G1 Phase and Regulation of Cell Proliferation

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CHAPTER 41 G1 Phase and Regulation of Cell Proliferation

During the G1 phase of the cell cycle, each cell makes a key decision: whether to continue through another cycle and divide or to remain in a nondividing state either temporarily or permanently. During development of metazoans, this is the time when cells exit the cell cycle as the first step toward forming differentiated tissues. In adults, strict regulation of the timing and location of cell proliferation is critical to avoid cancer.

Cells enter G1 phase at the end of a proliferation cycle, after completing mitosis. To make an unbiased decision whether to proliferate or differentiate, the cell must erase the bias toward proliferation that was carried over from the preceding cell cycle. This is accomplished by inactivating cyclin-dependent kinases (Cdks [see Chapter 40]) through proteolytic destruction of cyclin subunits and synthesis of inhibitory proteins. The absence of Cdk activity turns on a regulatory network that represses the transcription of many genes that promote cell-cycle progression. While this repressive network is active, the cell cannot proceed through the cell cycle. The repression can be switched off if the cell is continuously stimulated by growth-promoting signals from the surrounding medium, extracellular matrix, and other cells (see Chapters 27 and 30). These stimuli can trigger another round of DNA replication and mitosis, but first, the cell must pass a major decision point in G1 called the restriction point (Fig. 41-1). Factors that promote the growth of cell mass are referred to herein as growth factors, and factors that promote cell-cycle proliferation are referred to herein as mitogens.

In metazoans, many cells cease cycling in the G1 phase, either temporarily or permanently, exiting the cell cycle into a state known as G0 (Fig. 41-1). This frequently accompanies their acquisition of specialized, differentiated characteristics. Occasionally, it is desirable in tissues for cells in G0 to reenter the cell cycle to replace cells lost through death. These cells reenter the cycle in G1 phase. During the G1 phase, cells also screen continuously for damage to their DNA. If damage is detected, the cell either stops cycling or undergoes apoptosis (see Chapter 46).

This chapter describes how cells regulate their progress through the G1 phase, exit into the G0 phase, and return to the cycle. It also considers some of the points at which defects in growth control lead to cancer.

The G0 Phase and Growth Control

Most cells in multicellular organisms are differentiated (adapted to carry out specialized functions) and no longer divide. They typically form specialized tissues, each of which has a distinctive structural organization that is important for function. Unscheduled cell division can severely disrupt the organization of such tissues (Fig. 41-2). Accordingly, tissues strictly regulate both the location and the frequency of cell division. These divisions normally occur at a low rate, producing new cells in numbers just sufficient to replace those that die. Under special circumstances, however, such as in response to wounding (see Fig. 32-11), the rate of cell division increases dramatically. This highlights an important constraint on cell-cycle control in multicellular organisms: To make organized tissues, cells must exit from the cell cycle, but some cells must also retain the ability to reenter the active cell cycle when needed to repair injuries or replace worn-out cells.

Cells that stop cycling to differentiate normally do so in the G1 phase. Such cells are said to have left the cycle and entered a nonproliferating state called G0 (Fig. 41-1). G0 may last hours or days or even for the life of the organism, as it does for most neurons. It is important to note that nondividing cells are not dormant: G0 cells continue to expend energy for many ongoing processes. Because of turnover, all cells must continuously synthesize housekeeping proteins. They must also expend energy to maintain intracellular pH and ionic composition and to power intracellular motility. In addition, many specialized G0 cells consume large amounts of energy to synthesize and secrete protein products and generate action potentials. Energy metabolism is particularly dramatic in muscle cells that are responsible for all body movements. Thus, most G0 cells should be regarded as active cells that just happen no longer to be engaged in cell division.

How do cells stop cycling and enter the G0 phase? First, cells may receive external signals that stimulate withdrawal from the cell cycle. This often initiates differentiation of tissues. Second, cells may find themselves in an environment with insufficient mitogens to drive proliferation. Such conditions trigger many cell types to undergo suicide by apoptosis (see Chapter 46), but other cells enter a nondividing state. Third, at least in cell culture, cells that have divided more than a critical number of times or that have received certain types of unfavorable input from their environment undergo senescence, entering a viable but nondividing state. Senescence is a terminal G0 state from which cells normally cannot exit. One particularly interesting signal that can lead to senescence is stress caused by a critical shortening of the telomere regions of the chromosomes. In fact, overexpression of telomerase (see Fig. 12-15) can, when combined with suitable mitogenic stimuli, prevent cells from undergoing senescence in tissue culture.

Transforming growth factor–β (TGF-β) is an example of an external signal that arrests progress through the cycle and regulates differentiation and tissue morphogenesis (Fig. 41-3). TGF-β acts through a receptor serine/threonine kinase that activates the Smad transcription factors (see Fig. 27-10) and increases the expression of the Ink4 class Cdk inhibitor, p15Ink4b, by up to 30-fold (see Appendix 40-1). Like other Ink4 family members, p15Ink4B specifically inactivates Cdk4–cyclin D and Cdk6-cyclin D complexes. p15Ink4B binding also displaces CKI class inhibitors from the Cdk4–cyclin D complexes, permitting them to transfer to Cdk2–cyclin E complexes in the nucleus and further inhibit cell-cycle progression.

