Article:
The Study of Life -Cell Cycle Regulation
One of the most fascinating stories in science is the explanation of how a single cell, a newly fertilized egg, developes into a community of cells organized into a complex living creature. During the development of a complex creature such as a human being, as many as 1015 cells may be created. Consider that the process started out as a single cell, a fertilized egg which then became two cells, and then two became four, and so on, an exponential progress. The math part is easy to understand - it's not too difficult to see how one cell became so many. But this just raises more questions. For example, how do cells know when to stop growing? And, why do we grow more (and differently) in some areas than others? And, oddly enough, how do cells know when they should die (undergo apoptosis).
CELL CYCLE REGULATION
One of the most fascinating stories in science is the explanation of how a single cell, a newly fertilized egg, develops into a community of cells organized into a complex living creature. During the development of a complex creature such as a human being, as many as 1015 cells1 may be created. Consider that the process started out as a single cell, a fertilized egg which then became two cells, and then two became four, and so on, an exponential progress. The math part is easy to understand - it's not too difficult to see how one cell became so many. But this just raises more questions. For example, how do cells know when to stop growing? And, why do we grow more (and differently) in some areas than others? And, oddly enough, how do cells know when they should die (undergo apoptosis).2
In order to appreciate recent breakthroughs in our understanding of this process, it is necessary to review the concept of the cell cycle. High school biology texts usually focus on the mitosis portion of the cell cycle Mitosis is broken down into the prophase (where chromosomes coil into short rods, nuclear membrane breaks down, spindles form), metaphase (chromosomes attach to spindles and line up in center of cell), anaphase (each chromosome separates from its copy and is pulled to opposite ends of the cell), and telophase (each side of cell now has a complete set of chromosomes and nuclear membranes form around them, chromosomes uncoil so that protein synthesis may begin). A cell cycle actually consists of five stages, labeled G1, S, G2, M and D (Figure 1). The G1, S, and G2 stages were once thought to be part of a single interphase, a period of rest between cell divisions. This idea arose because it is only during the M and D stages that visible activity occurs.3 We now know, however, that most DNA, RNA and protein is made during interphase which is actually the period when cellular metabolic activity is at its highest. The construction (synthesis) of new DNA and RNA is mostly confined to the S Stage. The cell then moves into the G2 stage (a sort of rest period just before mitosis occurs), then to M state (mitosis), then to D stage (cell division) and finally to G1, the first stage, another "rest" period. A typical animal cell in tissue culture may take 24 hours to go through this cycle.
TheCell Cycle
It may help to think of the various biochemical regulatory processes as mere "tools" that the cell uses to regulate the cell cycle. This is no different than viewing a hammer and nails as a technique in building a house, or mathematical formulas as tools in understanding why stars shine, magnets or computers work. This article discusses these tools and attempts to give the reader an idea of the tasks that they perform. Some of these tools work directly on our genes and proteins. Others work indirectly, by acting on other tools. Some of these tools, such as transcription factors have already been discussed in prior articles (see Transcription Factors - Genetic Switches, The DOUBLE HELIX, Vol. 5:1, September 1996). This article will focus upon two tools or techniques responsible for overall control of the cell cycle -- cyclin-dependent kinases and proteolysis -- and one tool (actually a set of related tools) that limit DNA synthesis to the S phase of the cell cycle -- replicators and initiator proteins. Cyclin-Dependent Kinases
Yeast cells (Saccharomyces cerevisiae) are the most primitive eukaryotic cells. It was through studies of these cells that we learned of the role of the first of these "tools" (or techniques), the group of chemical compounds known as cyclin-dependent kinases, or Cdks. These proteins are the key regulators of the eukaryotic4 cell cycle. While yeast has its own distinctive set of Cdks, multicellular eukaryotes show a Cdk inventory that is remarkably consistent (i.e. conserved) through the phyla. The targets of cyclin-Cdk complexes are proteins which have a direct effect on transcription 5 of cell cycle genes. Fruit flies and vertebrates share these target molecules.
Cyclins are grouped by letters, e.g. A-, B-, D- , and E- types, as well as their kinase partners such as Cdk1, Cdk2, Cdk4, or Cdk6, all of which are present in species ranging from fruit flies to man. Different combinations of these cyclins and kinases regulate different portions of the cell cycle. By way of example, D-type Cyclins partnered with Cdk4 or Cdk6 regulate progression through The G1 phase of the cell cycle. The complex (a chemical combination) of an E cyclin with Cdk2 (called cyclin E-Cdk2) regulates entry into S phase, cyclin A-Cdk2 regulates progression through S phase, and cyclins A and B in complexes with Cdk1 or Cdc2 regulate entry into M phase.
