Introduction to the Cell Cycle

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CHAPTER 40 Introduction to the Cell Cycle

The cell cycle, is the series of events that leads to the duplication and division of a cell. Research on the molecular events of cell-cycle control revealed that variations of similar mechanisms operate the cell cycles of all eukaryotes from yeasts to humans. Furthermore, the components that regulate cell growth and division also play key roles in the cessation of cell division that accompanies cell differentiation. Control of the cell cycle is of major importance to human health because cancer, which is usually caused by perturbations of cell-cycle regulation, affects 46% of males and 38% of females in the United States.

Although animal cells have a wide variety of specialized cell cycles, the cells in the stratified epithelium that forms skin illustrate the most common lifestyles (Fig. 40-1). The basal layer of the epithelium is composed of stem cells that divide only occasionally. (See Box 41-1.) They can activate the cell cycle on demand and then return to a nondividing state. When specific signals induce stem cells to proliferate, one daughter cell usually remains a stem cell and the other enters a pool of rapidly dividing cells. These dividing cells populate the upper layers of the epithelium, stop dividing, and gradually differentiate into the specialized cells that cover the surface.

Figure 40-1 a Light micrograph of a section of skin, a stratified squamous epithelium, stained with hematoxylin and eosin. B, Diagram showing the different types of cell cycles at the various levels of this epithelium.

Similarly, the nervous system contains a few stem cells and a few dividing cells, but most neurons, once differentiated, can live for more than 100 years without dividing again. Like stem cells, fibroblasts of the connective tissue (see Fig. 28-2) are typically nondividing, but they can be stimulated to enter the cell cycle following wounding or other stimuli (see Fig. 32-11).

Figure 40-11 the original identification of a cyclin. Newly synthesized proteins (labeled with ³⁵S-methionine) in fertilized sea urchin eggs were separated by SDS polyacrylamide gel electrophoresis. It was noted that the protein labeled A (which was named cyclin) first accumulated, was greatly reduced at the metaphase/anaphase transition, and then began to accumulate again. Protein B, which is not involved in cell-cycle regulation, accumulated progressively over this time. “Cleavage index” refers to the percentage of dividing cells observed in the microscope at varying times after fertilization.

(From Evans T, Rosenthal ET, Youngblom J, et al: Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33:389–396, 1983.)

Principles of Cell-Cycle Regulation

The goal of the cell cycle in most cases is to produce two daughter cells that are accurate copies of the parent (Fig. 40-2). The cell cycle integrates a continuous growth cycle (the increase in cell mass) with a discontinuous division or chromosome cycle (the replication and partitioning of the genome into two daughter cells). The chromosome cycle is driven by a sequence of enzymatic cascades that produce a sequence of discrete biochemical “states” of the cytoplasm. Each state arises by destruction or inactivation of key enzymatic activities characteristic of the preceding state and expression or activation of a new cohort of activities. Later sections of this chapter explain these mechanisms.

Figure 40-2 introduction to the cell-cycle phases. A, Diagrams of cellular morphology and chromosome structure across the cell cycle. B, Time scale of cell-cycle phases. C, Length of cell-cycle phases in cultured cells.

Phases of the Cell Cycle

In thinking about the cell cycle, it is convenient to divide the process into a series of phases. Recognition of cell-cycle phases began in 1882, when Flemming named the process of nuclear division mitosis (from the Greek mito, or “thread”) after the appearance of the condensed chromosomes. It initially appeared that cells were active only during mitosis, so the rest of the cell cycle was called interphase (or resting stage) (Box 40-1).

BOX 40-1 Selected Key Terms

M phase: Cell division, comprising mitosis, when a fully grown cell segregates the replicated chromosomes to opposite ends of a molecular scaffold, termed the spindle, and cytokinesis, when the cell cleaves between the separated chromosomes to produce two daughter cells. In general, each daughter cell receives a complement of genetic material and organelles identical to that of the parent cell.

Interphase: The portion of the cell cycle when cells grow and replicate their DNA. Interphase has three sections. The G₁ (first gap) phase is the interval between mitosis and the onset of DNA replication. The S (synthetic) phase is the time when DNA is replicated. The G₂ (second gap) phase is the interval between the termination of DNA replication and the onset of mitosis. In multicellular organisms, many differentiated cells no longer actively divide. These nondividing cells (which may physiologically be extremely active) are in the G₀ phase, a branch of the G₁ phase.

Checkpoints: Biochemical circuits that regulate cell-cycle transitions in response to the physiological condition of the cell and the state of its environment. Checkpoints detect the presence or absence of external signals telling the cell to proliferate, damage to the DNA, and problems that arise during DNA replication and chromosome segregation.

Once DNA was recognized as the agent of heredity in the 1940s, it was deduced that DNA must be duplicated at some time during interphase so that daughter cells can each receive a full complement of genetic material. A key experiment identified the relationship between the timing of DNA synthesis and the mitotic cycle (Fig. 40-3) and defined the four cell-cycle phases as they are known today (Fig. 40-2).

