THE CELL CYCLE STAGES OF THE CELL CYCLE Animal development from a single-cell zygote to fertile adult requires many rounds of cell division. Cell cycle is a complement of genetic, biochemical and morphological events from cell birth till its division, differentiation or death. The cell cycle is an ordered set of periods that includes interphase and the division, culminating in cell growth and separation into two daughter cells. The interphase stages are G1-S-G2-M, where the G1 stage stands for "GAP 1"; the S stage stands for "Synthesis" (when DNA replication occurs), the G2 stage stands for "GAP 2". The M stage stands for "mitosis", and is the period when nuclear division (karyokinesis) and cytoplasmic division (cytokinesis) occur. Mitosis is further divided into the following phases: prophase, prometaphase, metaphase, anaphase and telophase. INTERPHASE Interphase generally lasts at least 12 to 24 hours in mammalian tissue. During this period, the cell is constantly synthesizing RNA, producing protein and growing in size. There are a lot of activities during this period: the cell obtains nutrients, makes ATP, grows, reads its DNA, and conducts other "normal" cell functions including preparation for cell division. G1 (presynthetic or postmitotic) stage of interphase is a period of cell growth in which proteins, carbohydrates, and lipids typical for a given cell type are synthesized, but DNA is not replicated (the "G" refers to the "gap" or break in DNA synthesis during this stage). So, chromosomes are single-chromatid (in a diploid cell the number of chromosomes (n) is equal to the number of chromatids (c): 2n=2c), decondensed, with transcriptionally active parts (euchromatin) and inactive parts (heterochromatin). At some point, if the cell is going to divide, DNA replication begins. The initiation of DNA replication ends G1 and begins the S period of interphase (from S=DNA synthesis). During S phase, the entire nuclear complement of DNA is duplicated. The chromosomes thus become bichromatid (each chromosome consists of 2 identical DNA that make 2 sister chromatids, united at centromere (2n=4c). Histone and nonhistone chromosomal proteins associated with DNA, as well as proteins required for a successful replication and repair are synthesized. This period is also marked by duplication of centrosomes. Doubling of a centrosome is similar to DNA replication in two respects: the semiconservative nature of the process and the action of cdk2 (cyclin-dependent kinase) as a regulator of the process. But the processes are essentially different in that centrosome doubling does not occur by template reading and assembly. The mother centriole just aids in the accumulation of materials required for the assembly of the daughter centriole. Aberrant numbers of centrosomes in a cell have been associated with cancer. As replication is completed, S ends and the cell enters the final stage of interphase, G2. G2 (postsynthetic or premitotic) is characterized by control of replication quality, DNA repair, 1

synthesis and accumulation of a group of proteins necessary for progress through mitosis (tubulin, factors for cell division). Most cell types remain in G2 only briefly; and at the end of G2, which marks the end of interphase, mitosis begins. After mitotic division is complete, the two cell products each enter G1 of the next cell cycle. G0 is a state of cell cycle arrest. This is the time when a cell quits dividing. It is a period of proliferative rest. Some cells may stay in G0 permanently. This is the case of highly differentiated cells, such as cells of nervous system, muscles, crystalline, which have lost their proliferation properties. Other cells may be temporally resting in G0: liver cells, lymphocytes – differentiated cells with low proliferation activity. The main genetic events performed during interphase and absent during division are: gene expression (transcription, translation), replication and repair. MITOSIS Mitosis is the type of division of somatic cells, which assures the growth of the organism and tissue regeneration. Cell growth and protein production stop at this stage in the cell cycle. All of the cell's energy is focused on the complex and orderly division into two similar daughter cells. Mitosis is much shorter than interphase, lasting perhaps only one to two hours. Prophase Chromatin in the nucleus begins to condense and becomes visible in the light microscope. Chromatin condensation is mediated by the condensin complex, which reorganizes chromosomes into their highly compact mitotic structure. The nucleolus disappears. Centrioles begin moving to opposite ends of the cell. The mitotic spindle is formed. Since the genetic material has been duplicated in an earlier phase of the cell cycle, the chromosome consists of 2 identical copies of DNA, called sister chromatids, which are attached to each other at the centromere. The replicated sister chromatids are “glued” together by a specific proteic complex called cohesin, which maintain chromatids together till they split at anaphase. Prophase accounts for approximately 3% of the cell cycle's duration. An important organelle in mitosis is the centrosome, the microtubule organizing center (MTOC) in animal cells. The centrosome is copied only once per cell cycle so that each daughter cell inherits one centrosome, containing two centrioles. The centrosome replicates during the S phase. During prophase, the two centrosomes have their microtubule-activity increased due to the recruitment of γ-tubulin. The centrosomes are pushed apart to opposite ends of the cell nucleus by the action of molecular motors (motor proteins: dyneins and kinesins that use microtubules and hydrolysis of ATP for movement). The nuclear envelope breaks down to allow the microtubules to reach the kinetochores (a DNA–protein complex required for attachment of mitotic 2

