Cell cycle regulation is important for the development of multicellulair organism. Loss of this control may cause cancer. The cell cycle has four phases: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitoses (M). In the S phase the complete genome can be duplicated and subsequentely the two copies are divided in two daughter cells in M phase. In the G1 the cells increase in size, produce RNA and synthesize protein. This phase is a important cell cycle control mechanism because it ensures that everything is ready for the DNA syntheses. The second control checkpoint is the G2, this checkpoint determines if the cell can proceed to M and divide.
www.cellsalive.com/cell_cycle.htm Authorization for DNA synthesis and chromosome segretation is tightly controlled by cyclins and their associated kinase activities that becomes active in the G1 and G2. The activity of Cyclin D and E are necessary at the checkpoint G1 and the activity of Cyclin A and B1 are important to go in M after the G2 checkpoint. Mitogenic and anti-mitogenic stimilu can stimlulate or block the cell cycle progression by targeting these cyclins. (Bron floris) .
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The retinoblastoma proteins, consisting retinoblastoma protein (pRb), p10 and p130, are essential for the cells to go from G1 to S phase by activating E2F transcription factors.
In figure 1 the cell cycle is shown with the different cyclin-cdk complexes also the pRb is shown in this figure.
Figure 1 Cell cycle
The pRb can be hyper- or hypophosphorylated, where pRb in hypophosporylation can bind and inhibit E2 factors. Since pRb is dephoshorylated late in mitoses it needs to be phosphorylated during G1, to activate E2F subsequentely allowing the cell to entry into S phase. E2F, a essential DNA synthese factor, is a transcription factor to transcribe genes. Cyclin D and E are important for the phosphorylation of pRb, what depends on the mitogen. Normal growth conditions reduce the activity of Cyclin D and E, Cdk4/6-Cyclin D phosphorylates pRb, so it inactivates pRb, whereby E2F becomes active, shown in figure 2. The two homologues of pRb, consisting p107 and p130, are also involved in the E2F regulation activity. These three homologues gave a different binding site, together called the pocket domain. In cancer cells the G1/S checkpoint is loss in most cases caused by loss of pRb.
Figure 2 phosphorylation of pRb
The cyclin-CDK complexes are regulated by cyclin depented kinase inhibitors (CKIs). CKI phosporylates CDK whereby the active binding site of CDK becomes inactive. Different kinds of CKIs are know, p21 and p27 are one of them and are mainly active in the G1/S checkpoint. Bron: Ablation of the retinoblastoma gene family deregulates G1 control causing immortalization and increased cell turnover under growth-restricting conditions jan hermen.
Cyclin dependent kinsase inhibitors
The cyclin dependent kinase inhibitors (CKIs) is composed of Cip/Kip proteins such as p21 and p27.
The cyclin depented kinase inhibitor p21 promotes cell cycle arrest in response to many stimuli. The tumour suppressor protein p53 mediates the DNA damage induced checkpoint through the transactivation of various growth inhibitors or apoptetic genes. One of these proteins is p21, what mediates p53 depented G1 growth arrest.
Additionly some studies indicates that p21 act as a master effector of multiple tumour suppressor pathway. Recent studies suggest that besides the role in bloking cellular proliferation and its ability to promote differentiation it can also under certain conditions promote cellulr proliferiation and oncogenicity. P21 is often mutated in human cancers, but the role depends on the cellular context and circumstances, whereby it can act as a tumour supressor or as an oncogene.
Binding to PCNA, p21 competes for PCNA with DNA polymerase-delta and several other proteins involved in DNA-synthesis, thus p21 directly inhibits DNA synthesis. P21 also inhibits CDK activity indirectly by interfering with the activating phosphorylation of CDK1 and CDK2. The inhibition of CDK2 activity causes inhibition of cell cycle progression, which is required not only for the phosphorylation of rb (consequent active E2F), but also for the firing of replication origins and the activity of proteins directly involved in DNA synthesis. Although this activity is shared by other CDK inhibitors such as p27 and p57, biochemical and genetic evidence suggest that they have distinct roles in tumorgenis (bron 18 van p21 review). Nevertheless, p21 is uniquely positioned to function as a central inhibitor of CDK2. In response to variety of cellular and environmental signals CDK2 is activitated to promote tumor suppressor activities. Besides inhibition of CDK2, p21 also inhibits the kinase activity of CDK1, that may be the crucial target of p21 in tumorgenesis, leading to growth arrest in the G2 phase of the cell cycle.