Figure 40-3 To determine whether cells synthesize DNA during a defined portion of the cell cycle or constantly throughout the entire cycle (as is the case in bacteria, for example), Howard and Pelc fed a radioactive component of DNA (³²P) to onion root tip cells, spread the cells in a thin layer on a microscope slide, washed away the ³²P that had not become incorporated into DNA, and overlayered the slide with photographic emulsion. After incubation in the dark, the emulsion was developed like film and examined with a light microscope. The nuclei of cells that were engaged in active DNA replication during the period of exposure to ³²P incorporated the radioactive label into DNA and exposed the photographic emulsion above them. Two possible outcomes were predicted. If cells synthesized DNA constantly during interphase, then all cells would incorporate the radioactive label. Conversely, if each cell synthesized DNA only during a discrete portion of the cell cycle, then only those cells that were engaged in active replication during the period of exposure to ³²P would expose the photographic emulsion. When the slides were examined, 20% of the interphase cell nuclei were labeled, proving that cells synthesize DNA only during a discrete portion of interphase. Mitotic cells were unlabeled. Assuming that the cells traverse the cycle at a more or less constant rate, it was possible to calculate the length of the synthetic phase. Overall, the time between successive divisions—the generation time—was about 30 hours in the root tip cells. If about 20% of the cells were labeled, then about 20% of the 30-hour generation time must be spent in DNA synthesis. Thus, 0.2 × 30, or 6 hours, was spent in replication.

(Drawing based on the work of Pelc HA Sr: Synthesis of DNA in normal and irradiated cells and its relation to chromosome breakage. Heredity Suppl 6:261–273, 1953.)

Each cell is born at the completion of the M phase, which includes mitosis, the partitioning of the chromosomes and other cellular components, and cytokinesis, the division of the cytoplasm. The chromosomal DNA is replicated during S phase (synthetic phase). The remaining two phases are gaps between mitosis and the S phase. The G₁ phase (first gap phase) is the interval between mitosis and DNA replication. The G₂ phase (second gap phase) is the interval between the completion of DNA replication and mitosis. All cycling cells have an M phase and an S phase; however, some early embryos have minimal G₁ and G₂ phases. The following sections describe the stages of the cell cycle in order, starting just after the birth of the cell.

G₁ Phase

The G₁ phase is typically the longest and most variable cell-cycle phase. When cells are “born” at cytokinesis, they are half the size they were before mitosis, and during G₁, they grow back toward an optimal size. During this time, many genes involved in cell-cycle progression are switched off so that the cell cannot initiate a new round of proliferation. This repressive system is termed the restriction point. If the supply of nutrients is poor or if cells receive an antiproliferative stimulus such as a signal to embark on terminal differentiation, they delay their progress through the cell cycle in G₁ or exit the cycle to enter G₀ (see the next section). However, if appropriate positive stimuli are received, cells overcome the restriction point block and trigger a program of gene expression that commits them to a new cycle of DNA replication and cell division. Faulty restriction point control may result in cell proliferation under inappropriate conditions. Cancer cells often have defects in restriction point control and continue to attempt to divide even in the absence of appropriate environmental signals.

G₀ and Growth Control

Most cells of multicellular organisms differentiate to carry out specialized functions and no longer divide. Such cells are considered to be in the G₀ phase. G₀ cells are not dormant; indeed, they are often actively engaged in protein synthesis and secretion, and they may be highly motile. The G₀ phase is not necessarily permanent. In some cases, G₀ cells may be recruited to reenter the cell cycle in response to a variety of stimuli. This process must be highly regulated, as the uncontrolled proliferation of cells in a multicellular organism can lead to cancer.

S Phase

Chromosomes of higher eukaryotes are so large that replication of the DNA must be initiated at many different sites, termed origins of replication. In budding yeast, the approximately 400 origins are spaced an average of 30,000 base pairs apart. An average human chromosome contains about 150 ×10⁶ base pairs of DNA, about 10 times the size of the entire budding yeast genome, so many more origins are required. Each region of the chromosome that is replicated from a single origin is referred to as a replicon.

Proliferating diploid cells must replicate their DNA once and only once each cell cycle. Each origin of replication is prepared for replication by the formation of a prereplication complex (a process that is referred to as licensing) during G₁. As each origin “fires” during S phase, the prereplication complex is dismantled and cannot be reassembled until the next G₁ phase. This ensures that each origin fires only once per cell cycle. The cyclic nature of origin licensing is driven at least in part by fluctuations in the activity of cyclin-dependent kinases (discussed later).

During replication, the duplicated DNA molecules, called sister chromatids, become linked to each other by a protein complex called cohesin (see Fig. 13-19). This pairing of sister chromatids is important for their symmetrical segregation later in mitosis (see Fig. 44-16).

G₂ Phase

In most cells of metazoans, G₂ is a relatively brief period during which key enzymatic activities that will trigger the entry into mitosis gradually accumulate and are converted to active forms. When their activities reach a critical threshold level, the cell enters mitosis. In parallel, the chromatin and cytoskeleton are prepared for the dramatic structural changes that will occur during mitosis. If unreplicated or damaged DNA is detected during G₂, a mechanism called a checkpoint delays entry of the cell into mitosis.

