The human body, composed by tens of trillions of cells, developments from one single cell, the zygote. New cells come from cells that already exist by cell division. The cells grow in number when cells that arise by cell division is more than cells that is lost through cell death or differentiation. Cell proliferation is the process that cells grow in number. Cell division supply cells for body development, renewal and wound repair. The process of cell division is known as the cell cycle, which is highly orderly and tightly regulated. During the cell cycle, a cell duplicates its contents including DNA and then divides into two. In this section, we will introduce mitotic cell cycle with animal cell as an example. If not specified, all the points are on animal cells.

Mitosis is the process of nuclear division featured by condensed chromosomes and spindle apparatus. The duplicated chromosomes are equally distributed to the two daughter cells. But the division could be either symmetric or asymmetric, i.e., the fate of the two daughter cells may be either the same or different. Mitosis is continuous but, for ease of description, artificially divided into five stages, prophase, prometaphase, metaphase, anaphase, and telophase.

Prophase is the first stage of mitosis and featured by several coordinated events. The nucleolus breaks down. The duplicated chromosomes begin to be disentangled and condensed into pairs of chromosomes, called sister chromatids, that remain linked, by intact cohesions, at the centromeric region, where kinetochores assemble. Kinetochores are protein complexes that serve as the sites for spindle microtubule attachment. Endocytosis and exocytosis stop at this stage. The microtubules become mitotic asters and move to opposite sides of the nucleus with duplicated centrosomes. The two pairs of centrosomes become the spindle poles.

Prometaphase is the second stage of mitosis and started with breakdown of nuclear envelope that is then retracted into the endoplasmic reticulum. Nuclear pores and lamina disassemble. Microtubules from the spindle poles search the chromosomes and some of the microtubules get associated with the kinetochores. The two kinetochores of a sister chromatid pair are linked with microtubules to a different spindle pole and then become aligned to the equatorial plate, i.e., the plane at the middle of the spindle.

When all chromosomes are aligned to the equatorial plate, the cell enter metaphase. At this stage, the cell will check whether all chromosomes are attached to the spindle correctly. The chromosomes are most condensed at this stage.

Anaphase is the fourth stage of mitosis and is induced by activation of APC/C (anaphase-promoting complex/cyclosome, an E3 ubiquitin ligase). Activation of APC/C will ultimately result in hydrolysis of cohesin by separin, and thus each pair of sister-chromatids get separated and could be pulled to respective poles. Meanwhile, the spindle poles also move farther apart.

Telophase is the last stage of the division of nuclear division, when the two sets of chromosomes arrive at the spindle poles. The chromosomes decondense, the nuclear envelopes are reconstructed around each chromosome set and the nucleoli reappear. The telophase, and also the mitosis, end at completing of new nuclei formation.

During late anaphase and telophase, the contractile ring, a thin band composed of actin filaments and myosin-II filaments, assembles underneath the cell membrane on the equatorial plate. The contractile ring is attached to the cell membrane and will eventually contract and divide the cell into two. Such a process that divide the cytoplasm is called cytokinesis.

Amitosis is cell division that does not happen in a mitotic way. Generally, cell division without formation of spindle or similar structure could be referred to as amitosis. Binary fission is more frequently used than amitosis in the case of referring prokaryotic cell division. In higher eukaryotic organism, amitosis is very rare and may only occur under some special conditions and in cells undergoing ploidy reduction. Amitosis may results in a random distribution of chromosomes in the daughter cells.

Most eukaryotes can reproduce sexually with two gametes, specialized reproductive cells, egg and sperm in most species including animal, from two parents respectively. A gamete carries half of the sets of chromosomes compared to somatic cells. A typical organism conducting sexually reproduction are diploid, i.e., with pairs of highly similar but nonidentical chromosomes, homologous chromosomes. A gamete has only one copy of each pair of chromosomes and is haploid. The process producing gametes is called meiosis, which conducts one round of chromosome replication followed by two rounds of successive cell division, namely, meiosis I and meiosis II. Both meiosis I and meiosis II form meiotic spindles and can be divided into sequential stages, prophase, prometaphase, metaphase, anaphase, and telophase. There is an interphase, without chromosome replication, between Meiosis I and Meiosis II.

Prophase I, the prophase of Meiosis I, is a complex and lengthy stage that may last hours, days, months, or even years in different species. In human female, oocytes pause at prophase I prior to birth and resume meiosis just prior to ovulation. During prophase I, homologous chromosomes interact side by side with each other by a process called pairing and get their DNA exchanged by events called crossovers. Prophase I is divided into five sequential stages according to the morphological changes:

Leptotene. Chromosomes condense and homologs pair and genetic recombination (DNA exchange) begins. The chromatids could be distinguished under electron microscope but not under light microscope at this stage and thus the chromosomes look like thin threads.