spindle at centromere) on the chromosomes, marking the end of prophase. During prometaphase the chromosomes are captured by the microtubules. Prometaphase The nuclear membrane dissolves completely, marking the beginning of prometaphase. Proteins attach to the centromeres creating the kinetochores. Microtubules attach at the kinetochores and the chromosomes begin moving to the cell equator. Metaphase During this the stage of mitosis in which condensed and highly coiled chromosomes align in the middle of the cell before being separated into each of the two daughter cells. Metaphase accounts for approximately 4% of the cell cycle's duration. The centromeres of the chromosomes arrange themselves on the metaphase plate (or equatorial plate), an imaginary line that is equidistant from the two centrosome poles. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Anaphase During anaphase the longitudinal cleavage of centromeres occurs. As a result of chromatids disjunction the single-chromatid chromosomes separate. Each chromatid moves to opposite poles of the cell, the opposite ends of the mitotic spindle, near the microtubule organizing centers. During this stage, errors (such as chromatids nondisjunction, transversal cleavage or anaphase lag) could happen, resulting in abnormal number or structure of chromosomes in daughter cells. Anaphase begins abruptly and accounts for approximately 1% of the cell cycle's duration. At this point, a protease known as separase cleaves cohesin, a protein responsible for holding sister chromatids together. The movement of chromatids is achieved by the shortening of spindle microtubules from kinetochores sites by depolymerization at their plus ends. No motor protein is involved in chromatid movement. Molecular motors are however required for microtubule reorganization during late anaphase. Telophase During telophase, the effects of prophase and prometaphase events are reversed. Two daughter nuclei form in the cell. The nuclear envelopes of the daughter cells are formed from the fragments of the nuclear envelope of the parent cell. As the nuclear envelope forms around the chromosomes, the nucleoli reappear. The DNA decondensation processes begin. Telophase accounts for approximately 2% of the cell cycle's duration. Cytokinesis (distribution of the cytoplasm) usually occurs at the same time that the nuclear envelope is reforming, yet they are distinct processes. In animal cells, a contractile ring, made of non-muscle myosin II and microfilaments, assembles equatorially. Myosin II uses the ATP energy to move along the actin filaments, constricting the cell membrane to form a cleavage furrow. The cleavage furrow thus develops where the metaphase plate used to be, pinching off the separated nuclei. Each daughter cell has a complete copy of the genome of its parent cell (2n=2c), and mitosis is complete. REGULATION OF THE CELL CYCLE Cell external signals and cell intrinsic (internal) information together determine whether cells enter a division cycle. In general, external signals affect this decision only until cells commit to go through the entire cycle, at a time in G1 known as "Restriction point" in mammals. From there on, progression through the cell cycle is controlled intrinsically by the cell-cycle machinery. 3

The collective results from studies in various eukaryotes have demonstrated that progression through the cell-division cycle is driven by activation and inactivation of cyclin-dependent kinases (cdk), which trigger the transition to subsequent phases of the cycle. CDKs are small protein kinases that require association with a cyclin for their activation. Members of the cyclin family of proteins are thus key regulators of the cell cycle. Cyclins are grouped into several classes: Cyclin D family members are G1 phase cyclins that regulate the entry of cells into G1 from Go. Cyclin D is upregulated by growth factor and external signals. Cyclin D couples with Cdk4 and Cdk6. Cyclin D-Cdk4 facilitates the expression of cyclin E. Cyclin E and Cyclin A are able to bind Cdk2 and promote the cell cycle progression through G1/S transition. Cyclin E stimulates replication complex assembly. Cyclin A activates DNA synthesis. Cyclins B1 and B2 are M-phase cyclins. Cyclin B1 and cyclin B2 and their catalytic partner, Cdk1 are components of the MPF (M phase/maturation promoting) factor that regulates processes that lead to assembly of the mitotic spindle and sister-chromatid pair alignment on the spindle. Period G1 S-phase Mitosis

Cyclins D cyclins cyclins E and A mitotic cyclins (B cyclins)

Cyclin-dependent kinases (Cdks) Cdk4, Cdk6 Cdk2 Cdk1

These are short-life proteins. Their levels Their levels in the cell remain fairly stable, but in the cell rise and fall each must bind the appropriate cyclin (whose with the stages of the cell cycle. levels fluctuate) in order to be activated.

