Cell Cycle 101: Checkpoints As Surveillance Mechanisms

Foreword

Have you ever questioned how a wound heals? How does an embryo become a baby? How does a cancerous tumor grow? At the heart of these questions lies cell replication and division. The remarkable ability of a cell to grow and divide into two identical daughter cells underpins the success of life. This is, however, only conceivable because eukaryotic cells (that is, with nuclei) go through the cell cycle, a set of well-defined and tightly synchronized events. This process is divided in two main stages: the interphase, whereby a cell grows; and the mitotic phase, by which cells replicate (mitosis) and divide (cytokinesis). During this process deoxyribonucleic acid (DNA), also recognized as the "molecule of life," is accurately replicated and faithfully distributed to newly generated daughter cells. DNA is the molecule that carries and conveys hereditary material or genetic information from parents to offspring, thus holding all the information needed to construct and sustain an organism. The cell cycle thus functions as the master regulator of tissue growth, development and proliferation. It also ensures that old cells are removed in a timely manner as we age and replaced by new cells that proliferate in a tightly controlled fashion. This keeps the total number of viable cells constant, which is essential for life as we know it.

The Cell Cycle 101 series attempts to elucidate the most important aspects of the cell cycle: from its history and discovery to the underlying mechanisms that control cell division. This series aims to explore the intricacies of cell cycle control in development and the consequences of its failure in disease.

Therefore, this series is divided into the following chapters:

1. Cell Cycle 101: History and Discovery

2. Cell Cycle 101: How One Cell Becomes Two

3. Cell Cycle 101: Checkpoints as Surveillance Mechanisms

4. Cell Cycle 101: Cell Cycle Regulation and Its Engines

5. Cell Cycle 101: Deciding When to Exit

6. Cell Cycle 101: DNA Proofreading and Repair Mechanisms

7. Cell Cycle 101: Cell Cycle Deregulation as a Hallmark of Cancer

Cell Cycle 101: Checkpoints As Surveillance Mechanisms

Cellular components must be duplicated and divided with exceptional accuracy and reliability throughout countless generations. This is particularly relevant for the genetic information held in the DNA of the chromosomes, whose near-perfect transmission is essential for the survival and evolution of species. Appropriate cell cycle control is essential to guarantee that each daughter cell receives equal copies of a mother cell's DNA; hence, the cell cycle separates chromosomal duplication and segregation in time. While DNA replication takes place in the S phase, chromosomal segregation and mitosis take place later in the M phase. These two phases are interspersed with two gap phases, resulting in a complete cell cycle with the G1-S-G2-M phases in that order (Alberts et al., 2003; Morgan, 2007). Cell cycle events in most eukaryotic organisms are not only closely related but also interdependent, with activation of late events relying on completion of early events, a concept first discovered by Leland Hartwell in the mid-1970s (Hartwell et al., 1974; Hartwell & Weinert, 1989). For instance, before attempting to distribute the chromosomes into daughter cells, chromosomal duplication should be initiated and completed (Elledge, 1996; Hartwell & Weinert, 1989; Murray, 1992). Such an ordered, timely and reproducible sequence of events must entail several regulators throughout the cell cycle, which will be explored in more detail in the next chapter of this series. While such regulatory networks are sufficient to maintain the robustness of the overall process as well as the reproducibility of the sequence of cell cycle events, they are insufficient to support cell cycle progression when errors occur.

