Unit V: Regulation of Cell Growth and Cell Death
Life is pleasant. Death is peaceful. It’s the transition that’s troublesome.
—Isaac Asimov (American science fiction writer and biochemist, 1920–1992)
Arguably the most important occurrences within the life of a cell are its generation from a progenitor followed by its demise, either through a natural or through a pathological process. Regulation of both processes is critical to ensure that the appropriate number of cells is available to carry out their function within the organism. Safeguards are also needed to protect against uncontrolled growth, which may result in malignancy and the death of the organism in which the cell is a participant. This unit begins with a description of the cell cycle, the orderly sequence of biochemical events that culminates with the generation of two new cells from one parent cell.
Cells that enter the active phases of the cell cycle often do so after resting in interphase for varying periods of time, depending on the cell type. Once a cell commits itself to dividing and to passing on its genetic information, it enters into a critical transition period, which, if unsuccessful, will mean that the cell has failed to reproduce itself and will likely die. Curiously, if the outcome of the cell cycle is a success, the parent cell will cease to exist, with two exact replicas replacing it.
The second chapter in this unit concerns regulation of the cell cycle and the third focuses on abnormal cell growth. Checks and balances within the cell cycle are necessary to enable the orderly process of cell duplication to occur. As our understanding of abnormal cell growth progresses, improved treatments to halt unregulated growth may be developed. The fourth chapter in this unit focuses on cell death, particularly on the physiological process of apoptosis, where collateral damage is minimized. This unit concludes with the final chapter, an exploration of aging and senescence of the cell and the organism.
Multicellular organisms are composed of a variety of specialized cells organized into a cellular community. When an organism requires additional cells, either for growth or to replace those normally lost, new ones must be produced by cell division, or proliferation. Somatic cells are generated by the division of existing cells in an orderly sequence of events. They duplicate their contents and then divide to produce two identical daughter cells. This sequence of duplication is known as the cell cycle and is the essential mechanism of eukaryotic reproduction.
Cell division occurs throughout the life of the organism, although different cell types divide more or less often than others. Cells display remarkable variation in their proliferative capacity, depending on the cell type and the age of the individual. For example, neonatally derived fibroblasts can complete close to fifty rounds of division, but fibroblasts isolated from adults can complete only approximately half as many cell cycles.
Homeostasis, or stable system maintenance, requires that as cells die off or are lost (e.g., through abrasion or sloughing), they are replaced by tissue-type specific cells. Cellular turnover is a normal function. The turnover times for some cells in the adult body are slow or nonexistent (in the endocrine and central nervous system, for example), whereas other cells turnover very rapidly. Each individual has approximately 2 × 1013erythrocytes.
Because the half-life for an erythrocyte is approximately 115 days, the human body must replace approximately 1011 new red blood cells every day! The most abundant leukocytes, neutrophils, have a half-life of approximately 10.5 h, which means the body needs to replace approximately 6 × 1010 neutrophilsper day. Cells within the epithelia also turnover rapidly. The lifespan for cells that line the stomach is 3 to 5 days and that for enterocytes that line the small bowel is 5 to 6 days.
For a cell to generate two daughter cells, complete copies of all of the cell’s constituents must be made. Genetic information contained within multiple chromosomes must be duplicated; the cytoplasmic organelles and cytoskeletal filaments must be copied and shared between the two newly formed daughter cells. The cell cycle may be broadly divided into three distinct stages: interphase, mitosis, and cytokinesis. Interphase is the period between successive rounds of nuclear division and is distinguished by cellular growth and new synthesis of DNA. It can be further subdivided into three phases called G1 phase, S phase, and G2 phase (Figure 20.1).
Division of genetic information occurs during the stage of the cell cycle known as mitosis. Mitosis can be divided into five distinct phases called prophase, prometaphase, metaphase, anaphase, and telophase and assures that each daughter cell will have identical complete functional copies of the parent cell’s genetic material. The third stage, cytoplasmic division or cytokinesis, culminates with the separation into two distinct daughter cells that enter interphase.
FIGURE 20.1.Stages of cell division and the cell cycle.
All cells, whether actively cycling or not, spend the vast majority of their lives in interphase. Interphase is an eventful and important part of the cell cycle and comprises G1, S, and G2 phases (Figure 20.2). Cellular growth and DNA synthesis occur during interphase, resulting in a duplication of cellular materials so that there are sufficient materials for two complete new daughter cells.
G1 and G0 phases
Named for the gap that follows mitosis and the next round of DNA synthesis, G1 phase is generally both a growth phase and a preparation time for DNA synthesis of S phase (Figure 20.3). RNA and protein synthesis also take place during G1 phase. In addition, organelles and intracellular structures are duplicated and the cell grows during this phase. The length of G1 phase is the most variable among cell types. Very rapidly dividing cells, such as growing embryonic cells, spend very little time in G1 phase.
On the other hand, mature cells that are no longer actively cycling are permanently in G1 phase. Those cells in G1 phase that are not committed to DNA synthesis are in a specialized resting state called G0 phase (pronounced G-zero phase). Some inactive or quiescent cells in G0 phase may reenter the active phases of the cell cycle upon proper stimulation. The restriction point is located within G1phase and, if passed, will commit a cell to continuing into DNA synthesis within S phase. The restriction point is critical for cell cycle regulation and is detailed in Chapter 21.