M Phase

During M phase (mitosis and the subsequent cytokinesis), chromosomes and cytoplasm are partitioned into two daughter cells. Mitosis is normally divided into five discrete phases.

Prophase is defined by the onset of chromosome condensation inside the intact nucleus and is actually the final part of G₂ phase. In the cytoplasm, a dramatic change in the dynamic properties of the microtubules decreases their half-lives from ˜10 minutes to ˜30 seconds. The duplicated centrosomes (centrioles and associated pericentriolar material in animal cells) separate and form the two poles of the mitotic spindle.

Prometaphase begins when the nuclear envelope breaks down (in higher eukaryotes) and chromosomes begin to attach randomly to microtubules emanating from the two poles of the forming mitotic spindle. Chromosomes may also nucleate some spindle microtubules. Once both kinetochores on a pair of sister chromatids are attached to opposite spindle poles, the chromosome slowly moves to a point midway between the poles. When all chromosomes are properly attached, the cell is said to be in metaphase.

The exit from mitosis begins at anaphase with the abrupt separation of the two sister chromatids from one another. The metaphase-anaphase transition is triggered by the proteolytic degradation of molecules that regulate sister chromatid cohesion. During anaphase, the separated sister chromatids move to the two spindle poles (anaphase A), which themselves move apart (anaphase B). As the chromatids approach the spindle poles, the nuclear envelope reforms on the surface of the chromatin. At this point, the cell is said to be in telophase.

Finally, during telophase, a contractile ring of actin and myosin assembles as a circumferential belt in the cortex midway between spindle poles and constricts the equator of the cell. The separation of the two daughter cells from one another is called cytokinesis.

Checkpoints

The cell cycle is highly regulated, and checkpoints control transitions between cell-cycle stages. Checkpoints are biochemical circuits that detect external or internal problems and send inhibitory signals to the cell-cycle system. There are four major types of checkpoints. The restriction point in the G₁ phase is sensitive to the physiological state of the cell and to its interactions with the surrounding extracellular matrix. Cells that do not receive appropriate growth stimuli from their environment do not progress past this point in the G₁ phase and may commit suicide by apoptosis (see Chapter 46).

DNA damage checkpoints operate in G₁, S, and G₂ phases of the cell cycle. In general, these checkpoints block cell-cycle progression, but they can also trigger cell death by apoptosis. The DNA replication checkpoint detects the presence of unreplicated or stalled DNA replication forks. This checkpoint shares some components with the DNA damage checkpoints but has the additional feature that it specifically stabilizes stalled replication forks so that they can be repaired. During mitosis, the spindle assembly checkpoint (also called the metaphase checkpoint) delays the onset of chromosome segregation until all chromosomes have attached properly to the mitotic spindle.

The checkpoints in G₁, S, and G₂ use common strategies to regulate cell-cycle progression (Fig. 40-4). DNA damage is detected by sensors. These activate transducers, which are often protein kinases but may also be transcriptional activators. The transducers act on effectors, which ultimately block cell-cycle progression and may also fulfill other functions. Two key protein kinases, ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad9 related (ATR), lie at the head of the pathway and may act as sensors of DNA damage. They activate two transducer kinases Chk1 and Chk2 and also stabilize a transcription factor called p53 that induces the expression of a cohort of genes involved in halting cell-cycle progression as well as genes that trigger cell death by apoptosis. Chapters 41 and 43 discuss these proteins in detail. In general, DNA damage checkpoints block cell-cycle progression by inhibiting the cyclin-dependent kinases by a variety of mechanisms.

Figure 40-4 Elements of the DNA damage checkpoint system.

The Biochemical Basis of Cell-Cycle Transitions

Transitions between cell-cycle phases are triggered by a network of protein kinases and phosphatases that is linked to the discontinuous events of the chromosome cycle by the periodic accumulation, modification, and destruction of several key components. This section provides a general introduction to the most important components of this network.

Cyclin-Dependent Kinases

Genetic analysis of the cell cycle in the fission yeast Schizosaccharomyces pombe identified a gene called cell division cycle–2⁺ (cdc2⁺) that is essential for cell-cycle progression during both the G₁ S and G₂ M transitions (Box 40-2). The product of this gene, a protein kinase of 34,000D originally called p34^cdc2, is the prototype for a family of protein kinases that is crucial for cell-cycle progression in all eukaryotes. This mechanism of cell-cycle control is so well conserved that a human homolog of p34^cdc2 can replace the yeast protein, restoring a normal cell cycle to a cdc2 mutant yeast. Boxes 40-3 and 40-4 present a number of the key experiments and experimental systems that led to the identification of the molecules that drive the cell cycle.

BOX 40-2 Use of Genetics to Study the Cell Cycle

Studies of the distantly related budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (see Fig. 2-9) have been extremely important for understanding the cell cycle for several reasons. First, the proteins that control the cell cycle are remarkably conserved between yeasts and mammals. Second, both yeast genomes are sequenced and annotated, simplifying characterization of novel gene products. Third, genetic analysis is facilitated, as both yeasts grow as haploids, and both efficiently incorporate cloned DNA into their chromosomes by homologous recombination.

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