Cell Cycle 101: Deciding When To Exit

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: Deciding When to Exit

Previous articles in this 101 series have examined the intricacies of the cell cycle, with great emphasis on the importance of the faithful distribution of DNA from a mother to the two genetically identical daughter cells. However, what happens to the daughter cells that are formed after each cycle of cell division is not straightforward, as it depends on the type of cell that has divided. A new round of cell division can be commenced immediately for rapidly dividing cells (Boward et al., 2016; Mercadante & Kasi, 2023). By contrast, other cell types may remain dormant in tissues and take longer to enter a new cycle of division, replicating only as needed to replace damaged cells. Dermal fibroblasts, the predominant cell type in the dermis (the inner layer of skin), which help the skin heal after injury (Plikus et al., 2021), as well as cells from other internal organs such as the liver (Y. Li et al., 2021) and lungs (Kotton & Morrisey, 2014) are among them. Some cells even cease dividing. For instance, following embryonic development, neurons, the building blocks of the brain and nervous system, cease proliferating and instead go quiescent (Purves et al., 2001). Adult neurons exit the G1 phase of the cell cycle and enter a resting phase, known as the G0 phase. This may seem odd, however, this is due to the inability, or rather limited ability, of neurons to regenerate after damage.

Considering the limitations of neurons compared to skin cells, one might be surprised that an organ as important as the brain seems so unprepared for damaging events. The fact is that neurons are structurally far more sophisticated than skin cells. As a result, the central nervous system adopted alternative self-repair strategies that differ from those of other organs. Rather than rebuilding new neurons, healthy neurons will strive to functionally compensate for the lost ones. However, not all neurons are generated during embryonic development. Remarkably, the adult brain retains some neural stem cells during development (Purves et al., 2001). Stem cells serve as the body's raw materials, capable of transforming into a variety of different cell types with specialized and distinct functions. This underpins adult neurogenesis, in which some neurons are de novo generated, yet this is strictly limited to a few regions of the brain (Boldrini et al., 2018; Purves et al., 2001).

Figure 1 - The structural architecture of a nerve cell (blue) is far more complex than that of a skin cell (red) ("Illustration of human", n.d.).

Cell Cycle Departure: The G0 phase

Cells can exist in two states throughout their lives: a cycling and active state or a non-cycling and quiescent (G0) state. The G0 phase is a dormant cellular state that occurs outside of the replicative cell cycle. Traditionally, cells were assumed to enter G0 due to adverse external conditions such as nutrient deprivation, which restricted the resources required for development. Hence, it was regarded as a period of rest (Lajtha, 1963). It is now recognized that G0 assumes numerous forms and occurs in response to a plethora of stimuli. When Howard & Pelc (1986) first characterized the four stages of the cell cycle, they found that not all cells in a population proliferate at the same pace. These were the first studies to suggest the existence of an additional stage in the cell cycle, today known as the G0 stage. Two contrasting theories held that non-proliferating cells were either in an extended G1 phase or in a cell cycle phase distinct from G1 (Patt & Quastler, 1963). Only later did subsequent research substantiate the existence of a restriction point in G1 phase (described in more detail earlier in this series) that marks the point of commitment to enter replication (Pardee, 1974). Cells must either commit to replication, or exit the cell cycle and enter G0, to which access is restricted. Cells are dormant or inactive during the G0 phase as they are neither dividing nor preparing to divide. They are, however, metabolically active and continue to execute their assigned biological functions (Pranzini et al., 2022; Su et al., 1996).

The Diversity of G0 States

While some cells inside organs and tissues are cycling and dividing, most cells do not and remain dormant. This dormant and inactive phase can be transient, referred to as quiescence, or permanent, with cells unresponsive to stimuli with no possibility of cell cycle reentry, which includes both cellular senescence and terminal cellular differentiation.

Figure 2 - The mitotic cell cycle and arrest. Quiescent cells leave the cell cycle but retain the ability to resume replication. This reversible state contrasts with terminally differentiated or senescent cells, which fail to re-enter the cell cycle (Sun, 2021).

Quiescence is a reversible proliferative arrest in which cells stop dividing but maintain the ability to resume the cell cycle in response to suitable stimuli. While dormant cells no longer multiply, unless stimulated by proper external cues, they remain metabolically active (Fukada et al., 2007; Hüttmann et al., 2001). Often triggered by unfavorable external conditions such as nutrient deprivation, quiescence serves as a coping strategy to protect cells from damage while preserving their proliferative potential and hence their ability to continue replicating when appropriate conditions are restored (Marescal & Cheeseman, 2020). However, not only suboptimal growing conditions drive quiescence. Animal cells often remain in the G0 state unless appropriate cues stimulate them to divide. Dermal fibroblasts, for example, are halted at G0 until activated to divide and assist in wound healing (Mitra et al., 2018). The capacity to switch between a quiescent and a proliferative state while preserving viability is critical for tissue homeostasis and coping with potentially life-threatening conditions. Alternatively, cells may stop proliferating permanently, without the opportunity to further re-enter the cell cycle.

