Encyclopedia of Cancer. Springer-Verlag Berlin Heidelberg 2016
Cell cycle checkpoints are the control mechanisms that stop cell progression during particular stage of the cell cycle to check and ensure the accurate completion of earlier cellular processes and faithful transmission of genetic information before cell division.
Cell growth and division proceeds through an ordered set of events called cell cycle, which is divided into four distinct phases namely G1 (the first gap phase), S (DNA synthesis), G2 (the second gap phase), and M (mitosis). G1 and G2 are two gap phases that accumulate nutrients, perform biosynthesis, and monitor cell state to get ready for DNA synthesis and mitosis, respectively. DNA replication occurs in S phase and the duplicated chromosomes are separated into two identical sets during mitosis (M phase). Followed by cytokinesis, the mother cell is divided into two daughter cells that are genetically identical to each other. The cell cycle is highly regulated and each phase is monitored by surveillance mechanisms to maintain cellular integrity and faithful transmission of genetic information from mother cell to daughter cell. If a crucial process has not been completed or if a cell has sustained damage, progression into the next cell phase would be prevented. These mechanisms that capable of delaying the cell cycle at specific time points are now referred to as checkpoints, which were first identified in the late 1980s.
Various stresses can activate the checkpoint and cause cell cycle arrest, such as nutrient deprivation, mitogenic stimuli, and cytotoxins. However, the most important function of checkpoints is to monitor DNA damages and coordinate repair. Cells are under constant attack by DNA-damaging agents arising from endogenous or exogenous sources such as UV and the reactive oxygen species that inevitably generates during metabolism. These attacks can interfere with DNA replication, transcription, and other cellular functions and finally lead to genome instability. As repairing damaged DNA takes time, it is essential to activate specific checkpoint machinery to temporarily stall the cell cycle progression. In case the damages cannot be dealt with, the checkpoint can also activate other mechanisms such as apoptosis to target the cell for destruction.
Multiple checkpoints have been identified from lower eukaryotes to human. Despite variations in molecular details, the controlling mechanisms of different organism share some conserved features in that they are tightly regulated through the interaction of specific protein kinases and adaptor proteins. The transition from one phase of the cell cycle to the next is driven by a group of kinases called cyclindependent kinases (CDKs), which become active when bound by their cyclin partners. CDKs phospharylate specific downstream substrates to alter their biochemical function and elicit specific cellular responses. The level of cyclins and CDKs fluctuate during the cell cycle that is controlled by complex negative-feedback loops. Through the oscillation of cyclin-CDKs, cellular processes within the cell cycle such as DNA replication, chromosome segregation, and cell division are precisely modulated.
Fig. 1. The cell cycle checkpoints in mammalian cells
Simple eukaryotes such as yeast has only one CDK (Cdc28 in Saccharomyces cerevisae and Cdc2 in pombe), whereas higher eukaryotes have multiple CDKs, and through different combination of CDKs and cyclins, to control different aspects of the cell cycle. For example, S-phase is controlled by cyclin A in combination with CDK2, whereas progression into mitosis is regulated by cyclin B-CDK1 in mammalian cells. So far 16 eukaryotic cyclins and up to nine CDKs have been discovered.
CDK activity is also negatively controlled by certain families of inhibitory proteins, and the cell-cycle progression is determined by the relative abundance of positive and negative regulators. The core cell cycle control protein/enzyme machineries sense stress/damage and trigger the cell cycle arrest are not conserved between different eukaryotes. Below describes the major checkpoints in mammalian cells as shown in Fig. 1.
The G1 checkpoint is located at the end of the G1 phase that ensures everything is ready for DNA synthesis. It is the major restriction point to decide whether the cell continue for a further round of cell division. Under unfavorable environmental conditions, it signals the cell to temporally withdraw from the cell cycle and enter into a resting phase called G0. Once passing this checkpoint, the cell would tend to complete the whole cycle. During G1 phase, the cells may also irreversibly withdraw from the cell cycle into terminally differentiated or senescent states.
One of the control pathways acting in G1 checkpoint is through the regulation of the tumor suppressor retinoblastoma protein (Rb) and the transcription factor called E2F. The hypophosphorylated form of Rb is active and represses cell cycle progression by inhibiting E2F, which is necessary for S phase entry. Phosphorylation of Rb blocks its inhibition on E2F and brings about the G1 phase progression or G1-S transition. In early G1 phase, increased expression of cyclin D in conjunction with CDK4 or CDK6 (depending on the cell types) leads to Rb phosphorylation. In late G1 phase, Rb is phospharylated by cyclin E/CDK2 complex. Phospharylation of Rb and subsequent release of E2F facilitates the transcription of late G1 genes to get ready for DNA synthesis and S-phase entry. Besides this positive regulation, G1 checkpoint is also negatively regulated by a family of proteins called cyclin-dependent kinase inhibitors (CKIs), which have a function in inhibiting the cyclin/CDK complexes. In mammalian cells, there are two major families of CKIs – INK4 family (selectively for CDK4 and CDK6) and the CIP/KIP family (has a broader range of inhibition).
