The details of the cell cycle may vary among different types of cells, however, certain requirements are universal. First, to produce a pair of genetically identical daughter cells, the DNA must be faithfully replicated, and the replicated chromosomes must be segregated into two separate cells (Fig 17-1). Most cells also double their mass and duplicate all their cytoplasmic organelles in each cycle. Thus, a complex set of nuclear and cytoplasmic events must be coordinated. In this lecture we'll study how this coordination comes about.

1. The General Strategy of the Cell Cycle.

• For a typical mammalian cell, the cell cycle lasts about 24 hrs. The cycle is divided into two major periods: mitosis, the process of nuclear division, and interphase, which comprises the time between successive mitosis. In early mitosis (i.e. prophase and prometaphase) the nuclear envelope breaks down, the contents of the nucleus condense into visible chromosomes, and the cell's microtubules reorganize to form the mitotic spindle. Then, the cell seems to pause at metaphase, in which the duplicated chromosomes are aligned on the mitotic spindle, poised for segregation. At this point, the cell "can decide" whether to stop cell division or not, however, past metaphase, there is no return and the process is taken till two daughter cells form. Anaphase marks the beginning of chromosome segregation, which will be followed by telophase and eventually, by cytokinesis, the separation of the two cells by division of the plasma membrane. This marks the end of the mitotic period, also known as the M phase. The M phase may last only for ~ 1hr, the other ~23 hr the cell spends in interphase. Towards late interphase is when the DNA is replicated. (Fig 17-2).
• The portion of interphase in which DNA replication occurs is known as the S phase (S=synthesis). Cells in S phase can be recognized by supplying them with 3H-thymidine, which only gets incorporated into DNA, or with bromo-deoxyuridine (BrdU), a T analog that can be recognized with a specific antibody (Fig 17-5). The interval between the completion of mitosis and the start of S phase is called G₁ phase (G=gap). During G₁ the cell monitors its environment and its own size, then, decides at the appropriate time to enter S phase. G₁ is by far the most variable cycle period timewise among different types of cells. Cells in G1, if they have not committed themselves to DNA replication, can enter G₀ (G zero), in which the cycle stalls with no S phase. G₀ can last from days to years and is typical of fully differentiated cells. After the S phase and before mitosis, cells are in G₂ phase. G1, S, G2 and M are the traditional subdivisions of the standard cell cycle (Fig 17-3).
• (Fig 17-9) There is an independent cell cycle control system made up of proteins that are different from the effector proteins that directly perform mitosis, G₁, DNA replication, or G₂. Brakes that can stop the cycle at specific checkpoints (a.k.a restriction points) regulate the control system. At checkpoints, feedback signals conveying information about the effector processes, or extracellular signals, can delay progress of the control system itself, so as to prevent it from triggering the next effector process before the previous one is finished. The two major checkpoints occur at G₁, just before entry into S phase, and at G₂ shortly before mitosis. There is an additional checkpoint before the exit from mitosis that corresponds to the point of no return at metaphase, of which we talked above. In yeast this checkpoint is call Start. This is also the point where cells enter G₀ if the conditions are appropriate.

• The G₂ checkpoint senses unreplicated DNA, which generates a signal that leads to cell cycle arrest, unless DNA replication is complete. Progression through the cycle is also stopped at the G₂ checkpoint in response to DNA damage, such as that resulting from irradiation. This allows time for DNA repair before the M phase. The molecular mechanisms by which unreplicated or damaged DNA signals arrest at this checkpoint are still unknown.

• DNA damage arrests the cycle at G₁ too, which allows time for repair before going into the S phase. At G₁, damaged DNA induces the rapid synthesis of the p53 protein, which then signals cell cycle arrest. Mutations in the p53 gene are the most common genetic alterations in human cancers, illustrating the critical importance of cell cycle regulation in the life of multicellular organisms.