Cellular senescence is a persistent and terminal stage of growth arrest in which cells cease to multiply despite adequate growth conditions and mitogenic stimuli, that is signals known to drive mitosis and cell division (Di Micco et al., 2021). Senescence-inducing stimuli are myriad and include permanent DNA damage, cellular stress, and activation of mutated genes that have the potential to cause cancer (oncogenes). While senescent cells can no longer divide, they are still functional and able to perform many normal cellular tasks (Burton & Krizhanovsky, 2014; Rodier & Campisi, 2011). Senescence is often a biochemical alternative to self-destruction of a damaged cell through apoptosis. By preventing the proliferation of potential cancer cells, senescence not only plays a role in normal development while maintaining tissue homeostasis, but also limits tumor progression (McHugh & Gil, 2018). However, cellular senescence can impair tissue repair and regeneration, thereby contributing to aging (Kumari & Jat, 2021). For example, senescent cells have been found in a variety of age-related diseases, including atherosclerosis, diabetes, and lung disease (Chandrasekaran et al., 2017; Muñoz-Espín & Serrano, 2014).

Figure 3 - Senescence is a highly dynamic process associated with permanent cell cycle exit and multiple cellular and molecular changes and distinct phenotypic alterations (Crouch, 2022).

Cellular terminal differentiation is the process by which stem cells acquire a specialized function and must be coordinated with cell cycle exit to ensure proper organogenesis (O’Farrell, 2011). The balance between cell cycle progression and entry into quiescence is essential for normal tissue development, size, and shape. Stopping development at an appropriate adult size has been selected throughout evolution because humans have intricate body designs that work well at a certain size (O’Farrell, 2009). During development, some of the earliest stages of tissue differentiation are accompanied by differentiation-associated quiescence. This quiescence persists into adulthood in some organs, as stem cells continue to replace some tissues that turnover (such as the skin) or upon injury, to repair damage (Slack, 2007; Slack & Dale, 2021). Once mature, differentiated cells become refractory to proliferative signals - even those that induced proliferation prior to differentiation (Buttitta & Edgar, 2007). Despite being permanently arrested, these cells which include cardiac muscle cells, blood cells, and neurons retain viability and functionality, and although they do not replicate, growth can still take place (O’Farrell, 2011). For instance, while cardiomyocyte proliferation underlies embryonic heart growth, adult cardiomyocytes enter a quiescent phase with further heart growth reliant on increasing cardiomyocyte size (Laflamme & Murry, 2011; F. Li, 1996). This is due to the fact that if cardiomyocytes continued to divide in order to generate muscle tissue, the contractile structures required for heart function would be compromised. Also during neuronal development, neurons engage in a proliferative quiescence once embarking on the road to differentiation, yet neurons frequently grow considerably following differentiation (O’Farrell, 2011).

Figure 4 - Stem cells are the body's raw materials, the cells from which all other cells with specialized functions arise (Batra, 2022).

Senescence and Disease

The concept of therapeutically targeting senescence dates back to the year 2000 when Jan van Deursen engineered a transgenic mouse strain that produces low levels of a mitotic checkpoint protein. The consequent chromosomal instability, he predicted, would lead to the development of cancer. However, hardly any mice developed cancer. Instead, they seemed to age prematurely (Baker et al., 2004). The mice's eyes were riddled with cataracts, their skin was wrinkled, their muscles had shrivelled, and their organs were clearly overloaded with senescent cells. The cells had stopped reproducing but refused to die, evolving into a “zombie-like” state of cellular life (Hayflick & Moorhead, 1961). Senescence was originally recognized by Smith & Peireira-Smith (1996) as a cancer defense mechanism. It has been hypothesized that cells undergo senescence to face replicative stress and avoid developing cancer. It is noteworthy that a considerable number of anticancer treatments, including chemotherapy and radiation therapy, elicit senescence in cancer cells by inducing DNA damage, inadequate mitogenic signaling or oxidative stress, the latter being characterized by an imbalance in the production of reactive oxygen species and antioxidant defenses (Saleh et al., 2020; Wang et al., 2020). Therefore, pro-senescence (senescence-inducing) therapy serves as a primary antitumoral strategy to halt proliferation and avoid ensuing genomic instability (Nardella et al., 2011).

However, despite escaping cancer, van Deursen’s transgenic mouse model underwent accelerated aging. This was driven by a rise in the production of a toxic cocktail of signaling molecules (interleukins and chemokines), growth hormones, enzymes, and other chemicals that promoted inflammation and damaged neighboring cells, a condition known as senescence-associated secretory phenotype (SASP). Because SASP disrupts tissue homeostasis, there is a reduction in tissue regeneration and repair, which drives aging (Coppé et al., 2008). Senolytic therapy, or the selective destruction of senescent cells by small molecule inhibitors (senolytics), was originally employed to alleviate age-related symptoms and improve healthy longevity (Baker et al., 2011; Childs et al., 2015). However, a two-step senescence-targeted therapeutic method for cancer treatment that combines a senescence-inducer (pro-senescence therapy) with a senolytic drug, to differentially destroy cancer cells which have been previously compelled to enter senescence, has recently been proposed (Sieben et al., 2018).