Zygotene. This stage is featured by association of homologous chromosomes, i.e., the chromosome pairing process called synapsis. At this stage, synaptonemal complex, a protein structure forming between homologous chromosomes, gets assembled and genetic recombination events are occurring. Under light microscope, the chromosomes look like paired threads.

Pachytene. The homologs get the paring finished and are synapsed along the entire lengths. This stage can last for days or longer.

Diplotene. The chromosomes undergo desynapsis, condensation and shortening while the synaptonemal complexes disassemble. After the synaptonemal complexes dissembled, the sister chromatids remain tightly connected and homologous chromosomes are hold together by inter-homolog connections, called chiasmata (chiasma for singular), located at sites which crossing-over once occurred. The pairs of homologous chromosomes are called bivalents or tetrads. At this stage, oocytes undergo active transcription to store up materials for early embryonic development.

Diakinesis. The meiotic spindle is assembled, nucleolus disappear, nuclear envelope breaks down and the chromosomes get arranged on the metaphase plate. In most eukaryotes, including human being, chiasmata can still be seen in this stage as they are necessary for holding the homologous chromosomes together. This stage may be also referred as prometaphase I.

At metaphase I, all the bivalents are arranged on the plate and two homologs of each bivalent are randomly connected to opposite spindle poles as the kinetochores of sister chromatids are arranged side-by-side. The chiasmata disappear while meiosis goes into anaphase I, when the homologous chromosomes get separated and transported to opposite spindle poles. The cohesion at the centromeres is protected by a protein called shugoshin and thus the chromatids are still attached to each other. During telophase I, the nuclear envelope may reform at this stage. The number of chromosomes of the daughter cells from meiosis I is reduces by half. After a brief interkinesis, also called interphase II, the cells undergo meiosis II, which is similar to mitosis.

The cell cycle, mitotic cell cycle if not specified hereafter, is divided into interphase and M phase. M phase (M for Mitosis), the landmark of cell cycle, consists of mitosis and cytokinesis. The interphase, the rest of the cell cycle between M phases, is divided into three sequential subphases, the first gap (G1) phase, the synthetic (S) phase, and the second gap (G2) phase. Some of the cells may exit the cell cycle and, and most of them, do not divide any longer. Such cells are called to be in G0 phase. Some cells in G0 phase may, in some cases, reentry cell cycle. The time length of the cell cycle and the time fraction of each phase varies in different cell types and conditions. Rapidly duplicating human cells take about 24 hours to get through the whole cell cycle, 9 hours for G1, 10 hours for S, 4.5 hours for G2, and 30 minutes for M phase.

G1 phase is typically the longest and most length-variable cell cycle phases, during which the newborn cell grows toward an optimal size and many cell cycle activities are repressed. Such a repressive control system is known as the restriction point. If the nutrients are not enough or the cells are suppressed by antiproliferation signals, the cells may delay the cell cycle in G1 or enter G0. RNAs and proteins required for DNA synthesis are made during this stage. G1 phase may last for minutes, hours, days, months and even longer.

Once a cell passes the restriction point, it enter the S phase, when the cell duplicates its chromosomes exactly once. The replication of DNA is initiated at hundreds to thousands of different sites, more than 100,000 in human. Each chromosome region replicated from a single origin is known as a replicon. The centrosome get duplicated and histone at S phase. The S phase end at the completion of DNA replication.

G2 phase is relatively brief in most cells. During this phase, a cell checks against DNA damage. As the sister chromatids are still held together, if one get damaged, the other cold serve as a template for the repair. DNA damage may cause a delay, G2 delay, of the transition into M phase.

Cell cycle is tightly controlled and timed by cell-cycle control system, whose key components are cyclins and cyclin-dependent kinases (CDKs). Cell-cycle control system interact with other biological processes such as cellular metabolism and circadian clock to get coordinated with other biological processes.

The protein concentration of some cyclins oscillates with the cell cycle and thus cyclins are so named. All cyclins contain at least one 100-amino-acid domain named cyclin box that could bind to CDKs. Cyclins are present or activated only during the stage at which they activate specific CDKs and function and therefore categorized to four classes, G1/S-cyclins that trigger progression through restriction point in late G1 phase, S-cyclins that help stimulate chromosome duplication during the whole S phase, M-cyclins that stimulate entry into mitosis at the end of G2 phase, and G1-cyclins that play a role in regulating G1/S-cyclins.

