Tumor suppressor genes are segments of DNA that code for negative regulator proteins, the type of regulators that, when activated, can prevent the cell from undergoing uncontrolled division. A cell that carries a mutated form of a negative regulator might not be able to halt the cell cycle if there is a problem.
Tumor suppressors are similar to brakes in a vehicle: malfunctioning brakes can contribute to a car crash. Mutated p53 genes have been identified in more than one-half of all human tumor cells.
This video reviews the ways that cancer is a by-product of broken DNA replication:. Cancer is the result of unchecked cell division caused by a breakdown of the mechanisms that regulate the cell cycle. The loss of control begins with a change in the DNA sequence of a gene that codes for one of the regulatory molecules. Faulty instructions lead to a protein that does not function as it should.
Any disruption of the monitoring system can allow other mistakes to be passed on to the daughter cells. Each successive cell division will give rise to daughter cells with even more accumulated damage. Eventually, all checkpoints become nonfunctional, and rapidly reproducing cells crowd out normal cells, resulting in a tumor or leukemia blood cancer. Answer the question s below to see how well you understand the topics covered in the previous section.
This short quiz does not count toward your grade in the class, and you can retake it an unlimited number of times.
Use this quiz to check your understanding and decide whether to 1 study the previous section further or 2 move on to the next section. In order to make sure everything goes right, there are checkpoints in the cycle: Figure 1.
Learning Objectives Identify important checkpoints in cell division Explain how errors in cell division are related to cancer. Both the initiation and inhibition of cell division are triggered by events external to the cell when it is about to begin the replication process. An event may be as simple as the death of a nearby cell or as sweeping as the release of growth-promoting hormones, such as human growth hormone HGH.
Crowding of cells can also inhibit cell division. Another factor that can initiate cell division is the size of the cell; as a cell grows, it becomes inefficient due to its decreasing surface-to-volume ratio. The solution to this problem is to divide. Dwarfism : Commodore Nut right was a famous circus performer afflicted with dwarfism.
This was a result of a lack of Human Growth Hormone. Whatever the source of the message, the cell receives the signal, and a series of events within the cell allows it to proceed into interphase.
Moving forward from this initiation point, every parameter required during each cell cycle phase must be met or the cycle cannot progress.
The cell cycle is controlled by three internal checkpoints that evaluate the condition of the genetic information. It is essential that the daughter cells are exact duplicates of the parent cell. Mistakes in the duplication or distribution of the chromosomes lead to mutations that may be passed forward to every new cell produced from an abnormal cell. To prevent a compromised cell from continuing to divide, internal control mechanisms operate at three main cell cycle checkpoints. A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable e.
The integrity of the DNA is assessed at the G1 checkpoint. Proper chromosome duplication is assessed at the G2 checkpoint. Attachment of each kinetochore to a spindle fiber is assessed at the M checkpoint.
The G 1 checkpoint determines whether all conditions are favorable for cell division to proceed. The G 1 checkpoint, also called the restriction point in yeast , is a point at which the cell irreversibly commits to the cell division process. External influences, such as growth factors, play a large role in carrying the cell past the G 1 checkpoint.
The cell will only pass the checkpoint if it is an appropriate size and has adequate energy reserves. At this point, the cell also checks for DNA damage. A cell that does not meet all the requirements will not progress to the S phase. The cell can halt the cycle and attempt to remedy the problematic condition, or the cell can advance into G 0 inactive phase and await further signals when conditions improve. If a cell meets the requirements for the G 1 checkpoint, the cell will enter S phase and begin DNA replication.
This transition, as with all of the major checkpoint transitions in the cell cycle, is signaled by cyclins and cyclin dependent kinases CDKs. Cyclins are cell-signaling molecules that regulate the cell cycle. Immunohistochemistry of paraffin-embedded human breast cancer tissue slide using AP Cyclin B1 Antibody at a dilution of under 10x lens.