Hartwell discovered this missing piece of the cell cycle puzzle while studying the budding yeast Saccharomyces cerevisiae (S. cerevisiae), which divides via a budding process in which smaller daughter cells pinch off, or bud, the mother cell. As a result, S. cerevisae exhibits asymmetrical growth giving rise to daughter cells that are smaller than the parental cell. As the cell cycle advances, the bud grows, DNA replicates, and cells enter mitosis before dividing to form another cell. Cell cycle progression is closely tied to the bud size, in such a way that cells without a bud are in G1, cells with a small bud are in S phase, and cells with a large bud are in G2 (Hartwell, 1967). Hartwell was able to isolate thermosensitive cell cycle mutants by mutagenesis (a laboratory technique whereby DNA mutations are designed to produce libraries of mutant genes, proteins or even strains) what he called cell division cycle (cdc) mutants (Hartwell, 1967, 1973; Hartwell et al., 1974). His interest was sparked by the cdc28 mutant that failed to divide being arrested in G1 before entering S phase, as evidenced by the mutant's inability to form a bud, which is a daughter cell. This prompted Hartwell's discovery that when DNA is damaged, the cell cycle ceases, giving rise to the term "checkpoint". Hartwell hypothesized that cells contain surveillance mechanisms (checkpoints) that constantly control the advancement and completion of biological events. These checkpoints must be always active and widespread throughout the cell cycle and only the completion of an event may deactivate the related checkpoint, enabling the cell cycle to proceed (Hartwell et al., 1974; Lau et al., 2021). Simultaneously, Paul Nurse was experimenting with the fission yeast S. pombe, which earned its name from the fact that its cell division resembles that of bacteria. They feature a rod-like structure that becomes longer as the cell cycle progresses and eventually divides through the middle when it reaches the size of two cells. The length of the cell is therefore closely related to the advancement of the cell cycle: small cells are in G1 and large cells are in G2. Paul Nurse, like Lee Hartwell, discovered numerous genes required for cell cycle progression, which he also designated cdc genes (Nasmyth & Nurse, 1981; Nurse et al., 1976). Hartwell and Nurse were awarded the Nobel Prize in Physiology and Medicine in 2001, together with Timothy Hunt, who discovered cyclins, which are proteins which are broken down throughout different phases of the cell cycle and have a key function in its regulation.

Figure 1 - Leland Hartwell, Timothy Hunt and Paul Nurse were jointly awarded the 2001 Nobel Laureate in Physiology or Medicine (Nobel Prize Outreach, 2001).

Cell Cycle Checkpoints: Safeguarding the Order of Cell Cycle Events

High replication fidelity, as well as overall stability and error-free conveyance of genetic material to succeeding generations, are crucial for survival at both the uni- and multicellular levels. Checkpoints have typically been characterized as molecular signaling pathways that induce cell-cycle delay or arrest in response to DNA damage, providing additional time for cells to repair damage (Hartwell & Weinert, 1989). Checkpoints are surveillance mechanisms that ensure the timing, integrity, and accuracy of cell cycle events. Such monitoring systems comprise a network of regulatory proteins that coordinate cell cycle progression in response to external and internal cues. External cues include the availability of nutrients and growth factors, while internal cues relate to specific features within the cell, such as cell growth to the appropriate size, chromosomal replication and integrity, and successful segregation during mitosis (Amara et al., 2013; Elledge, 1996). Each checkpoint thus serves as a potential endpoint throughout the cell cycle, during which cellular conditions are examined, with progression through the various stages of the cell cycle occurring only when favorable conditions are met.

When cells are subjected to stressful conditions, several checkpoint mechanisms prevent them from progressing through the cell cycle (Hartwell & Weinert, 1989). The three major checkpoints being: the G1 checkpoint at the G1/S transition, also known as the Start (in yeast) or restriction point (in mammals); the G2 checkpoint at the G2/M transition; and the spindle assembly checkpoint (SAC) at the metaphase-to-anaphase transition of mitosis (Alberts et al., 2003). In response to cellular stress, proliferating cells that are in the G1 or G2 phases can activate checkpoints that impose either temporary or permanent cell-cycle arrests in G1 or G2, followed by re-entry into the S phase or mitosis (M phase). Cells that are subjected to stress during DNA replication, on the other hand, only temporarily slow their passage through S phase, and if damage is not repaired during this delay, they depart S phase and arrest later when they reach the G2 checkpoint. Because of this, mammalian S-phase checkpoints have traditionally been presumed to have a modest function in comparison to the more effective G1 and G2 checkpoints (Bartek et al., 2004). Failure to repair DNA damage, entering mitosis with non-replicated DNA, or starting anaphase prior to chromosomal alignment on the mitotic spindle results in the formation of dead, aneuploid, or mutated cells. Under extreme stress, DNA damage checkpoints can lead to permanent cell-cycle withdrawal (a process known as cellular senescence) or apoptosis, the process of programmed cell death (Chao et al., 2017; Elledge, 1996; Zhou & Elledge, 2000). Failure to properly activate DNA damage checkpoints can lead to genomic instability as unrepaired DNA damage can be passed on to the next generation. While in unicellular species these defects impact the organism's ability to reproduce, in multicellular organisms aneuploidy and mutations can lead to unregulated cell growth which may ultimately result in cancer (Murray, 1994). The detection of such abnormalities via cell cycle checkpoints activates specific mechanisms in the cell to ensure the accurate transmission of its genetic information including: repair mechanisms that fix spontaneous or environmental-driven errors in DNA replication and chromosomal alignment; mechanisms that delay or pause the cell cycle until repair is complete; and death of damaged cells to prevent them from generating offspring (Hartwell & Weinert, 1989; Murray, 1992).