Cyclin levels is regulated by multiple mechanisms including transcription and proteolysis. Transcription of cyclins could be either stimulated by proliferation signals or promoted by preceding cyclins or themselves. After finishing their function, cyclins are poly-ubiquitinated by E3 ubiquitin ligase and then degraded by proteasome, which is irreversible and can cause rapid decrease of cyclins.

In human, there is 30 genes of cyclin family that is divided to 16 types, of which cyclin A, cyclin B, cyclin D and cyclin E are well known to play roles in cell cycle control. Cyclins have other non-canonical functions such as transcriptional regulation and DNA repair. These functions may be independent of CDKs. Most of the cyclins do not oscillate with the cell cycle.

Cyclin-dependent kinases (CDKs) are serine/threonine kinases characterized by needing to bind to a cyclin to activate their kinase activity. A cyclin-CDK complex can phosphorylate multiple substrates that play roles in cell cycle and/or other biological processes including transcription modulation. CDKs are much more conserved than cyclins. All CDKs have an ATP-binding pocket, a cyclin-binding domain, and a T-loop motif that blocks the active site without cyclins.

In human, there is 21 genes encoding CDKs, named CDK1-20, of which four, CDK1, CDK2, CDK4, and CDK6, are well known to play roles in cell cycle regulation. CDKs is regulated by multiple mechanisms besides cyclin binding. The catalytic activities of most CDKs are also modulated by interactions with CDK inhibitors (CKIs) and phosphorylation of inhibitory and activating phosphorylation sites. CKIs inhibit activity of cyclin-CDK complexes. There are two families of CKIs, the INK4 family members including p16, p15, p18 and p19 that primarily inhibit CDK4 and CDK6, and the CIP/KIP family members including p21, p27 and p57 that broadly interfere with cyclin A-, B-, D-, E-CDK complexes. Phosphorylation by inhibitory kinases including WEE1 interferes with ATP-binding and inhibits the cyclin-CDKs activity, while phosphorylation by CDK-activating kinases (CAKs) stabilizes the complex, enhances substrate binding and optimizes the kinase activity.

Some CDKs play non-canonical roles such as kinase-dependent and -independent transcription regulation, DNA-damage repair, and regulating metabolism. Some of these processes are related to cell cycle control while some of the processes is not. For example, G1-cyclin-CDKs activate the neurons to enter G1 for DNA repair without moving further to S phase.

Cyclin-CDK complexes employs global regulatory strategies to regulate specific cell-cycle events. They may have hundreds of targets including regulatory proteins. The cyclins and CDKs well known in cell cycle control system are listed in table 7.1. Table 7.1 Well Known Cyclins and CDKs in Cell Cycle Control System of Mammals Cyclin-CDK Complex Cyclin Type CDK partner Function G0-CDK Cyclin C CDK3 G0/G1 transition G1-CDK Cyclin D CDK4, CDK6 Regulating G1/S-Cyclin G1/S-CDK Cyclin E CDK2 G1/S transition S-CDK Cyclin A CDK2, CDK1 Controling DNA synthesis M-CDK Cyclin B CDK1 Entry into M phase

In actively proliferating cells, levels of Cyclin Ds, D1, D2, and D3, do not unusually oscillate as other cyclins. In early G1 phase, the G1-CDKs phosphorylate and inactivate Rb (retinoblastoma protein) that transform E2Fs (E2F transcription factors) to repressive state, which activates E2Fs. The activated E2Fs activate the transcription of many genes including G1/S-cyclins, S-cyclins and proteins taking parts in chromosome duplication. Myc can promote cell proliferation by several mechanisms including increasing the transcription of cyclin Ds. Cyclin D also binds to PCNA and inhibits DNA synthesis.

Active G1/S-CDK phosphorylates p21 and p27 and initiates their degradation by SCF (Skp, Cullin, F-box containing complex), which permits rapid accumulation of CDK activities, which further phosphorylates Rb and fully activates E2Fs. The phosphorylation stat of Rb then are maintained by S-CDKs and M-CDK. E2Fs also promoter their own transcription, so once a cell enter S phase, it cannot go back.

S-CDK activates DNA helicases and promotes the assembly of the replication fork, which initiates the DNA replication. S-CDKs also help prevent re-replication. At the beginning of S phase, Cyclin D is phosphorylated by GSK3β (Glycogen synthase kinase 3β) and exported to the cytoplasm.