• The checkpoint at metaphase monitors the alignment of chromosomes on the mitotic spindle, thus ensuring that a complete set of chromosomes is distributed accurately to the daughter cells.
• It is equally important that the genome is replicated once per cycle. Thus, once DNA replication is ended, control mechanisms must exist to prevent a new S phase prior to mitosis. Experiments in which cells in different phases of the cycle were isolated and then fused to each other provided initial insight about the coupling of S phase to M phase. Whereas nucleus from G₁ cells progressed normally to S phase when fused to a S phase cytoplasm, G₂ nucleus could not start DNA synthesis even in the presence of a S phase cytoplasm (Fig 17-21). One model to account for this replication block is that a licensing factor required for DNA replication binds to chromosomes during mitosis. The factor is inactivated during the S phase and is unable to enter the nucleus again until the nuclear envelope breaks down in mitosis, thereby preventing replication until after G₁ in the next cycle. The nature of this factor is unknown.
• The cell-cycle control system is based on two families of proteins: the cyclin-dependent protein kinases (Cdk) and the cyclins. Cyclins bind and activate Cdk's, which phosphorylate selected proteins on Ser/Thr residues thereby inducing downstream effector cell cycle processes (Fig 17-10). Cyclins owe their name to the fact that they undergo a cycle of synthesis and degradation with each division cycle. There are mitotic cyclins, which bind Cdk molecules during G₂ and are required for entry during mitosis, and G1 cyclins, which binds to other Cdk molecules during G₁ and are required for entry into S phase. In yeast the same Cdk is involved in the entry to both M and S phase. The cyclic assembly, activation, and disassembly of cyclin-Cdk complexes are the pivotal events that drive the cell cycle (Fig 17-11).

2. Regulators of Cell Cycle Progression.

• MPF: a Dimer of Cdc2 and Cyclin. Experiments in three different model organisms, frogs, yeast, and sea urchin, contributed to the identification of the first cell cycle regulator, M phase-promoting factor (MPF). Frog oocytes are arrested in G₂ until hormone stimulation triggers their entry in the M phase of meiosis. In 1971, investigators found that such arrested oocytes could be induced to enter M phase by microinjection of cytoplasm from hormone-stimulated-oocytes. Thus a cytoplasmic factor in hormone-treated oocytes (MPF) was sufficient to relieve the block. Further studies showed that MPF could also promote the G₂-to-M phase transition in mitotic cells, which pointed to its activity as general regulator of this transition. We'll here more about these experiments in one of the student presentations.