FIGURE 20.3. G1 and G0 phase.
Synthesis of nuclear DNA, also known as DNA replication, occurs during S phase (Figure 20.4). Each of the 46 chromosomes in a human cell is copied to form a sister chromatid. ATP-dependent unwinding of the chromatin structure by DNA helicase exposes the binding sites for DNA polymerase that will catalyze the synthesis of new DNA in the 5? to 3? direction. Multiple replication forks are activated on each chromosome in order to ensure that the entire genome is duplicated within the time span of S phase. Upon completion of DNA synthesis, chromosome strands are condensed into tightly coiled heterochromatin. The time for completion of this process is relatively constant among cell types. Actively cycling cells spend approximately 6 h in S phase. DNA replication is described in detail in Chapter 7.
FIGURE 20.4. S phase.
The gap between the completion of S phase and the start of mitosis, known as G2, is a time of preparation for the nuclear division of mitosis (Figure 20.5). This safety gap allows the cell to ensure that DNA synthesis is complete before proceeding to nuclear division in mitosis. Also contained with the G2 is a checkpoint where intracellular regulatory molecules assess nuclear integrity (see Chapter 21.III). Typically, this phase lasts for approximately 4 h.
FIGURE 20.5. G2 phase.
Mitosis or nuclear division is a continuous process and can be divided into five phases based on progress made to a specific point in the overall nuclear division. Dividing cells spend about 1 h in mitosis (Figure 20.6). After completion of nuclear division, cytokinesis occurs, involving cytoplasmic division and resulting in the formation of two separate daughter cells from the one parent cell.
FIGURE 20.6.M phase.
In prophase, the nuclear envelope remains intact while the chromatin that was duplicated during S phase condenses into defined chromosomal structures called chromatids (Figure 20.7A). Chromosomes of mitotic cells contain two chromatids connected to each other at a centromere. Specialized protein complexes, called kinetochores, form and associate with each chromatid. Mitotic spindle microtubules will attach to each kinetochore as chromosomes are moved apart later in mitosis.
The microtubules of the cytoplasm disassemble and then reorganize on the surface of the nucleus to form the mitotic spindle. Two centriole pairs push away from each other by growing bundles of microtubules forming the mitotic spindle. The nucleolus, the organelle within the nucleus where ribosomes are made, disassembles in prophase.
For illustrative purposes, three chromosomes have been drawn.
The disassembly of the nuclear envelope marks the beginning of prometaphase (Figure 20.7B). Spindle microtubules bind to kinetochores and chromosomes are pulled by the microtubules of the spindle.
Metaphase is characterized by chromatids aligned at the equator of the spindle, halfway between the two poles (Figure 20.7C). The aligned chromatids form the metaphase plate. Cells can be arrested in metaphase when microtubule inhibitors are used (see also Chapter 21). Karyotype analyses used to determine the overall chromosome composition and structure most often require cells in metaphase.
In anaphase, the mitotic poles are pushed further apart as a result of polar microtubules elongating (Figure 20.7D). Each centromere splits in two and paired kinetochores also separate. Sister chromatids migrate toward the opposite poles of the spindle.
The last phase of nuclear division, telophase, is characterized by kinetochore microtubule disassembly and mitotic spindle dissociation (Figure 20.7E). Nuclear envelopes form around each of the two nuclei containing the chromatids. The chromatids decondense into dispersed chromatin and nucleoli reform in the daughter nuclei.
Early in the 20th century, researchers observed that cancerous tumors arising in rodents could be serially transplanted indefinitely. By midcentury, cancer cells were shown to be immortal in tissue culture. In the 1960s, Dr Leonard Hayflick made the startling observation that normal noncancerous cells have a limited replicative capacity and are mortal. Hayflick discovered that human umbilical cord fibroblasts stopped dividing after about 50 divisions in culture—a phenomenon that has come to be known as Hayflick’s limit. Fibroblasts cultured from adults were able to divide far fewer times. Replicative capacity depends upon the number of cell divisions and not on the age of the cells
Contributing to Hayflick’s limit is the irreversible shortening of telomeres (a hexameric DNA repeat sequence, TTAGGG, at the end of each human chromosome) each time a cell divides. Although telomerase, a complex of RNA and protein, helps maintain and repair telomeres by adding telomeric repeats, telomeric material is eventually lost, contributing to cellular senescence or aging. Telomeres normally help move chromosomes to opposite poles within the cell during telophase. When telomeres become too short, chromosomes can no longer segregate, and the cell can no longer divide
Some tissues require continuous cell replacement, such as skin and gut epithelia and erythrocytes. These cells derive from progenitor stem cells that do not exhibit Hayflick’s limit. Other cells that are subject to Hayflick’s limit rarely divide, such as cells of the endocrine system, or not at all, such as neurons, during adult life.