During G2 phase, the transcription of cyclin B increases and M-CDK gets accumulated, however, M-CDK is phosphorylated by WEE1 and inactivated. During G2/M transition, CDK1 is dephosphorylated by CDC25 and activated. M-CDK can activate CDC25 and inhibit WEE1, which promotes abrupt and complete transition to M phase. The M-CDK is first discovered in frog eggs that it stimulates oocyte maturation, so it also called maturation-promoting factor (MPF). After the attachment of chromosome to the spindle gets completed, cyclin B is ubiquitinated by APC/CCDC20 and destroyed by proteasome. After cyclin B is degraded, CDH1 is no longer phosphorylated by M-CDK and APC/CCDH1 gets activated. APC/CCDH1 activity is required for M-G1 transition and DNA replication fork initiation.

Check point pathways are surveillance mechanisms to avoid unnecessary and unprepared division and ensure faithful chromosome duplication and transmission to daughter cells.

In late G1 phase, restriction point (also known as R point) blocks a cell from entering S phase, the key decision to division. Mitogens activate signaling pathways leading to activated gene expression of transcription factors including c-Myc, which activate expression of cyclin D. Mitogens also inactivate GSK. When cells cultured in a dish cover the surface completely, the signaling initiated by cadherin results in expression of p27 and arrest of their cell cycle, which is called contact inhibition.

There are several DNA-damage checkpoints for DNA integrity. Many types of DNA damage can activate the serine/threonine kinases ATM (for Ataxia Telangiectasia Mutated, only responsible for double-strand breaks) and/or ATR (for Ataxia Telangiectasia and RAD3-related protein), which then activate pathways leading to DNA repair, cell cycle arrest until the damages get repaired, or cell death in the face of extensive DNA damage. At G1 phase, DNA damage activates p53 which then activates the expression of p21 that inhibits G1-CDKs. These pathways include the G1 checkpoint, the G1/S checkpoint, the S-phase checkpoint pathways and the G2 checkpoint (also called G2/M checkpoint).

DNA-replication checkpoint, often referred to S/M checkpoint, ensures that the DNA are completely replicated. Any stalled DNA-replication fork can trigger the ATR pathways. ATR is responsible for single-strand DNA that may exist in many, if not all, DNA damage types. Spindle Assembly Checkpoint Spindle assembly checkpoint (SAC) ensures that a cell does not enter anaphase if the chromosomes are not correctly arranged on the plate. The SAC is default activated and is inactivated only when all kinetochores are properly attached to the spindle. SAC proteins accumulating at each kinetochore assemble mitotic checkpoint complexes (MCCs) that inactivate APC/CCDC20 E3 ligase. SAC proteins are displaced when the kinetochore is under tension of microtubules from two spindle poles. When all the kinetochores are properly attached and all SAC protein complexes are displaced, MCCs are no longer assembled and APC/CCDC20 is no longer inhibited. APC/CCDC20 is activated by M-CDK and then ubiquitinates proteins that should be degraded, including cyclin B and securin.

The spindle position checkpoint prevents cytokinesis until the spindle is correctly positioned. This checkpoint ensures that the nucleus is equally partitioned into two daughter cells. This checkpoint is best studied in budding yeast. Animals may have some other mechanism to ensure the equal partition.

Cell division supply cells for body development and homeostasis. In some tissues of an adult, stem/progenitor cells proliferate continuously to renew the tissue. For example, differentiated epidermal cells normally survive only two to four weeks and epidermal progenitor cells proliferate to supply new cells. In many tissue of an adult, cells normally do not proliferate and can remain functional for a long time or even the whole life of its host. Some of these cells, e.g., hepatocytes can enter cell cycle when the tissue lost its mass, while some, e.g., heart muscle cells and neurons, have very limited, if any, regenerative capacity. If the mechanisms maintaining normal proliferation malfunction and cause uncontrolled cell division, a tumor would occur. A malignant tumor is called cancer. Cancer cells are able to spread to other place of the body and give birth to new tumors.

A cell that is misregulated and undergo uncontrolled division may develop to tumor, a complex cell organization of different cell types that cooperate to optimize the growth of the tumor. The rate of cell growth, the proportion of cells dividing within the tumor, is tested to reflect the aggressiveness of the tumor. A cancer with higher growth rate is more aggressive. Tumor cells do not exhibit contact inhibition when they are cultured in a dish.

The malignancy of cancer is due to DNA mutations that affect the expression or activity of the proteins that control or regulate cell cycle. The genes associated with cancer can be categorized as proto-oncogenes (e.g., FOS, Myc and RAS) that promote cell proliferation, tumor suppressor genes (e.g., p53, RB and GAP) that normally protects cells from becoming cancer, apoptotic regulatory genes (e.g., BCL2 and CASP8) that normally control apoptosis, and DNA repair genes (e.g., BRCA1 and XPA) that ensure genome integrity. The p53 gene is mutated or deleted in about half of human cancers.

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