Figure 2 - Chromosomal instability (CIN), a form of genomic instability commonly seen in tumor cells, is often the result of defective checkpoint activity (McGranahan, 2012).

G1 Restriction Point: Commitment Dilemma

The primary regulatory processes leading to proliferation take place during the G1 phase of the cell cycle. Growth factors are required to initiate and maintain the transition from G1 to S phase, with growth factor deprivation inhibiting the onset of S phase in normal cells. The restriction point is the moment at G1 when commitment occurs and the cell no longer needs growth factors to complete the cell cycle (Pardee, 1974). The G1 checkpoint, also known as the START (in yeast) or restriction point (in mammals), is the point at which the cell irreversibly commits to the cell division process. Thus, this represents the most fundamental turning point in the life of a cell. Once a cell passes the G1 checkpoint and enters S phase, it is irrevocably committed to division (Johnson & Skotheim, 2013; Morgan, 2007). That is, a cell that successfully completes the G1 checkpoint will complete the full cell cycle and give rise to two daughter cells, barring unforeseen issues such as DNA damage or replication errors that must occur later in the cell cycle. External factors such as growth factors and nutrients play a crucial role in pushing the cell past the G1 checkpoint. In addition to appropriate resources and cell size, the G1 checkpoint also checks for genomic DNA damage. If a cell does not fulfill all the conditions, it will not be permitted to advance to the S phase (Dorsey et al., 2018; Johnston et al., 1977). The cell can either halt the process, delaying the cell cycle in G1 while attempting to resolve the error, or it can exit the cell cycle and enter a reversible G0 phase. The G0 phase is a form of quiescence, or resting state, in which cells dwell until environmental circumstances are re-established (LibreTexts Biology, 2022). Cells at this stage receive and respond to appropriate signals, such as growth factors, prompting them to re-enter and advance through the cell cycle.

After committing to division, cells enter S phase during which DNA is duplicated. Considering the intricacies underlying DNA replication, as well as the different types of errors and defects that occur spontaneously in each cell during this process, the S phase is perhaps the most genetically vulnerable part of the cell division cycle (Ren & Wu, 2004). Consequently, during the S phase of the cell cycle, DNA replication is also tightly controlled, and activation of the intra-S phase checkpoint due to DNA damage generally leads to the blockage of DNA synthesis. In response to DNA damage, activation of such a checkpoint triggers further activation of DNA repair systems that arrest or halt cell cycle progression (Melo & Toczyski, 2002; Paulovich & Hartwell, 1995; Ren & Wu, 2004).

Figure 3 - The G1/S and the intra S-phase checkpoints regulate cell cycle progression in response to different stimuli, yet both respond to DNA damage inducing cell arrest (Adapted from BioRender Templates, 2023).

G2/M Checkpoint

Following DNA replication in S phase, the cell enters the G2 growth phase. Cells are especially prone to DNA damage during the S phase. Abnormalities must be repaired not just in G1 and G2 cells. During G2, the required mitotic proteins are synthesized and the cell again undergoes regulatory mechanisms to ensure the correct status for entry into the proliferative mitotic cycle (M). The transition from G2 to M is drastic: there is an all-or-nothing effect and the transition is irreversible (Stark & Taylor, n.d.; Wang et al., 2009). This is beneficial for the cell since entering mitosis is a critical step in a cell's life cycle. If it does not fully commit, the cell will face multiple challenges, partially dividing, which will most likely result in cell death (Dasso & Newport, 1990; Elledge, 1996; Hartwell et al., 1974). Since entry into mitosis is a significant and costly commitment for the cell, it stands to reason that systems are in place to prevent premature entry into this stage. It has been demonstrated that errors in preceding phases, such as having non-replicated DNA regions, hinder cell cycle advancement in the G2 phase (Dasso & Newport, 1990). This implies that G2 also comprises a DNA damage checkpoint. The cell is examined once again for regions of damaged DNA or incomplete replication. Notably, like the G1 phase, the G2 phase comprises cell size checks, which are required for cells to coordinate growth with cell cycle progression. The size of new daughter cells after mitosis influences cell cycle pace: larger daughter cells advance faster through G1 and/or G2, while smaller cells delay exiting these growth phases (Barnum & O’Connell, 2014).