Cell Cycle Checkpoints The cell has several systems for interrupting the cell cycle if something goes wrong. Since DNA is the main cell component that assures the molecular processes and cell life, DNA integrity is controlled at specific DNA damage checkpoints, which arrest the cell cycle till the DNA is repaired. DNA damage checkpoints sense DNA damage both before the cell enters S phase (a G1 checkpoint, assuring that everything is ready for DNA synthesis: DNA is not damaged, the internal and external cell environments are favorable, cell grows normally), as well as after S phase (a G2 checkpoint, to determine if the DNA is properly replicated, cell is grown enough and can now proceed to enter M mitosis). If the damage is so severe that it cannot be repaired, the cell selfdestructs by apoptosis. One of the cell cycle checkpoints occurs during prometaphase and metaphase – spindle checkpoint. Only after all chromosomes have become aligned at the metaphase plate, when every kinetochore is properly attached to a bundle of microtubules, does the cell enter anaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase, even if most of the kinetochores have been attached and most of the chromosomes have been aligned. DNA replication and chromosome distribution are indispensable events in the cell cycle control. Cells must accurately copy their chromosomes, and through the process of mitosis, segregate them to daughter cells. The checkpoints are surveillance mechanism and quality control of the genome to maintain genomic integrity. Checkpoint failure often causes mutations and genomic arrangements resulting in genetic instability. Genetic instability is a major factor of birth defects and in the development of many diseases, most notably cancer. All the checkpoints examined require the services of a complex of proteins. Mutations in the genes encoding some of these have been associated with cancer; that is, they are oncogenes. This should not be surprising since checkpoint failures allow the cell to continue dividing despite damage to its integrity. Examples of checkpoint proteins: The p53 protein senses DNA damage and can stop progression of the cell cycle in G1 (by blocking the activity of Cdk2). The p53 protein prevents a cell from completing the cell cycle if its DNA is damaged or the cell has suffered other types of damage. When the damage is minor, p53 4

arrests the cell cycle until the damage is repaired. If the damage is major and cannot be repaired, p53 triggers the cell to commit suicide by apoptosis. These functions make p53 a key player in protecting against cancer; that is, it is an important tumor suppressor gene. More than half of all human cancers do, in fact, harbor p53 mutations and have no functioning p53 protein. Cells with functional p53 arrest in G1 or G2 when exposed to γ-irradiation. The Rb (retinoblastoma) protein integrates the signals reaching the cell to determine whether it is safe for the cell to complete the passage from G1 of the cell cycle to mitosis. The Rb protein also plays a role in mitosis itself: it is needed for proper chromosome condensation starting in prophase, as well as their proper attachment to the spindle. Failure of Rb function during mitosis can lead to aneuploidy (abnormal number of chromosomes) and chromosome breakage. Cells that fail to replicate all their chromosomes do not enter mitosis (G2 checkpoint arrest). Operation of this checkpoint control involves the recognition of unreplicated DNA and inhibition of MPF activation. Although the ability of unreplicated DNA to inhibit entry into mitosis is well documented, little is yet known about the proteins that mediate this checkpoint control. Restriction Points The eukaryotic restriction point is a particular checkpoint where it is proposed that cells are arrested under growth limiting conditions and DNA damage. Progression through the cell cycle restriction point makes the cell independent of external stimuli. The 2 main restriction points are: R1 - G1/S (control of the readiness for DNA synthesis) and R2 - G2/M (control of replication quality and readiness for cell division). APOPTOSIS Apoptosis, programmed or physiological cell death, is a normal process of the development and health of multicellular organisms. Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion. This makes apoptosis distinct from another form of cell death called necrosis, in which uncontrolled cell death leads to lysis of cells, inflammatory responses and, potentially, to serious health problems. Apoptosis, by contrast, is a process in which cells play an active role in their own death (which is why apoptosis is often called as cell suicide). The following cells undergo apoptosis: mutant, transformed cells; old cells; cells with abnormal receptors, recognized as foreign; cells that have lost contacts with neighbor cells; excessive number of cells. Upon receiving specific signals instructing the cells to undergo apoptosis a number of distinctive changes occur in the cell. Proteins known as caspases are typically activated in the early stages of apoptosis. These proteins cleave key cellular components that are required for normal cellular function including structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes. The caspases also activate other degradative enzymes such as DNases, which begin to cleave the DNA in the nucleus, causing its fragmentation. The breakdown of chromatin in the nucleus often leads to nuclear condensation and in many cases the nuclei of apoptotic cells take on a "horse-shoe" like appearance. Further proteolytic processes lead to cell fragmentation and formation of apoptotic bodies (small vesicles), which undergo phagocytosis by neighbor cells. There are a number of mechanisms through which apoptosis can be induced in cells. In some cases the apoptotic stimuli comprise extrinsic signals such as the binding of death inducing ligands to cell surface receptors called death receptors. In other cases apoptosis can be initiated following intrinsic signals that are produced following cellular stress. Cellular stress may occur from exposure to radiation or chemicals or to viral infection. It might also be a consequence of growth factor deprivation or oxidative stress caused by free radicals. In general intrinsic signals initiate apoptosis via the involvement of the mitochondria.

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