Figure 4. The CCND1 gene, located on 11q13, has been reported to be overexpressed in mantle cell lymphoma MCL due to the chromosomal translocation. CCND1 has been shown to interact with tumor suppressor protein Rb and the expression of this gene is positively regulated by Rb. Over-expression of CCND1 correlates with the early onset of cancer and risk of tumor progression and metastasis. Figure 5.
Immunohistochemistry of paraffin-embedded human tonsillitis tissue slide using AP KI67 antibody at a dilution of under 40x lens. It is involved in the control of the cell cycle. It is essential for meiosis but is not required for mitosis. It has 2 isoforms produced by alternative splicing.
Figure 6. A class of proteins termed mediators has also been implicated in the transduction of the DNA damage signal. The prototype of this class of proteins is the RAD9 gene of Saccharomyces cerevisiae. BRCT-repeat containing proteins have been identified in mammals, and some of them are thought to have functions similar to those of RAD9. All of these proteins have been implicated in checkpoint response after DNA damage and are thought to be involved in the recognition of the damage and the recruitment of additional proteins that facilitate downstream signaling and repair.
Two extensively studied kinases of the phosphoinositide 3-kinase PI3K -related family of proteins, ATM and ATR, are located immediately downstream of the damage sensors. ATM is the gene mutated in patients suffering from AT, a disease associated with immune deficiency, cerebellar degeneration and an increased predisposition to cancer Rotman and Shiloh, , ; Shiloh, At the cellular level, ATM mutation is associated with gross chromosomal rearrangements, radioresistant DNA synthesis, as well as a reduction in G1 and G2 arrest.
Phosphorylation of these proteins follows in AT cell-delayed kinetics but is not absent and is due to the activation of alternative signaling pathways. This essential role of the protein in the life cycle of the cell and the organism compromises genetic studies. It is not entirely clear how ATM and ATR are activated in response to the various stimuli and, in fact, a direct-sensor role is occasionally attributed to these proteins Tibbetts et al.
On the other hand, direct stimulation by DNA in vitro remains controversial Banin et al. It will be particularly interesting to see whether similarly functioning ATM partners also exist. Despite similarities, important differences also distinguish ATM and ATR, the most prevalent probably being the kinetics of activation and the types of damage to which they respond best.
Existing data support the hypothesis that ATM is the main determinant of the early checkpoint response induced by IR damage, whereas ATR responds later to processed IR-induced lesions, as well as to lesions induced by UV and by blocked replication forks Zhou and Elledge, ; Nyberg et al.
CHK2 is phosphorylated by ATM in response to ionizing radiation, and this phosphorylation is required for its activation Matsuoka et al.
Phosphospecific antibodies recognizing phosphoserine of CHK1 reveal that this site is strongly phosphorylated in response to hydroxyurea and UV light and moderately in response to IR Kim et al.
The outline specifies the roles of the above kinases in checkpoint signaling and indicates alternative strategies utilized by the cells as part of the overall response to DNA damage. Accurate replication of the genome is paramount to genomic stability. Therefore, organisms have developed a complex network of regulatory processes to impose strict regulation upon DNA replication under both normal circumstances, as well as following DNA damage.
Once the division cycle is initiated, surveillance mechanisms monitor the order and quality of events and halt progression if DNA damage is encountered Hartwell and Weinert, ; Hartwell and Kastan, ; Nurse, , ; Elledge, ; Paulovich et al. Initiation of cell cycle progression is affected by the activation of two key regulatory kinases, CDK4 and CDK2, in association with D-type cyclins and cyclin E, respectively.
A further contribution for this transition is provided by a pathway involving the Myc proto-oncogene, a transcription factor Amati et al. Thus, cyclin E-CDK2 is positioned at the convergence point of two regulatory pathways contributing to the initiation of the cell cycle in nonirradiated cells. As we describe next, the same kinase is the target of two branches of the DNA damage-induced process that delays cell cycle progression in G1 Bartek and Lukas, a , b.