Although cyclin-dependent kinases are the key cell cycle regulators, their concentrations must be controlled if they are not to do too much or too little of whatever their assignment is and end up disrupting, instead of coordinating the cell cycle. Activation of the kinases is, in fact, carefully controlled through the process of phosphorylation which can either stimulate or inhibit their activity. Concentrations of the activating cyclin molecules are regulated through the processes of transcription (which increases their concentrations) and proteolysis (a destructive process which reduces concentrations). More will be said about proteolysis below.
Where do the cell cycle regulators come from? For embryos that develop in large eggs, nutrients and cell cycle control molecules are packed into the egg during oogenesis (creation of the egg). This allows early embryonic development to proceed rapidly as the egg is compartmentalized into smaller and smaller cells. In mammals, where the embryo is nurtured by the mother, the process is different. Cells tend to increase their mass during each division cycle, and differentiation occurs as this process goes on.
In the case of eggs, at least one of the maternal (there is no input from the father at this point in time) cell cycle regulators must be kept inactive to keep the cell from cycling it is fertilized. Different organisms have developed6 different strategies to arrest the cell cycle at this point. For example, in vertebrates, the kinase c-Mos causes the cell cycle to freeze in metaphase of the second meiotic division (this is referred to as meiotic arrest). Fruit flies use the mechanical tension exerted by the spindle on paired meiotic chromosomes.
Fertilization unfreezes this meiotic arrest which allows maternal gene products (packaged with the egg) to trigger the cell cycle and explosive mitosis and cell proliferation to begin. Eventually these maternal cell cycle activators are degraded and, in order for the cell cycle to continue, quantities of the activator proteins must be produced through transcriptional copying during mitosis as the embryo develops. From this point on, cell proliferation is controlled by genes in the developing zygote which permits exquisite control over the activation and inhibition of control proteins, allowing them to be used selectively, depending on the cell type.
As an example, in fruit flies the maternal cell cycle activator Cdc25 activates Cyclin A-Cdk1 and Cyclin B-Cdk1 kinases, which trigger mitosis. Cdc25 is initially present in large quantities in the egg, which leads to explosive cell growth. When Cdc25 is degraded (through proteolysis), explosive cell division slows down and interphases to become longer. Cycles that occur after the maternal Cdc25 is degraded are regulated at G2 and M phase changes through surges of transcription of embryonic Cdc25. This process is controlled by the zygotic Cdc25string gene. Cells destined for the same fate, such as cells that are to form the nervous system, express Cdc25string at the same time.
This gene is one of several that belong to a well-studied group of genes that encode transcription factors (proteins that act like gene switches, turning genes on or off ). Some of these transcription factors act on the regulatory region of the Cdc25string, a part of the gene contains pattern information in a way similar to that of major developmental controllers like the homeobox genes.7 To sum up, fruit fly studies have demonstrated the existence of homeobox-like genes that generate transcription factors (gene switches) that control regulator genes (like the Cdc25string gene) that, in turn, control the activation of cyclin-dependent kinases (Cdks) that directly affect the cell cycle.
Once the zygote takes over production of its own cell-cycle activators, how does it know when to stop? In other words, how does it know that it has essentially reached the adult form? In fruit flies, most embryonic cell cycle S phases occur immediately after mitosis, with no intervening G1 rest periods. Somehow, after the final embryonic mitosis, cells arrest for the first time in G1. This occurs as the result of the carefully timed inactivation of Cyclin E-Cdk2, which normally stimulates DNA replication. Research indicates that Cyclin A may also require inactivation at this point. Embryos with too much Cyclin E-Cdk2 activity go through an extra cell cycle and die as a result. It is still not known exactly why Cyclin E transcription stops at this point. Studies of a particular fruit fly gene, roughex, demonstrate a role in synchronizing cell cycles at the G1 phase. Cells with a mutation of this gene skip G1 and go directly into another S phase, resulting in a condition called hyperplasia, where excessive cell production leads to excessive tissue formation. Another fruit fly gene, rca-1, may also play a role in ending embryonic cell proliferation and facilitating entry into the G1 phase. It is sensitive to concentrations of Cyclin A and may activate when cyclin A activity has reached certain levels. A similar gene, which has homologies in mammals, has been identified in the nematode worm, Caenorhabditis elegans. The answer to the puzzle of what times the transition from embryonic cell division to adult cell division is not simple or entirely understood. Nature seems to have provided a number of genetic mechanisms that work independently toward the same result.