In addition to its role in stopping cell-cycle progression in response to TGF-β, the CKI inhibitor p27Kip1 helps to arrest the cell cycle when normal cells become crowded by neighboring cells (contact inhibition; see Fig. 30-8) or when the environment lacks mitogens. Genetic analysis in mice indicates that p27Kip1 regulates cell-cycle progression during development. Indeed, mice that lack p27Kip1 are 30% larger than their normal littermates by several weeks of age. This increase in size occurs at least partly because cells in many organs undergo extra rounds of division.

An analogous mechanism appears to arrest the cell cycle during the differentiation of muscle cells. The transcription factor MyoD is a master regulator of muscle differentiation. MyoD activates transcription of the CKI inhibitor p21Cip1, which helps to arrest proliferation and start muscle differentiation (Fig. 41-3). p21Cip1 stops cell-cycle progression in at least two ways. First, by binding Cdk-cyclin complexes, it blocks them from promoting cell-cycle progression. Second, p21Cip1 binds to the DNA replication factor proliferating cell nuclear antigen (PCNA [see Chapter 42]) in a way that blocks chromosomal replication but not repair of DNA damage.

Several of these mechanisms, including increased expression of both p16Ink4a and p21Cip1, are responsible for permanent cell-cycle arrest of aged cells (senescence). Furthermore, formation of heterochromatin permanently inactivates some genes required for proliferation. This is accomplished when inhibitory proteins of the Rb and E2F families (see later) bind to promoters and recruit histone methyltransferases (see Fig. 13-9).

Once cells exit the cycle, multiple redundant pathways block reentry by reinforcing the primary inhibition of Cdk activity. In addition, a specialized histone variant H1o replaces histone H1 in G0 cells, resulting in more condensed chromatin, which represses transcription and replication generally. However, not all gene expression is suppressed in differentiated cells, many of which synthesize large amounts of specific proteins (e.g., digestive enzymes secreted by the pancreas).

Exit from the G0 Phase

Cells in the G0 phase may reenter the growth cycle in response to specific stimulation, often induced by injury or normal cell turnover. Cultured fibroblasts are favored for studies of this process, as they readily enter the G0 phase when deprived of serum (i.e., mitogens and growth factors) and rapidly reenter the cell cycle when serum is restored. The pattern of gene expression induced by serum in culture reproduces that found in wounded tissues. When a living tissue is wounded (see Fig. 32-11), fibroblasts are exposed to serum. In response, they divide and colonize the wound, where they lay down new extracellular matrix to repair the damage.

Serum stimulates three waves of gene expression in cultured fibroblasts in G0 (Fig. 41-4). The first wave includes more than 100 “immediate early” genes, including transcription factors of the Jun, fos, myc, and zinc finger families (see Chapter 15) that activate numerous downstream genes required for cell growth and division. Other immediate early genes encode tissue remodeling factors, cytokines (growth factors), extracellular matrix components (fibronectin), plasma membrane receptors (integrins), and cytoskeletal proteins (actin, tropomyosin, vimentin), as well as activities involved in angiogenesis (blood vessel formation), inflammation, and coagulation. These proteins facilitate the movement of fibroblasts into wounds and initiate the repair of tissue damage.

Expression of a second wave of “delayed early” genes precedes the onset of the S phase. Genes activated after the onset of the S phase are referred to as “late” genes. Both delayed early gene transcription and late gene transcription require synthesis of proteins, including the transcription factors that are encoded by immediate early genes. Delayed early genes encode a variety of proteins that are required for cell growth and proliferation, including cyclin D and several other proteins that regulate cellular proliferation.

These waves of transcription in response to mitogens enable the G0 cells to pass through a “gate” and reenter the active cell cycle. This gate is a specialized form of the restriction point, a critical aspect of G1 control that regulates the proliferation of all normal cells.

The Restriction Point: A Critical G1 Decision Point

All eukaryotes have a mechanism that operates during the G1 phase to ensure that cells proliferate only when the environment is supportive and the chromosomes are undamaged. Whether cells also monitor their size is controversial. Healthy yeast cells do not embark on a round of DNA replication and division until they reach an appropriate minimum size (actually, they probably measure their ribosome content and ongoing rate of protein synthesis). This is important because after cell division, the daughter cell (bud) is smaller than the mother. The daughter cell needs more time to grow before it divides, if the population is to maintain a constant cell size.

The influence of cell size on the division cycle was first demonstrated in an elegant microsurgery experiment (Fig. 41-5). Two Amoeba proteus cells were grown under identical conditions in parallel cultures. Each day, a portion of the cytoplasm was amputated from one amoeba, and the other was left untouched as a control. Under those circumstances, the cell that suffered the amputations did not divide for 20 days. During this time, the control amoeba divided 11 times. When the amputations were stopped, the amoeba that had been operated on divided within 38 hours. The interpretation of this experiment was that the repeated amputations prevented the experimental amoeba from ever attaining a size sufficient to undergo division. Evidence suggests that some types of human cells have a similar size control while others do not.