Our current understanding of the molecular mechanism underlying DNA damage-induced delay in G1 is summarized in Figure 2. The checkpoint response in this phase of the cell cycle has two kinetically distinct components. An analogous mechanism based on protein degradation in response to IR has also been reported for cyclin D1 Agami and Bernards, This putative component of the checkpoint response is not shown in Figure 2 and would operate independently of ATM.
The central element of the second branch of the IR-induced G1 component of the DNA damage checkpoint is the stabilization of the P53 protein and the activation of its transcriptional activity. It remains to be seen whether P21 is the only critical target downstream of P53 in this G1 response. A concomitant phosphorylation of Ser20 by a similarly activated CHK2, and possibly CHK1, disrupts the normal interaction between P53 and MDM2 that targets in nonirradiated cells P53 for ubiquitination and proteasome-mediated degradation.
This leads to the observed P53 increase in abundance after IR. Furthermore, MDM2 is itself transcriptionally activated by P53 generating a negative feedback loop that keeps P53 in check, and dynamic changes in the subcellular localization of P53 and MDM2 provide additional levels of regulation for this complex response Melchionna et al.
The above dual response is carefully designed for inducing a delay in G1, starting from a single set of damage-responsive kinases and targeting predominantly a single kinase of the cell cycle engine. Despite significant progress in understanding the checkpoint itself, the molecular interphase with genetic instability and DNA repair remain unknown. The fact that P53 is frequently mutated or deleted in human tumors suggests an important role of the G1 checkpoint in maintaining genomic integrity.
Although it is widely accepted that cell cycle delays induced by checkpoint activation facilitate repair, there is no direct evidence that the G1 checkpoint promotes DNA DSB repair. Of the two pathways implicated in the repair of DNA DSBs, NHEJ is fast compared with the G1 delay and has not been linked to the checkpoint response, while homology-directed repair is thought not to occur efficiently in G1 cells.
Alternatively, the correlation between the G1 checkpoint and genomic integrity might be via the elimination of DNA damage containing cells by apoptosis, and it is well known that P53 is involved in the regulation of this process Figure 1 Hickman et al. It will be important to put into perspective the different aspects of the G1 checkpoint with the repair of DNA DSBs, genomic stability and apoptosis.
Moreover, although the vast majority of cells in an adult organism are in G1 at any given time, damage registered during S can interfere with the functioning DNA replication machinery and lead to serious genomic abnormalities. Although the recognition that this effect reflects an active cellular response initiated by DNA damage is relatively recent Lamb et al.
The dose—response curve describing the inhibition of DNA replication after exposure to IR is biphasic with a radiosensitive component reflecting inhibition in the firing of replicon clusters and a radioresistant component deriving from inhibition in chain elongation Painter, ; Lavin and Schroeder, ; Larner et al.
The term radioresistant DNA synthesis, RDS, is frequently used to signify the absence or simply a reduction in this checkpoint. How the coordination of the two pathways is designed will be an interesting topic of examination in the future. Results support the function of MDC1 as a mediator sharing an intimate relation with H2AX and helping to recruit other repair or signaling proteins to the damage sites Stewart et al. Evidence accumulates for parallel pathways that cooperate to inhibit DNA replication after exposure to IR Falck et al.
A temporary arrest of mammalian cell division is one of the first effects of radiation to be documented and investigated Bernhard et al. Indeed, a long G2 delay on a DNA repair proficient background has been associated with radioresistance to killing McKenna et al. Figure 4 summarizes important molecular components of the G2 checkpoint. Activation of this kinase, by association with cyclin B, subcellular translocation events and a series of phosphorylation and dephosphorylation events, is essential in initiating mitosis Nurse, ; Norbury and Nurse, ; Morgan, Given the pivotal role of this kinase in the transition from G2 to M, its equally central role in the G2 checkpoint comes as no surprise.