Figure 5 - These mice are twins, born from the same litter. One (right) has been genetically altered and shows signs of premature ageing (Lu, 2020).

The Curious Case of Benjamin Button: The Backward Growing Mouse

F. Scott Fitzgerald's short story "The Curious Case of Benjamin Button" is based on an odd premise: a man who ages backwards, by physically getting younger as he grows older. This has given rise to the idea of the Benjamin Button effect, which aims to generate a visibly younger (and more stunning) yet natural-looking appearance through non- or minimally invasive procedures and therapies (Waldorf, 2017). Not only do our bodies age, but each individual cell does as well. Cell rejuvenation treatment, at its foundation, seeks to restore individual cells to their youthful state. As we age, our cells accumulate mutations and epigenetic markers, which are DNA alterations that determine whether a gene is switched on or off (Costa & Johannes, 2020). In 2006, Shinya Yamanaka discovered a protein cocktail (termed the Yamanaka factors) that converts adult cells, which have a specific function, into stem cells which have the unique capability of transforming into any cell type, which netted him the 2012 Nobel Prize for Medicine (Yamanaka, 2023). These factors essentially restore the DNA to its original pattern, eliminating epigenetic marks and resetting the biological clock of these cells to their younger state (Browder et al., 2022). Despite the fact that Yamanaka factors completely converted adult cells into stem cells, the 50-day reprogramming process resulted in a loss of cellular identity, which is reflected in a shift in cell differentiation towards a different mature cell type (Takahashi & Yamanaka, 2006). There is a fine line between remodeling the cells enough to become younger while preserving their cell type and function (retaining identity) and not converting them into induced pluripotent stem cells (losing identity), rendering them unsuitable for rejuvenation purposes (Conger, 2020).

Parallel lines of research have attempted to unveil the key to the fountain of youth. Rejuvenate Bio, a San Diego-based biotech company, employed gene therapy to inject some of the so-called Yamanaka factors into older mice, which exhibited extended lifespans (Cano Macip et al., 2023). The company claims that older people could one day reset their biological clock with an injection. At the same time, a team led by Harvard Medical School geneticist David Sinclair reversed age-related changes in genetically modified mice (Lu et al., 2020). Ocampo et al. (2016) previously showed that signs of aging can be erased in genetically aged mice briefly exposed to four key Yamanaka factors without erasing the identity of the cells (Ocampo et al., 2016). By delivering three of the four Yamanaka factors to damaged retinal cells of aged mice, Sinclair’s team observed that mice with poor vision and damaged retinas suddenly regained their sight, with vision sometimes rivaling that of their offspring. Remarkably, damaged neurons in mice's eyes were regenerated, and new axons, or projections, developed from the eye into the brain (Lu et al., 2020). Yamanaka factors appear to have restored mouse epigenomes to a more youthful state. This extraordinary study demonstrates that there is a backup copy of youthful memory stored in the body, bringing new hope to aging prevention and all aging-related diseases.

“If we reverse aging, these diseases should not happen. We have the technology today to be able to go into your hundreds without worrying about getting cancer in your 70s, heart disease in your 80s and Alzheimer’s in your 90s.” - David Sinclair, Life Itself.

Figure 6 - Cellular rejuvenation therapy, using four reprogramming molecules (Oct4, Sox2, Klf4 and cMyc, also known as “Yamanaka factors”) safely reverses signs of ageing in mice (Browder, 2022).

Last year, researchers successfully achieved partial in vivo reprogramming by expressing Yamanaka factors for a short period of time without causing carcinogenesis, partially regenerating physically aged mice and increasing longevity. By halting the reprogramming process midway, the innovative technique avoids the complete loss of cell identity (Browder et al., 2022). This allowed researchers to identify the ideal balance of reprogramming cells to make them biologically younger while still allowing them to restore specialized cellular tasks.

Conclusions

The precise control of cell proliferation is essential for tissue homeostasis and development, as improper control can result in disease with excessive proliferation or cell death. Cells use several growth arrest mechanisms, such as quiescence and senescence, to accomplish precise control. The fundamental machinery of the cell cycle responds to various internal and external inputs to decide whether to enter these growth-retarded phases or to proliferate. Recent research has uncovered the biological basis of these cell cycle decisions, highlighted the distinctive features of cell cycle entry from quiescence, identified endogenous DNA damage as a quiescence-inducing signal, and demonstrated how cancer can be targeted by inducing senescence while aging is driven by prolonged senescence. While pro-senescence drugs limit tumor growth by inhibiting proliferation and fibrosis and active tissue regeneration, anti-senescence therapeutics allow for the removal of accumulated senescent cells to restore tissue function and potentially enhance organ rejuvenation. Remarkably, cells that re-enter the cell cycle after chemotherapy are extremely aggressive and chemo-resistant and may contribute to cancer recurrence. As numerous anti-cancer treatment modalities induce senescence in tumors, it is important to understand the processes involved in senescence escape. Equally important is unraveling the mechanisms driving senescence, the key hallmark of aging, as deeper knowledge can pave the way for the development of innovative therapies and reduce the off-target effects that contribute to unwanted toxicity.

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