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Figure 41-5 a microsurgery experiment demonstrates that amoebae will not divide if they are kept from attaining a sufficient size. A, Control cell continues to divide. B, Experimental cell does not divide.

(Reference: Prescott DM: Relation between cell growth and cell division. II: The effect of cell size on cell growth rate and generation time in Amoeba proteus. Exp Cell Res 11:86–98, 1956.)

An essential aspect of growth control during the G1 phase involves monitoring the external environment for nutrient availability and for signals to proliferate (mitogenic signals) coming from other cells and from the extracellular matrix. In a classic experiment, when three populations of cells proliferating in culture were starved by deprivation of amino acids, serum, or phosphate, they stopped cycling in G1. When the missing ingredients were restored, all three populations of treated cells resumed the cell cycle and entered the S phase at about the same time. This was surprising because amino acids are needed to make protein, serum provides growth factors and mitogens, and phosphate is needed for synthesis of DNA phospholipids (needed to make membranes). This experiment was interpreted as evidence that all three types of starvation caused cells to arrest at an equivalent point in the G1 phase, termed the restriction point. The restriction point is defined as the point after which the cell cycle will proceed even if mitogenic factors are withdrawn (Fig. 41-6). This supremely important aspect of cell-cycle control prevents cells from dividing at inappropriate times and in inappropriate places. Defects in restriction point control are among the most common causes of cancer.

Genetic analysis of budding yeast also revealed a point in the G1 phase after which cells appear to be committed to completion of the cycle. Cells that are starved for nutrients arrest at, or just prior to, this point, termed START. The mammalian restriction point resembles yeast START in a number of aspects, but they are not exactly equivalent, owing to differences between animal and yeast cell cycles.

Regulation of Cell Proliferation by the Restriction Point

The restriction point is a molecular “gate” that regulates the expression of genes required for cell-cycle progression. The gate is based on proteins that are related to the retinoblastoma susceptibility protein (pRb) and a family of essential transcription factors known as E2F. Mammals have three Rb-related proteins (pRb, p107, and p130) and ten E2F family members, which together constitute a complex multifunctional network with particular pairs doing specific jobs. This account refers to the families generically as Rb and E2F. An alternative way around the restriction point gate depends on a potent transcriptional regulator called Myc to be discussed separately later.

Rb regulates the ability of E2F to activate genes required for cell-cycle progression. E2F forms a heterodimer with one of two DP family members and associates with the promoter region of its target cell-cycle genes (Fig. 41-7A). Rb binding blocks the ability of E2F to activate transcription. In addition, Rb recruits histone deacetylases, enzymes that remove acetyl groups from the amino-terminal tails of histones (see Fig. 13-9). This causes compaction of chromatin structure and represses genes required for cell-cycle progression.

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Figure 41-7 Regulation of cell-cycle progression by the E2F/DP/Rb complex. A, The E2F/DP/Rb complex recruits histone deacetylases (see Chapter 13) and represses specific genes that are required for cell-cycle progression. This blocks cell-cycle progression at the restriction point. B, Phosphorylation of Rb by Cdks alleviates this block and permits passage of the restriction point. cAMP, cyclic adenosine monophosphate; MEK, mitogen-activated protein kinase kinase; PKA, protein kinase A.

Phosphorylation of Rb by Cdks causes it to dissociate from E2F, allowing E2F/DP to activate, rather than repress, transcription of the target genes. Cells that can phosphorylate Rb pass the restriction point and complete a cell cycle, whereas cells that cannot phosphorylate Rb remain arrested in the G1 or G0 phase. The E2F/DP heterodimer remains bound to its target promoter regions after phosphorylated Rb dissociates from E2F. If activated as a result of DNA damage, E2F can also act as a potent inducer of cell death by apoptosis (see Chapter 46).

E2F/DP that is free of Rb is a potent transcription factor, promoting expression of genes that stimulate both reentry of G0 cells back into the cycle and their subsequent passage through the cell cycle. These target genes encode proteins that are required for DNA synthesis (DNA polymerase a1, accessory factors, and enzymes that synthesize nucleotide precursors; see Chapter 42), proteins that promote cell-cycle progression (cyclins E and A, Cdk1 and Cdc25), and proteins that regulate cell-cycle progression (pRb, p107, Emi1).

Passage through the restriction point therefore hinges on Cdk activation (Fig. 41-7B; see also Chapter 40 and Appendix 40-1), which leads to Rb phosphorylation and activation of E2F/DP dimers on the promoters of essential cell-cycle genes. The normal pathway of Rb phosphorylation is started by Cdk4–cyclin D and Cdk6–cyclin D (referred to hereafter as Cdk4/6–cyclin D) and is carried forward by Cdk2–cyclin E and Cdk2–cyclin A. Cdk activity in early G1 is regulated by adjusting the relative levels of the three D-type cyclins and the CKI class Cdk2 inhibitors p27Kip1 and p21Cip1. Regulation of cyclin D levels provides the crucial link between extracellular mitogens and the cell cycle.

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