRB is inactivated due to the fact that it has been phosphorylated. This causes the release of the regulatory proteins, and leaves them free to express genes which allow the cell to move from G1 to S. In many cancerous cells both copies of the gene that code for the pRB protein are mutated and the cell cycle is not regulated.
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Due to the nature of cancerous cells, they need a substantial blood supply to allow the cells of the tumour to survive and respire. This is achieved through a process called angiogenesis. This process is inhibited by a protein that is expressed by the p53 gene (thrombospondin), levels of this protein fall with presence of p53 mutations. On the other hand, angiogenesis is increased by an angiogenesis activation that is expressed by Ras oncogenes.
The process of apoptosis is regulated by Bcl2 and BAX proteins, with Bcl2 blocking apoptosis, and BAX promoting it. It involves the fragmentation of DNA in the cell, and the disruption of intercellular bodies, and the dissolving of the cell into small spheres or â€œapoptotic bodiesâ€Â which are then engulfed by phagocytotic cells. In many oncogenes, excessive quantities of Bcl2 are produced, suppressing cell apoptosis, and causing unregulated cell division.
Genes which control the checkpoints G1 to S, and G2 to M, if mutated may mean that the cell cycle may continue before the DNA damage has had chance to be repaired. This will lead to a build up of mutations in the genes of the cell, potentially those required for the control of cell replication, or those that encode cyclins, and the cell may be unable to leave the cell cycle and enter the G0 phase. A build up of mutations within cells is what causes cancerous cells to form. No one factor would be sufficient to cause cancer to develop, but many gene mutations in cells allow DNA damage to go unchecked and allow the cells to replicate in an unregulated manor.