In addition to the above pathway, another control of the G1 checkpoint is through the tumor suppressor p53 and its negative regulator MDM2. p53 Activation can cause G1 growth arrest via the CIP family member p21Cip1. This pathway, which also works in G2 checkpoint, plays an important regulatory role in DNA repair, senescence, and apoptosis.
Intra S-phase checkpoint
Strict control of S-phase is important to ensure the genome stability and precise transmission of genetic information. The intra S-phase checkpoints monitor DNA damage, coordinate DNA repair pathways, and cause transient and reversible inhibition of the DNA replication during the whole S phase. They are activated when the replication fork stalls which can help preventing the conversion of primary DNA damages into lethal lesions such as DNA double strand breaks.
There are two major checkpoint pathways in human that are initiated by the sensor proteins ATR or ATM, which delays the cell cycle either through the downstream signal cascade of Chk1(Chk2)/cdc25a/ CDK2 or ATM/MRN/SMC1. In the first pathway, it is often triggered by the formation of single-stranded DNA (ssDNA) in replication fork as a result of uncoupling between DNA unwinding and DNA synthesis. ssDNA signals the recruitment of ATR to the stalled forks then activates downstream mediator and transduces the signal to Chk1/2. Phospharylated Chk1/2 then activating other downstream proteins/ factors, such as cdc25 and CDK2/cyclin A, to control several cellular processes including cell cycle delay, prevention of late replication origins from firing, and the activation of DNA repair pathways. In the second pathway, ATM is recruited to sites of DNA damage by a component of the double strand break repair complex MBN. ATM then phosphorylates another component of the MRN complex called NBS1 as well as the cohesin complex SMC1 and lead to s-phase delay with mechanisms that are poorly understood.
G2/M checkpoint is located at the end of G2 phase, which controls the entry into mitosis. It checks a number of factors such as the completion of DNA replication and the genomic integrity before cell division starts. Genomic DNA often contains damaged parts prior to mitosis, which makes G2 checkpoint an important control in preventing transmission of damages to daughter cells. It is especially critical in repairing some lethal damages such as DNA double strand break, which can be repaired precisely by homologues recombination using the intact DNA sequences in sister chromatids as template.
The G2-M transition is regulated by the cdc2/Cyclin B complex. Under favorable conditions, it is activated by the mitosis promoting factor (MPF) for further cell progression. It is maintained in an inactive state by the tyrosine kinases Wee1 and Myt1 as the negative control. Once DNA damages are recognized by the sensory protein kinases DNA-PK or ATM, two parallel signal cascades can be activated and induce growth arrest by inactivating cdc2/Cyclin B. The first pathway is through Chk1/2 kinases and its downstream target cdc25, which prevents cdc2 activation and rapidly inhibits G2-M progression. A second pathway, which is slower, is through the tumor suppressor p53. P53 regulates multiple downstream players such as p21cip1, 14-3-3, and Gadd45, which inactivate cdc2-cyclin B by different mechanisms, to arrest the cell cycle progression.
The mitotic checkpoint, also known as the spindle assembly checkpoint, occurs at the metaphase/ anaphase transition to ensure that all the chromosomes are aligned at the mitotic plate and a bipolar spindle is formed.
The central element in this checkpoint is the anaphase promoting complex (APC) which is highly conserved across different eukaryotes. In its activated form, APC can target many cyclins for degradation, which in turn triggers the signal cascade leading to the cuts of the cohesin complex that holds sister chromatids together. APC is negatively controlled by MAD1/2, BUB1/3, BUBR2, and the centromere protein E (CENP-E). Under favorable cellular conditions when chromosomes are correctly aligned, the checkpoint signal that inhibits APC is silenced and renders the latter to target cyclin B for destruction and inactivates CDK1, thereby promoting exit from mitosis and initiating anaphase. Followed by cytokinesis, the cell splits into two cells and the daughter cells enter into G1 to start a new cell cycle.
Checkpoints and cancer
Checkpoint failure often causes accumulation of genome mutations and rearrangements, which is a major factor in the development of many diseases including cancer. Cell cycle arrest with its vital role in maintaining genome stability is the most important barrier to prevent uncontrolled proliferation, the hallmarks of cancer. Many tumor suppressors are in fact components of the cell cycle checkpoints, such as p53, p16, ATM, and BRCA1/2. Mutation or loss of these tumor suppressors is common in cancer cell, which provides growth advantages over adjacent normal cells that are regulated by growth signals.
Cell cycle checkpoints are also one of the most important targets in cancer drug development, which can enhance the efficacy of DNA damage related therapies. Most tumor cells have defects in their G1 checkpoint pathway, and therefore rely more on the efficient S and G2 phase checkpoints for repairing DNA damages and cell survival. Modulating the S and G2/M checkpoints has emerged as an attractive therapeutic strategy for anticancer therapy. Various inhibitors selectively targeting the key players in S or G2/M but not G1 checkpoints, such as Chk1, have been developed and showed promising effects in enhancing the conventional chemotherapy and radiotherapy. Checkpoint inhibition has become an area of intense interest in cancer biology and is continue growing.
Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer 9(3):153–166
Reinhardt HC, Yaffe MB (2009) Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr Opin Cell Biol 21(2):245–255
Sclafani RA, Holzen TM (2007) Cell cycle regulation of DNA replication. Annu Rev Genet 41:237–280