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p21 responds to a variety of stimuli to promote growth-inhibitory activities that depend primarily on its ability to inhibit the kinase activity of cyclin-dependent kinase 2 (CDK2). p21-induced cell cycle arrest also depends on its ability to inhibit CDK1. p21 can inhibit cellular proliferation independent of CDK2 inhibition by inhibiting proliferating cell nuclear antigen (PCNA), which is required for S phase progression.
Like p21 also p27 is binding to and regulating the activity of Cdks. P27 has been implicated in mediating several growth inhibitory signals including transforming growth factor-beta (TGF-beta) and contact inhibiton. (cyclins and cell cycle checkpoints Johnson and walker) In G0 and early G1, p27 is maximal active and it binds and inhibits cyclin E-Cdk2. Also the E2F transcription factor activates p27 promoter and therefore leading to feedback inhibition of E2F action. (p27 artikel) In early G1, p27 also promotes assembly and nuclear import of cyclin D. D-type cyclin Cdks are activated by mitogens to cause G1 progression. (p27 a barometer of signaling deregulation and potential predictor of response to targeted therapies) During G1 p27 is decreasing, this permits cyclin E-Cdk2 and cyclin A-Cdk2 to activate G1-S transition and take part in the initiation of DNA-replication. (p27 en a barometer of signaling ….)
P27 is integrating diverse signals into a final decision between proliferation and cell-cycle exit. The Cdk-inhibitory activity of p27 are controlled by its concentration, subcellular localization and phosphorylation status. p27 increases in response to cell density, differentiation signals following loss of adhesion to extracellular atrix and in response to growth inhibitory signaling by transforming growth factor. Although p27 is not a classic tumour suppressor like p53, it is also rarely mutated or deleted in human cancers, but it is frequently deregulated in cancer. In most human cancers p27 protein levels are reduced or the protein is mislocalized. The inactivation or acceralation in proteolyses of p27 in human cancers is caused by cooperation of oncogenic activation of receptor tyrosine kinases (RTK), phosphatidylinositol 3-kinase (PI3K), SRC, or Ras-mitogen activated protein kinase (MAPK) pathways. P27 artikel
Rb deficintie tumours
Loss of the activity of pRb can cause different tumours: retinoblastoma, lung, breast, and celvic carcinomas. Retinoblastoma is a rare diseases that occurs by young children, under the age of 5. It is a retinal tumour caused by a mutation in the retinoblastoma allele. Mutation in the Rb allele causes pRb loss and it is also seen in other tumours. The loss of Rb has different effects that will be descriped below.
Rb plays a role in the cell cycle re entry in quiescent stem cells, loss of rb function leads to exit from quiescence and an increased number of stem cells without loss of self-renewal capacity.
Also rb loss can initiated cacer by allowing fully differentiated cells to re-enter the cell cycle.
Rb seems to have a separate function in the ATR pathway, which responds to signle stranded DNA resulted from stalled replication forks and induces DNA repair. The Rb deficient cells fail to induce cell cycle arrest after UV-induced lesions, however the DNA repair mechanisms are rapidly engaged.
It is shown that the role of Rb in the response to DNA damage might depend on significantly on cellular context. Precence of RB seems to be beneficial to tumour progression,
Similar to apoptosis, loss of rb might increase autophagy-mediated cell death and presence of rb might be beneficial to early tumours under some conditions, although more studies are required to better understand the role of rb in authophagy.
Similar to its role in progenitor cells undergoing differentiation, the presence of rb in tumour cells could in theory promote their differentiation and thereby restrict their proliferative potential, which might explain why rb must be lost during the progression from a well differentiated to a poorly differentiated tumour. Because some anticancer strategies force some tumour cells to undergo differentiation, this aspect of rb function might have important therapeutic implications.
Loss of rb can lead to chromosome segregation defects through the misregulation of genes that are important for processes such as centromsome duplication and dna replication. Rb deficient cells fail to properly arrest in G1 upon dna damage and might replicate mutated dna leading to the accumulation of mutations.
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Loss of rb promotes angiogenesis. Emerging evidence shows that vascular endothelial growth factor and other angiogenic factors that are secreted by tumour cells to recruit endothelila cells are transcriptional targets of the rb-e2f pathway.