• Complexes of cyclins and Cdks. Both Cdc2 and cyclin B are members of large families of related proteins, with different members of these families controlling progression through distinct phases of the cell cycle. In yeast Cdc2 controls passage through Start in association with G₁ cyclins (Cln1, Cln2 and Cln3). Complexes of Cdc2 with B type cyclins (Clb's) regulate progression through S phase and entry in mitosis. In animal cells, complexes of Cdk2, Cdk4 and Cdk6 with D type cyclins regulate progression through the G₁ checkpoint. Cdk2/cyclin E complexes function later in G₁ and are required for the G₁ to S transition. Cdk2/cyclin complexes are then required for progression to S phase, and Cdc2/cyclin B complexes drive the G2 to M transition.
• Mechanisms of Cdk regulation. In addition to the regulation of Cdk's activity by association to its partner cyclin, at least three other mechanisms control Cdk's activity. (1) The activating phosphorylation of Cdks around position 160 is catalyzed by an enzyme called CAK (Cdk-activating kinase). CAK is itself composed of a Cdk (Cdk7) complexed with cyclin H. (2) Members of the Cdc25 family of protein phosphatases mediate the inhibitory dephosphorylation of T residues near the Cdk amino terminus. (3) A family of Cdk inhibitors (CKIs) binds to Cdk/cyclin complexes thereby inhibiting their activity. An example is the CKI called p16, which specifically arrests cells in G1 by binding to complexes of Cdk 4 and Cdk6 to cyclin D. The combined effects of all these Cdk control mechanisms account for checkpoint regulation and for the influence of extracellular signals on cell proliferation.
• Growth factors and D-type cyclins. As long as growth factors (e.g. EGF, PDGF, etc) are present through G₁, complexes of Cdk/cyclin D drive cells through the G₁ checkpoint. If growth factors are removed before this key regulatory point, the levels of cyclin D rapidly fall due to degradation and cells are unable to progress through G₁ to S, instead becoming quiescent and entering G₀. A key downstream substrate protein of the Cdk4/cyclin D complex is Rb, the product of a gene whose mutation was first described for retinoblastoma cancer patients, but that has been found associated with many other forms of cancer. Rb, like p53 (above), is a tumor suppressor gene, whose function is opposite to Cdk/cyclin complexes in that it acts as a break that slows down cell cycle progression. Rb couples the cell cycle regulation machinery with the DNA synthesis machinery. In its dephosphorylated state (during early G₁, and G₀), RB binds to members of the E2F family which in turn repress the transcription of selected genes included genes required to make dNTP's. Phosphorylation of Rb by Cdk4/cyclin D results in Rb dissociation from E2F protein. This form of E2F acts now acts as an activator of gene transcription.
• Inhibitors of cell cycle progression. A variety of factors (e.g. DNA damage, peptide factors) that inhibit cell proliferation act by inducing CKIs, in effect, the converse of the action of growth factors in inducing synthesis of the D-type cyclins. For example, DNA damage results in the elevation of p53, which activates transcription of the CKI p21. In addition to blocking cell cycle progression by binding to Cdk/cyclin complexes, p21 binds directly to a subunit of DNA polymerase d (a.k.a. PCNA, for proliferating cell nuclear antigen) thereby inhibiting DNA replication in S phase. TGF-b, is the best-characterized inhibitor of animal cell proliferation, and in epithelial cells acts by arresting the cell cycle at G₁. This action is mediated by induction of CKIs p15 and p27, which bind to Cdk4/cyclinD complexes inhibiting their activity. In the absence of active Cdk4, Rb phosphorylation is blocked and the cell cycle stops at G₁.

3. Development, Differentiation, and Programmed Cell Death (Apoptosis).

• Most cells in adult animals are arrested in the G₀ state. A few types of differentiated cells are no longer capable of proliferation (e.g. some CNS neurons), but most are able to proliferate as required to replace cells that have been lost because of injury or cell death. Some differentiated cells (e.g. white and red blood cells) have short life spans and are continually replaced via the proliferation of stem cells.
• Unappreciated until recently, programmed cell death (PCD) is a normal physiological form of cell death (as opposed to accidental death) that plays a key role both in the maintenance of adult tissues and in embryonic development. For example, ~ 5 x 10¹¹ blood cells are eliminate daily by PCD daily in humans, balancing their continual production from stem cells in the bone marrow. Virus-infected cells are eliminated by PCD, thereby preventing the production of new virus and the spread of virus infection in the host organism. Moreover, cells with damaged DNA are eliminated by PCD, which reduce the changes of them giving rise to cancers.

• PCD is an active process. It is characterized by a sequence of physiological events that together are called apoptosis. During apoptosis, chromosomal DNA is fragmented by cleavage between nucleosomes. The chromatin condenses and the nucleus breaks up into small pieces. Finally, the cell itself shrinks and breaks up into membrane-enclose vesicles called apoptotic bodies. These are ready recognized, phagocytosed and thus, removed from tissues by macrophages and neighboring cells.
• Regulators and effectors of apoptosis. Many cell death signals induce apoptosis through a common conserved (from worms to mammals) pathway. Apoptosis is negatively regulated (i.e. avoided) by members of the Bcl-2 family of proteins (Ced9 in worms), which act upstream of members of the ICE family of cysteine proteases (Ced3 in worms), which cleave one or more critical target proteins.