Cell cycle checkpoints
المؤلف:
Hoffman, R., Benz, E. J., Silberstein, L. E., Heslop, H., Weitz, J., & Salama, M. E.
المصدر:
Hematology : Basic Principles and Practice
الجزء والصفحة:
8th E , P187-189
2025-11-05
62
Competence
Cells require the presence of nutrients and growth factors to switch from quiescence to a state of proliferation. When cells sense that conditions are suitable for proliferation, they leave quiescence into the G1 phase and become competent to enter the cell cycle. G1 has been subdivided into segments and regulatory points based largely on the study of the proliferative response of cells to sequential application of different growth factors, nutrients, and metabolic inhibitors. From the standpoint of cell cycle regulation, a particularly important point in G1 is the restriction point, or R, which occurs near the G1–S boundary. The period after mitosis, when cells can enter quiescence, is termed G1pm (postmitosis), and the period between quiescence and S phase is termed G1ps (pre-DNA synthesis) (Fig. 1). Notably, nearly all of the variability in the length of G1 can be accounted for by the G1ps interval. Experiments have shown that, to leave quiescence and to enter the cell cycle, cells require growth signals either continuous for several hours during G1 or, alternatively, as two discrete pulses of approximately 1 hour in duration and with a pause of several hours in between. To become competent for cell cycle entry, initial activation of the MAPK pathway by mitogen signals is required that in turn activates essential metabolic programs. However, this initial increase in MAPK activity does not lead to induction of MYC and cyclin D; it leads only to the presence of growth signals several hours later that activate the PI3K pathway and MYC. Functions of MYC include transcriptional activation of CDK4 and Cyclin D, as well as downregulation of CDK inhibitors. The following increase in cyclin D–CDK4 complex activity leads to phosphorylation of the principal target RB during the G1 phase.
Fig1. CHECKPOINTS OF CELL CYCLE ENTRY. (A) Quiescence is a non-proliferative state in which viable cells have left the cell cycle and may remain for prolonged periods. In contrast, terminally differentiated cells have irreversibly exited the cell cycle during the process of differentiation. When cells sense that conditions are suitable for proliferation, they leave quiescence into the G1 phase and become competent to enter the cell cycle. G1 has been subdivided into segments, and a particularly important point is the restriction point, or R, which occurs near the G1-S boundary. The period after mitosis, when cells can enter quiescence, is termed G1pm (post mitosis), and the period between quiescence and S phase is termed G1ps (pre-DNA synthesis). When DNA damage is recognized in G1, the G1/S checkpoint becomes activated, which blocks cells from S phase entry, although they may have passed the restriction point. If damage is not repaired timely, cells will enter senescence where they remain viable but not capable of re-entering the cell cycle. (B) During quiescence, the CDK inhibitor p27 prevents Cyclin-CDK activity, and DREAM and RB bind and repress cell cycle genes. When prompted by growth signals, cells enter the competent state. Activation of RAS-MAPK and PI3K signaling pathways is followed by activation of Cyclin D-CDK4/6, leading to mono-phosphorylation of RB. The progressive decrease in p27 protein levels during G1 allows for activation of Cyclin E-CDK2, which multi-phosphorylate the mono-phosphorylated RB, leading to the release of the activating E2F transcription factors. When DNA damage is recognized in G1, p53 becomes activated, and the p53 target gene p21 promotes cell cycle arrest at the G1/S transition through inhibition of Cyclin E-CDK2 and activation of RB. Also, p16 can become activated by oncogenic stress, which leads to inhibition of Cyclin D-CDK4/6 and activation of the G1/S checkpoint. If stress signaling is not relieved, cells will enter a senescent state.
Restriction Point
In 1974, Arthur Pardee published the first report on the restriction point and defined it as a point at which cells become committed to entering the S phase, regardless of subsequent availability of growth factors or essential nutrients. He also correctly predicted that cancer cells undergo changes to become independent from growth fac tors and the restriction point. In the four decades that have passed since the initial description of the restriction point, many important insights have been gained that revealed the signaling events that con tribute to proliferation and growth. In addition to the key contributions of signaling by MAPK, PI3K, and MYC to enable cell growth, it also became clear that there are restraining activities that can inhibit cell cycle entry and progression. As mentioned earlier, cyclin D levels increase during the progression phase of G1, and cyclin D–CDK4/6 complexes monophosphorylate RB, which restricts the activating E2F transcription factors. Later in G1, cyclin E levels increase, and cyclin E binds specifically to CDK2. This leads to RB hyperphosphorylation and release of the activating E2Fs, enabling E2F-dependent gene expression. Once past this point, growth factors are no longer required for S phase entry. Therefore, expression and activation of cyclin E–CDK2 resulting in the hyperphosphorylation of RB enables a cell to pass the restriction point and become committed to cell cycle entry.