A related point is that abnormal proliferation that is induced by loss of rb is often accompanied by increased cell death, through 53 dependent and indepented mechanisms.
Besides loss of the cell cycle control checkpoint, there is also find genomic instability like aneuploidie (abnormality in chromosome number) and chromosomal rearrangement, interestingly this is also found in retinoblastoma. Therefore loss of Rb increases polypoidie (same chromose in one nuclei), abnormality in chromosome number and forming of micronuclei (chromosome fragments that are not incorporated into the nucleis at cell division bron groups.molbiosci.northwest.edu/Holmgren/glossary/definitions/Def-M/micronuclei.html
En bron tanja paper, loss of rb1 induces non proloferative retinoma: increasing genomic instability correlates with the progression to retinoblastoma.
In precancerous lesions and cancers, activated oncogenes induce stalling and collapse of DNA replication forks, which in turn lead to formation of DNA DSBs. A number of stalled DNA replication fork induces DNA replication stress (systemic state in the cell that leads to collapse of DNA replication forks). DNA replication stress can be defined as any systemic state in the cell that lead to collapse of DNA replication forks, that is, to dissoctiation of the replication proteins from the DNA. Pathways that are induced to complete DNA replication after fork collapse often involve recombinogenic process and formation of DNA DSBs. Genomic instability in human precancerous lesions and in cancer is induced by the oncogenes themselves.
Telomere erosion which can lead to transient surges in Genomic instability, when telomeres become critically short but before telomase experession is induced. Telomere erosion almost certainly contributes to genomic instability in human cancers, but wheter it contributes to genomic instability in human precancerous lesions is less clear.
Second mechanism involves permenant increases in genomic instabilitu induced by mutations in genes whose function is to preserve genomic integrity. Dna repair and cell cycle checkpoints. Human cancers has chromocomal instability in stead of microsatellite instabilitu caused by dna mis matchrepair genes.
Cin can lead to dnA DBSs it is possible that oncogeneinduced dna damage rather than inactivation of caretaker genes may contribute to cin in human cancer.
Genomic instability us cisiered critical for cancer development because it would be difficult otherwise for a normal cell to accumulate all the mutations necessary to become a cancer cell.
DNA DSBs in human precancerous lesions and cancers are continuous induced by activated oncogenes. Most oncogenes deregulate entry into the cell cycle and do so by directly or indirectly enhancing the activities of the CDKs that function in the G1 and S phase.
It is still unclear whether genomic instability induced by DNA replication stress has any role in cancer progression, but, in oral precancerous lesions, LOH at the common fragile site FRA3B predicted progression to cancer much better than any other marker, including p53 mutations and LOH at the INK4a/arf locus.
Our findings indicate that unscheduled and moderate expression of cdc25b during s phase is sufficient to induce replicative stress and genomic instability, since abnormal expression of cdc25b has been found in numerous cancers our results provide new insights into the molecular mechanisms of the involvement of this phosphatase in tumorgenesis.
Genomic instability is also observed in retinoblastomas (dimeras et al 2008) however it has also been suggested that interference with prb function could promote polyploidy, aberrations in chromosome number or micronuclei formation. How these alterations orginate in prb function is not entirely understood. It has been suggested that dna double strand breaks are not properly repaired in prb depleted cells. S phase cells exposed to genotoxic stress slow down replication fork progression to suppress the formation of dsbs this process required de-phospohrylation of prb and therefore the inability of prb-defective cells to decelerate s phase progression could promote dna damage.
It is known that normal cells shows G1 arrest, when cultured without mitogens. Cells without the Rb proteins (TKO Mefs) do not show a G1 arrest however they show a G2 arrest. So these cells go through S phase caused by a up-regulation of p21 and p27. In figure 3 the cell cycle is shown like in pRb loss, cultured without mitogens.
We also know that human retinoblastomas show genomic instability, but so far there is no link with DNA repair. The aim of this project is therefore if the loss of G1/S checkpoint in combination with a mitogen deprived condition will induce the genomic instability and form oncologic transformation.
Our hypotheses is that the unscheduled S phase in TKO-Mefs causes replicationproblems what results in DNA damage. To learn more about the S phase, we have to look closer to the this phase with and without mitogens.
Figuur 3 celcyclus van TKO-Mefs zonder groeifactoren.