The fundamental function of the G2 checkpoint is to ensure that all chromosomes have been duplicated and that the replicated DNA is not defective. When DNA is faulty, the G2/M checkpoint prevents cells from entering mitosis, providing these cells the opportunity and time needed to repair the damaged DNA before spreading genetic abnormalities to daughter cells, therefore preserving genomic stability (Hartwell & Weinert, 1989; McGowan, 2002). If the damage is irreversible, checkpoint signalling drives pathways that lead to apoptosis, that is programmed cell death (Cuddihy & O’Connell, 2003). This self-destruction process guarantees that damaged DNA is not passed on to daughter cells, which is key to preventing diseases like cancer (Wang et al., 2009).

Figure 4 - The G2/M checkpoint is critical to ensure that all chromosomes have been duplicated prior to mitosis. DNA damage and appropriate cell growth are also assessed at this stage (Adapted from BioRender Templates, 2023).

The Spindle Assembly Checkpoint (SAC)

During mitosis, one copy of each chromosome is transferred to each of the two daughter cells. To ensure appropriate segregation, each kinetochore (a particular disc-shaped DNA-protein complex) on the sister chromatids must be attached to opposing poles of the mitotic spindle (in a bipolar orientation), the structure that pushes the chromatids apart, so that an identical copy of each chromosome travels towards each pole of the cell (Santaguida & Musacchio, 2009). Only when all kinetochores are connected to microtubules does the mitotic spindle pull the sister chromatids further apart. Cells go to tremendous lengths to guarantee that each daughter cell only obtains one copy of each chromosome. The spindle assembly checkpoint (SAC), also known as the metaphase-to-anaphase transition, is essential for maintaining the integrity of chromosomal transfer by monitoring the attachment of kinetochores to microtubules (Nicklas, 1997; Schwartz & Shah, 2005). When abnormalities in the attachment state of kinetochores or in the microtubules dynamics are detected by the SAC, this surveillance mechanism is activated, preventing sister chromatid separation and hence the onset of anaphase (Hanahan & Weinberg, 2011; Rausch et al., 2012). This is achieved through induction of cell arrest prior to sister chromatid separation, that is during the transition from metaphase to anaphase and is critical for maintaining genomic stability (Nitta et al., 2004), Extended mitotic arrest caused by SAC activation is required for successful induction of death of aberrant cells during mitosis, a fundamental regulatory mechanism that prevents chromosomal mis-segregation and aneuploidy, that is the presence of an abnormal number of chromosomes in a cell (Holland & Cleveland, 2009).

Figure 5 - Once the genome is duplicated, proper chromosome segregation is ensured by the spindle assembly checkpoint (SAC). The SAC is best characterized for its role in preventing segregation prior to chromosome alignment (Adapted from BioRender Templates, 2023).

Consequences of Checkpoint Failure: Mitotic Catastrophe

Long recognized as a mechanism of cell death triggered by cells entering mitosis prematurely or erroneously, mitotic catastrophe can be driven by chemical or physical stress. While first portrayed as the primary cause of ionizing radiation-induced cell death, it is nowadays known to be elicited in response against therapy with chemicals that impair microtubule stability, numerous anticancer treatments, and mitotic failure caused by malfunctioning cell cycle checkpoints (Vakifahmetoglu et al., 2008). Several factors can cause checkpoint-mediated cell cycle arrest to fail. First, like other biological operations, checkpoints must have an inherent error rate. Second, like many signal transduction systems, they exhibit adaptability. This means that even if the damage is not repaired, the cell can resume the cell cycle after a period of arrest (Roberts et al., 1994). Finally, cells with damaged checkpoints may have an advantage when many genetic modifications are favored by selection.