G1/S Checkpoint
The G1/S DNA damage checkpoint can be viewed as a point in the cell cycle when the cell has become fully committed to entering into the S phase and past the restriction point but is unable to enter the S phase because cyclin E–CDK2 and cyclin D–CDK4 are inactivated by p21 and p16, respectively, and RB remains capable of binding to and repressing the activating E2Fs, thereby decreasing levels of factors required for DNA synthesis. In G1, DNA damage is recognized by ataxia-telangiectasia mutated (ATM) kinase, which in turn phosphorylates histone variant H2AX to recruit repair factors and CHEK2 kinase. CHEK2-mediated phosphorylation activates p53 and inactivates CDC25A, which is required for the activation of cyclin E–CDK2 complexes. The p53 target genes p21 and BTG2 further promote cell cycle arrest at the G1/S transition through inhibition of cyclin E–CDK2 and activation of RB. If the DNA damage or stress signals are repaired, cells can exit the G1/S checkpoint and re-enter the cell cycle.
S Phase Checkpoint
Under conditions that put DNA replication at risk, such as DNA damage or nucleotide depletion, the S phase checkpoint gets activated. Ataxia-telangiectasia and Rad3-related protein (ATR) is the main kinase that senses DNA damage during the S phase, and it phosphorylates CHEK1 kinase, which in turn activates p53. Similar to the G1/S checkpoint, inhibition of cyclin E–CDK2 is central to the S phase checkpoint. The inhibition of CDK2 activity blocks the loading of CDC45 onto replication origins and prevents the initiation of new origin firing. In addition to inhibiting cyclin E–CDK2, p21 directly interacts with PCNA to stop DNA replication. If the damage is repaired, cells continue DNA replication and cell cycle progression.
G2/M Checkpoint
The G2/M checkpoint prevents cells from initiating mitosis when DNA damage occurs during G2 or when cells progress into G2 with some unrepaired damage inflicted during previous S or G1 phases. The G2/M checkpoint also involves DNA damage recognition by ATM and ATR kinases and subsequent p53 activation through CHEK1 and CHEK2 kinases, and it ultimately requires activation of the p21 CDK inhibitor. If p21 is missing, both G1/S and G2/M checkpoints are abolished. In addition to p21, p53 induces GADD45A and 14-3-3 (stratifin (SFN)), which contribute to G2/M cell cycle arrest. 14-3-3 removes essential mitotic regulators from the nucleus and thereby promotes G2/M arrest. Moreover, inhibition of cyclin-CDK activity through p21 induces DREAM and RB-E2F complexes, which in turn repress the transcription of the cell cycle machinery.
Senescence
If damage is not repaired timely, cells will enter a senescent state in which they remain viable but not capable of re-entering the cell cycle. Telomere shortening, which signals cell aging, also is recognized as a type of DNA damage and can trigger senescence. RB is key in establishing the senescent state, which is activated downstream of p53 and the CDK inhibitors p16 and p21. During senescence, cells have com mitted to proliferation and presumably have passed the restriction point. In contrast to quiescence, senescent cells are unable to re-enter the cell cycle in response to external stimuli, such as growth signals.
Spindle Assembly Checkpoint
The spindle assembly checkpoint (SAC) involves the MAD (mitotic arrest deficient) proteins MAD1, MAD2, BUBR1 (MAD3), and BUB1. To complete mitosis, the cell strictly requires the activity of cyclin B–CDK1. The main effector of the SAC is the mitotic checkpoint complex (MCC), which consists of MAD2, BUBR1, and BUB3 and binds to CDC20, the substrate-specific cofactor of the APC/C that mediates degradation of cyclin B and securin. Inhibition of CDC20 prolongs prometaphase until all chromosomes have become correctly orientated on the metaphase plate. Unattached kinetochores recruit MCC through BUB1, leading to active MCCs. Only when kinetochores are attached correctly to the mitotic spindle is the MCC deactivated, which in turn activates APC/CCDC20 and allows for mitosis progression.
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