An essential and precisely controlled event in the initiation of mitosis is the removal of inhibitory phosphorylations from CDC2 on TYR15 and Thr14, added earlier in the cycle by Wee1 and Myt1, respectively Parker and Piwnica-Worms, ; Booher et al.
Several processes contributing to IR-induced G2 arrest inhibit the processes mediating the removal of these inhibitory phosphorylations. Not surprisingly therefore, central in the regulation of the G2 checkpoint is the inhibition of the CDC25C phosphatase Draetta and Eckstein, ; Peng et al.
Due to the central role of this phosphatase in the regulation of CDC2 activity, several pathways of regulation appear to converge at this site. The inhibition is mediated by binding of the phosphorylated form of CDC25C to the protein that is thought to render CDC25C catalytically less active and to cause its sequestration in the cytoplasm Peng et al. The relevance of cytoplasmic sequestration of CDC25C remains uncertain, however, as mammalian cells, in contrast to the fission yeast, sequester Cyclin B-CDC2 in the cytoplasm by active export until just before mitosis see below , and even in the fission yeast forced nuclear localization of CDC25 does not override the G2 arrest Lopez-Girona et al.
More recent results with HeLa cells point to CDC25A as an additional target of this pathway and, therefore, a determinant of the G2 checkpoint response Zhao et al. This phosphorylation promotes binding of the protein that enhances the inhibitory activity of the kinase towards CDC2 Lee et al. Subcellular compartmentalization is an important mechanism of cyclin regulation in higher eukaryotes even in the absence of DNA damage. A cytoplasmic retention signal at the N-terminal part of the protein, containing a hydrophobic nuclear export signal that binds the nuclear export factor CRM1 exportin 1 , appears responsible for the nuclear exclusion during S and G2 Pines and Hunter, ; Li et al.
After induction of DNA damage, Cyclin B remains sequestered in the cytoplasm, and in certain types of cells nuclear targeting causes premature mitotic events Jin et al. Both proteins are members of the Polo-like kinase PLK family that play a crucial role in several mitotic events including initiation and exit from mitosis, as well as centrosome function Glover et al.
A hallmark feature of this family of kinases is the presence of a highly conserved carboxyl terminal region that includes two blocks of strong similarity termed polo boxes.
A recent report identifies a novel phosphopeptide-binding domain in the carboxyl region of the protein that encompasses the two polo domains polo box domain, PBD. An additional exciting observation is that PLK1 protein stability is regulated by the checkpoint protein Chfr, which delays entry into mitosis when cells are under mitotic stress Scolnick and Halazonetis, ; Kang et al.
The mechanism of Chfr activation after IR is not known. However, these results indicate an intricate web of interactions between pathways ultimately leading to the silencing of CDC2 activity.
The involvement of P21 in the G2 checkpoint also implicates P53 in the response. The above-outlined network of interactions regulating progression through G2 after DNA damage is further enriched by the recognition that the actual mechanism of the checkpoint may also be determined by the phase in the cell cycle where radiation is given.
Recent results suggest the activation after DNA damage of two molecularly distinct checkpoints in G2 Xu et al. One occurs early after IR, pertains to cells in G2 at the time of irradiation, is ATM dependent, transient and dose independent, and is reflected by an abrupt reduction in mitotic index. The second, which occurs later, pertains to cells irradiated in earlier phases of the cell cycle, is ATM independent, dose dependent and reflected by an accumulation of cells in G2.
Earlier studies provided evidence for strong dependence of G2 arrest on the phase of the cell cycle at the time of radiation and demonstrated an order-of-magnitude longer delay for cells irradiated in G2.
How these results correlate with the above observations and the nature of the underlying molecular mechanisms are likely to be active areas of investigation in the coming years. The widely held view that checkpoints aid DNA repair is logical, supported by circumstantial evidence but not rigorously proven.
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