CYTOKINESIS—COMPLETION OF A CELL CYCLE
In order to create two distinct, separate daughter cells, cytoplasmic division follows nuclear division. An actin microfilament ring forms to create the machinery needed. Contraction of this actin-based structure results in the formation of a cleavage furrow that is seen beginning in anaphase (Figure 20.7F). The furrow deepens until opposing edges meet. Plasma membranes fuse on each side of the deep cleavage furrow and the result is the formation of two separate daughter cells, each identical to the other and to the original parent cell.
First discovered in the eggs of African clawed toad Xenopus laevis, aurora kinases, a family of serine/threonine kinases, play important functional roles during mitosis, specifically by controlling chromatid segregation. Three members of the aurora kinase family have been discovered in mammalian cells. Aurora A functions in prophase and is critical for proper formation of the mitotic spindle and for recruitment of proteins to stabilize centrosomal microtubules. Without Aurora A, the centrosome does not accumulate sufficient ?-tubulin for anaphase and the centrosome never fully matures.
Aurora A is also necessary for proper separation of centrosomes after the spindle has been formed. Aurora B functions in the attachment of the mitotic spindle to the centromere and also in cytokinesis for cleavage furrow formation. Aurora C is expressed in germ line cells, although its function remains to be elucidated. Elevated expression of all three members of the aurora kinase family has been observed in many human tumors. Inhibitors of aurora kinases are being assessed as anticancer therapies.
ASSESSMENT OF THE CELL CYCLE
Both the assessment of cell proliferation and the cell cycle are clinically important for the evaluation of disease progression. Equally important to both cell biology and drug-discovery research are methods used to evaluate cell proliferation and the role of agents that promote or retard the cell cycle. Although there are a number of tools and methods to assess proliferation, they can basically be divided into those used to analyze cell proliferation and those used to assess the cell cycle.
Assessment of cell proliferation
The proliferation of cells may be assessed either by measuring the synthesis of new (nascent) DNA or by the serial, halving dilution of labeled cytoplasmic proteins as the cell divides.
1. DNA synthesis: It may be assessed using modified analogs of thymidine, one of the nucleoside building blocks of DNA. In one experimental approach, radioactive tritiated (3H) thymidine is added into tissue culture medium in which cells are grown. Because thymidine is exclusively used for DNA synthesis, cells that are actively synthesizing DNA will incorporate 3H-thymidine and the accumulated radioactivity can be measured (Figure 20.8). A nonradioactive method to assess nascent DNA synthesis is to incubate the cells with the bromodeoxyuridine (BrDu) analog of thymidine. After the incubation period, cellular DNA is denatured by heat or acid and a labeled anti-BrDu monoclonal antibody is used as a probe to measure newly synthesized DNA.
FIGURE 20.8.Cell proliferation assessed by 3H-thymidine incorporation by stimulated lymphocytes.
2. Dilution of a cytoplasmic probe: It may also be used to assess cell proliferation. Cells are labeled with the succinimidyl ester of carboxyfluorescein diacetate (CFSE). The diacetate residues of CFSE allow it to readily diffuse across plasma membranes and into the cytoplasm. There, intracellular esterases cleave the acetate groups making the compound both fluorescent and membrane impermeant, thus trapping CFSE within the cell. The succinimidyl ester groups of CFSE readily and irreversibly bind to available amines (usually on lysine) on intracellular cytoplasmic and membrane proteins. As cells divide, their fluorescently labeled cytoplasmic proteins are divided equally between the two daughter cells. Each daughter cell has half the fluorescence of the previous generation, which can be measured by flow cytometry (Figure 20.9).
FIGURE 20.9.Cell proliferation by stimulated lymphocytes assessed by CFSE.
Cell cycle analysis
The amount of DNA contained within a cell is cell cycle dependent and ranges between 1n in G1 phase and 2n in G2 and M phases. Cell cycle distribution within a cell population can be assessed by flow cytometry and is a clinically important tool both in evaluating treatment therapies in lymphomas and leukemias and as a research tool in evaluating oncogene and tumor suppressor gene mechanisms. Briefly, any one of a wide variety of nucleic acid–binding fluorescent dyes may be used to label DNA. Fluorescence is proportional to the DNA content of the cell. Analysis of a flow cytometry histogram shows the proportion of cells within the population in G1, S, and G2 phases of the cell cycle (Figure 20.9).
- New cells to account for growth or to replace cells lost by injury or disease are produced from existing somatic cells by cell division.
- The sequence of duplication and division is known as the cell cycle.
- The cell cycle is divided into three stages: interphase, mitosis, and cytokinesis.
- All cells, including those cycling actively, spend the majority of their time in interphase, which is composed of G1, S, and G2 phases.
- Interphase is an eventful phase that includes cellular growth, protein and RNA synthesis in G1 phase, DNA synthesis in S phase, and preparation for mitosis during G2 phase.
- In mitosis, nuclear division follows interphase and culminates in two separate nuclei identical to each other and to the nucleus of the parent cell that was copied.
- Phases within mitosis include prophase, prometaphase, metaphase, anaphase, and telophase.
- Cytokinesis, or cytoplasmic division, occurs after mitosis and results in two distinct, separate daughter cells identical to each other and to the parent cell