Cancer cells often lack checkpoints, allowing for faster replication and genomic evolution (Hartwell & Kastan, 1994). As a countermeasure to inadequate checkpoint activation, higher eukaryotes have developed alternative mechanisms to eliminate mitosis-incompetent cells: one of which is mitotic catastrophe. Although not in itself a true mechanism of cell death, mitotic catastrophe anticipates antiproliferative mechanisms such as apoptosis, necrosis, and senescence, employing them to inhibit the proliferation of defective mitotic cells (Galluzzi et al., 2012; Mc Gee, 2015; Vitale et al., 2011). Unprepared entry into mitosis from interphase or prolonged mitotic arrest induced by activation of the spindle assembly checkpoint followed by morphological and biochemical changes in the cell are important factors driving mitotic catastrophe. Failure to perform cell death programs in response to an abnormal mitotic process is likely to result in cells dividing asymmetrically in the following round of cell division, leading to the production of chromosomal instability and aneuploid or tetraploid cells, all of which are typical features of tumor cells (Gordon et al., 2012; Holland & Cleveland, 2012). For instance, misallocated chromosomes during mitosis hamper cell functioning, lower cell fitness, and promote the development of cancer. In fact, one of the hallmarks of cancer cells is genomic instability, which leads to genetic alterations that can lead to enhanced tumor growth via genetic variation in the tumor cell. It has been demonstrated that malignancies with a higher degree of genomic instability are linked to poorer patient outcomes (Alberts et al., 2003). Hence, mitotic catastrophe arises both as a biological mechanism to prevent the proliferation of potentially malignant cells and as a form of cell death resulting from defective cell cycle progression or entry, which ultimately acts as a key mechanism for the reliable transmission of genetic information.

Figure 6 - Comparison of the morphology of the nucleus of cells undergoing (a-c) apoptosis or (d-f) mitotic catastrophe after irradiation-induced DNA damaged (Amé, 2009).

Conclusions

Hand-in-hand with an organism’s intrinsic capacity to reproduce comes the challenge of faithfully replicating its genomes through consecutive rounds of cell division. All life on Earth is constantly exposed to DNA-damaging chemicals, which can be both external (e.g. radiation) and internal (e.g. DNA replication or recombination) to the cell. Precise replication of the genome and regular monitoring of its integrity are critical for life and disease prevention. Cells respond to DNA damage by activating a complex network of so-called checkpoint pathways, which pause cell cycle progression and make the necessary repairs. This complex network of monitoring systems detects nutrients and growth factors, non-replicated and abnormal DNA structures, sends out an alarm signal, and responds by coordinating the actions of numerous DNA repair processes. In response to DNA damage, the G1/S and G2/M checkpoints are activated to prevent the transmission of damaged or incomplete chromosomes to daughter cells. DNA damage checkpoints give cells time to repair damaged DNA. If the DNA damage is irreversible, cells can enter senescence (stop growth) or die. The G1/S checkpoint prevents cells with damaged DNA from duplicating, while the G2/M checkpoint prevents cells with damaged DNA from dividing. The SAC, on the other hand, ensures proper chromosome segregation. While some inefficiency in monitoring the checkpoints is necessary, as if the checkpoints are too tight, the likelihood of developing chromosomal instability is reduced, but at the expense of limiting the cells' ability to replicate and thus survive; if the checkpoints are too permissive, the cells can proliferate in the presence of damage yet face the risk of developing chromosomal instability and cancer. The purpose of multicellular organisms is to find the appropriate balance between cell survival and the risk of chromosomal instability.

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Figure 3 - Adapted from “Cell Cycle Checkpoints Callout”, by BioRender.com (2023). https://app.biorender.com/biorender-templates/figures/all/t-62508cc9d943679dd656e5c0-cell-cycle-checkpoints-callout-layout

Figure 4 - Adapted from “Cell Cycle Checkpoints Callout”, by BioRender.com (2023). https://app.biorender.com/biorender-templates/figures/all/t-62508cc9d943679dd656e5c0-cell-cycle-checkpoints-callout-layout

Figure 5 - Adapted from “Cell Cycle Checkpoints Callout”, by BioRender.com (2023). https://app.biorender.com/biorender-templates/figures/all/t-62508cc9d943679dd656e5c0-cell-cycle-checkpoints-callout-layout

Figure 6 - Amé, J. C., Fouquerel, E., Gauthier, L. R., Biard, D., Boussin, F. D., Dantzer, F., de Murcia, G., & Schreiber, V. (2009). Radiation-induced mitotic catastrophe in PARG-deficient cells. Journal of cell science, 122(Pt 12), 1990–2002. https://doi.org/10.1242/jcs.039115