But, are fruit fly studies relevant to an understanding of vertebrate or even mammalian genetics? There are tissues in Drosophila known as "imaginal disks" (which eventually differentiate into wings and legs) that show a cell proliferation pattern similar to that of vertebrates. These cells have a lot in common with vertebrate cells in the way they proceed through embryogenesis. Unlike most fruit fly cells (which skip a G1 phase during embryogenesis), imaginal cells arrest in G1 during mid-embryogenesis and remain dormant until after the larva hatches. Their cell cycle reactivates when they acquire an influx of nutrition from feeding. These cells maintain a constant size as they proliferate which indicates that cell size is a limiting factor in their growth.
Cell-to-cell signaling plays an important role in this process. An immature disc cell transplanted into an adult grows to adult size and stops. The same phenomenon has been seen with certain vertebrate organ cells. One theory suggests that each cell carries a sort of "genetic map" of what its "neighborhood" should look like. Using some form of cell-to-cell communication, the cell "perceives" its location on this map and "knows" whether to grow (or not) and to divide to make more cells (or not to). Scientists are still trying to explain the mechanism as to how signaling proteins (used for cell-to-cell communications) effect transcription factors and control expression of cell cycle regulators such as Cyclins, Cdks and Cdk inhibitors. Many of the same signaling proteins are present in vertebrates. Experiments in mice have shown that when these signaling genes are knocked out in mice, dramatic mutations occur.
Proteolysis
Proteolysis, which is a destructive process, plays an important role in cell cycle regulation not only by regulating CDK activity, but by directly influencing chromosome and spindle formation. When cell cycle regulators undergo proteolysis (are destroyed), the cell cycle is either slowed or accelerated. But, how is proteolysis controlled? Or, restated, how are targets for destruction selected?
The target must first be marked. This is done by assembly of a ubiquitin chain which acts like a molecular "flag" which is attached to the protein to be degraded (broken down or destroyed). This process, known as ubiquitin tagging, sets the target up for attack by an the enzyme, 26S proteasome, process known as ubiquitin-mediated proteolysis. When the destructive process needs to be slowed down, ubiquitin chains can be selectively disassembled by certain enzymes (deubiquitinating enzymes) that can reverse the process. The number and location of ubiquitin chains on the target (also referred to as the substrate) guarantees efficient recognition by the proteasome enzyme. Yeast cells have 16 genes that code for deubiquitinating enzymes. Ubiqutin chains are assisted in attaching to their target by ubiquitin-protein ligase, also known as E3. Yeast cells have 13 genes that code for E3 enzymes.
For a eukaryotic cell to progress through the cell cycle requires cyclin-dependent kinase activity. In vertebrates, cyclins D and E function during the G1 phase, cyclins E and A during S phase, and A and B during mitosis (M phase). In order for a cell to move from the G1 (rest) phase into S (DNA/RNA synthesis), the G1 cyclins experience proteolysis (are destroyed). In yeast this is accomplished by the CDC34 gene. In a similar vein, proteolysis plays a key role the timing and initiation of each cell cycle transition.
In summary, the cell cycle can be considered as a kinase cycle, with the specific events of the cycle being the result of the activity of specific CDK molecules. The discovery that mitotic cyclins (which play a role in regulating mitosis) must be degraded (through proteolysis) in order for the cell to emerge from mitosis demonstrated the critical role of proteolysis in controlling CDK activity and driving the progress of a cell through the cell cycle. The recent discovery that proteolysis triggers anaphase through a mechanism that has nothing to do with CDK destruction shows that proteolysis also controls a step in the chromosome cycle.
Replicators and Inhibitors
How does the cell ensure that DNA replication is limited to S phase, and only occurs once per cell cycle? Replicators are small sections of DNA which are essential to the process of DNA copying. Initiators are proteins. ORC (origin recognition complex) is a key initiator protein in eukaryotic cells. ORC can be thought of as a platform for all protein-protein interactions that are regulated during the cell cycle. ORC binds to replicators. As a cell prepares to exit the G1 phase of the cell cycle, it forms a pre-replication complex (pre-RC) at the site that will be the origin of chromosome copying (replication). In yeast, the pre-RC is established after the cell goes through anaphase, and at no other time. Once chromosome copying begins (is initiated), the pre-RC is probably destroyed. ORC can be thought of as a platform for the assembly of a pre-RC that can be formed only at certain stages of the cell cycle. Understanding how the cell cycle controls initiation of DNA copying involves identifying the proteins that form the pre-RC and understanding how the pre-RC is activated by cell cycle regulators.
The results of the most recent research indicates that, during the G1 stage, chromosomes require a pre-RC which can only be constructed during a vary narrow "window" of opportunity. This pre-RC probably contains ORC and other proteins involved in initiation. In yeast, one of these other initiating proteins is Cdc6p. Cdc6p is an important part of this pre-RC but only has a life span of about five minutes. Therefore, if initiation is to occur, it has to occur during that brief period of time when Cdc6p is functional. Cyclin-dependent kinases play a role in this process by activating the initiation process and preventing formation of another pre-RC after initiation occurs.
Checkpoints
As the cell cycle moves through S phase into M phase, there are checkpoint controls that ensure proper timing and sequence of cell cycle transitions and high fidelity (accuracy) of DNA copying. These checkpoints also respond to damage by stopping the cell cycle to provide time for repair and induce transcription of genes that help repairs. Without them there is genomic instability. The genes for these controls are highly conserved 8through the phyla which is evidence for their fundamental role in the regulation of life chemistry.
Checkpoints are not actually places in the cell or specific phases of the cell cycle. The term refers to biochemical pathways that ensure dependency. For example, the DNA damage checkpoint is the mechanism that detects damaged DNA and generates a signal that arrests cells in the G1 phase, slows down S phase , arrests cells in the G2 phase and induces transcription of repair genes.
Most checkpoint pathways have been identified through analysis of cell cycle mutants in yeast. There are checkpoints that sense mating partners, coordinate cell size and cell cycle progression, inhibit mitosis while in G1, make nuclear division dependent upon budding (in yeast), restrict DNA replication to once per cell cycle, and make DNA synthesis dependent upon G1 cycles.
Mammals have the same cell cycle responses to DNA damage as yeast. Mammalian cells may also activate a death pathway (apoptosis). Cell elimination is a legitimate strategy for higher animals since the goal is not the survival of the damaged cell, but of the total organism. Three mammal genes control the DNA damage checkpoint. The p53 gene is the most widely studied and has been referred to as an tumor (cancer) suppresser gene. A cell containing a mutated p53 gene and damaged DNA is unable to arrest the cell cycle at the G1 phase. Cancer may be the result as damaged DNA is permitted to undergo replication. The p53 gene has been implicated in most human cancers. As the Human Genome Project continues, additional human checkpoint genes will be identified.
The final checkpoint that will be discussed in this article is the spindle assembly checkpoint. Remember that in mitosis, a bi-polar (having two distinct ends) spindle must form; chromosomes must attach to the spindles (they attach through the kinetochore, a protein that forms on the centromere of chromosomes); sister chromosomes bind to spindle fibers attached to opposite poles and are then pulled (or travel) to the metaphase plate. The spindle assembly checkpoint prevents anaphase (the actual separation of the chromosomes) from beginning too soon. A number of genes have been identified that produce proteins that seem to coordinate this process. The various stages in spindle formation and chromosome separation are chemically sensed by checkpoint genes. The "signals" to which these genes react are protein kinases. Too high a concentration of a certain kinase and the cell cycle will stop, too little and it accelerates. The overall checkpoint mechanism is extremely complex and involves the interaction of many proteins, not all of which are identified or understood at this point.
In summary, the development of an organism is regulated by complex interactions between proteins that function as cell cycle controllers, inhibitors and accelerators of cell cycle processes, and by checkpoints that act as watchdogs or crossing guards. Genes regulate the activity of other genes in a symphony of biochemical signals, very much like a form of communication. 1 Equivalent to the number 10,000,000,000,000,000. 2 For example, as part of the process where an human infant brain matures, many millions of brain cells must die off. This is perfectly normal and is essential for normal development. Apoptosis is also triggered in cells that get too "old" and have begun to collect mutations or defective DNA. It is one of the body's mechanisms to prevent cancer. 3 "M" stands for mitosis, "D" stands for division of the cell. 4 Large, complex cells containing a membrane-bound nucleus. 5 The process where the genetic code contained within DNA molecules gets translated (or written) onto molecules of messenger RNA (mRNA) as a step in manufacturing proteins. 6 The word "developed" is used in an evolutionary sense, referring to a biological mechanism that has evolved along with other inherited traits characteristic of a grouping or organisms. 7 A group of genes that control for the most fundamental structural information, e.g. front or back, top or bottom/head or tail, orientation. 8 This refers to the preserving of certain genes (and their related functions) that control processes considered fundamental to all forms of life ranging, e.g. from primitive invertebrate worms to humans.