Cell Cycle Proteins

 Cell Cycle Proteins Background

The cell cycle is a series of events in which a cell duplicates its contents and eventually divides in two, resulting in two identical “daughter” cells. Bacterial cells that lack nuclei undergo cell division by a process termed “binary fission”. Successful completion of the cell cycle and division in the nucleated cells of eukaryotes is somewhat more complex, however. In order to successfully divide and generate two identical daughter cells, a cell must first replicate each chromosome, replicate its centrosome, and then correctly segregate these into the daughter cells, ensuring that each daughter receives one copy of the entire genome. To ensure this faithful replication and segregation of the genome, eukaryotes have evolved a complex cell cycle, with many built-in checkpoints that ensure a cell is ready to proceed to the next step in the cycle.


Cell Cycle Regulation

The mammalian cell cycle is comprised of two primary stages: interphase and mitosis (M phase), during which the physical division of one cell into two occurs. Interphase is then subdivided into three distinct phases—G1, S, and G2— while M phase is generally subdivided into prophase, prometaphase, metaphase, anaphase, and telophase. The transition from one phase to another is regulated by the interplay of different proteins, and proceeds in an orderly fashion.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases that are key regulatory proteins for cell cycle progression. While the levels remain largely constant throughout the cell cycle, different CDKs are activated at specific stages of the cell cycle by cyclins, which (with the exception of cyclin D) do show cyclic cell cycle-regulated expression. As with CDKs, different cyclins are required at different phases. When CDKs are activated by cyclins, they phosphorylate target proteins, inducing downstream signaling. Inhibition of many of these regulatory proteins can lead to a disruption in the cell cycle. The first phase in interphase, G1 (“gap phase 1”), is controlled largely by the activity of three D type cyclins (cyclin D1 (CCND1), CCND2, and CCND3), and CDK4 and CDK6. The activation of CDK4 and CDK6 by cyclin D is required for entry into S phase. Cyclin D transcription is in turn activated by extracellular signals via many signaling pathways, including Wnt signaling, the Her2 pathway, and the growth factor-activated Ras/Map kinase signaling pathway.

Once CCND is expressed and can activate CDK4/6, the active complex begins to phosphorylate Rb, which plays a vital role in ensuring cells do not proceed in the cell cycle without proper signals telling it to do so. Phosphorylation of Rb relieves repression of the E2F family of transcription factors, allowing for the transcription of many genes required during S-phase, including cyclins E and A. If conditions are unfavorable (e.g. low nutrients or contact inhibition), Rb remains unphosphorylated , E2F remains repressed, and the cell may prolong G1, or exit the cell cycle entirely and enter an arrest phase termed G0. A cell may stay in G0 throughout its lifespan, or, given the appropriate signals, may reenter the cell cycle. During G1, the cell will eventually pass what is termed the “restriction point” (R). After passing the restriction point, the cell is committed to entering the cell cycle, regardless of endogenous or exogenous signals. When a cell is ready to exit G1, CCNE binding to CDK2 regulates progression into S phase.

S phase (“synthesis” phase) is the phase in which DNA replication takes place, and requires the activity of CDK2, CCNE, and CCNA. Pre-initiation replication complexes (pre-RCs) bind to multiple origin recognition complexes (ORCs) during G1 phase, and upon entry into S phase these ORC complexes are activated by CCNE-CDK2. At this point CCNE levels decrease while CCNA levels rise, allowing for CCNA-CDK2 complexes to take over the control of regulating DNA replication. In the second “gap phase”, G2, CDK2 is replaced in the complex with CDK1. The CCNA-CDK1 complex is responsible for ensuring the faithful replication of the genome and preparing the cell to enter mitosis.

Mitosis is the process of chromosome segregation and cell division. The events of mitosis are triggered by the activation of CCNB and CDK1 (Cdc2). CCNBCDK1 are present at high levels in G2, but are held inactive by phosphorylation via the protein kinase Wee1. Late in G2, the protein phosphatase Cdc25 is activated, which removes the inactivating phosphates on the CCNB-CDK1 complexes. CCNB-CDK1 activity leads to the activation of proteins that will guide the cell through mitosis. Exit of mitosis after sister chromatid separation requires the inactivation of the CCNB-CDK1 complex. The inactivation is largely accomplished by ubiquitin-dependent proteolysis of CCNB by the anaphase promoting complex/cyclosome (APC/C).

In the event that it is unfavorable for a cell to divide, there are mechanisms to prevent the cell from entering the cell cycle (prior to R), or to halt the cell cycle in the event that it would be hazardous to the cell to continue (e.g. when DNA damage is present). While CDKs can be activated by cyclins, their activity can also be inhibited by proteins called CDK inhibitors (CKIs). There are two families of CKIs, the INK4 family and the Cip/Kip family. The INK4 family includes p15, p16, p18, and p19, all of which inhibit CDK4 and CDK6 complexes, while the Cip/Kip family includes p21, p27, and p57, which inhibit the other CDKs. Internal and external signals can affect CKIs, including TGF-beta signaling which increases p15 and p27 expression, or the transcriptional activation of p21 via the p53 tumor suppressor gene in response to cellular stresses.


Cell Cycle and Cancer

Cancer is, at its root, a disease of the cell cycle—deregulation of the cell cycle is one of the most often noted hallmarks of tumor development. Tumor suppressors, such as p53 and the retinoblastoma protein (RB), function to repress or halt the cell cycle, promote apoptosis, or both. Oncogenes, such as BRCA1 and BCL2, serve to promote cell proliferation or block apoptosis, and are often transcription factors, growth factors or growth factor receptors, or apoptotic regulators. The tumor suppressor p53 is found mutated in almost 50% of all tumors, and the p53 pathway (including upstream regulators or downstream targets) has been proposed to be altered in perhaps all tumors. Another example is the BCL2 oncogene, which is involved in the initiation of almost all follicular tumors and is involved in chronic lymphocytic leukemia and lung cancer. Thus, understanding the cell cycle and its key regulatory components is important to understanding cancer, and perhaps in identifying therapeutic targets.


Methods to study the Cell Cycle

The study of the cell cycle usually requires having all or most of the cells at the same stage in the cell cycle. There are several methods to synchronize cells, which are mainly divided into two groups: induction techniques and selection techniques.

Induction techniques: Induction techniques frequently utilize an inhibitor of one stage of the cell cycle. The cells then accumulate at this stage and, upon release of the inhibitor; cells show some evidence of synchronized growth. The most frequently used inhibitors are those that act during DNA synthesis or during mitosis. Another technique involves the use of selective lethal agents, which kill cells in certain stages of the cell cycle. The minority of surviving cells is supposed to grow synchronously. Tritiated thymidine and hydroxyurea (HU) have been used in this manner, to kill cells in S phase.

Another induction technique involves nutritional or serum starvation. Removal of serum from the culture medium will induce cells to enter a quiescent state. After readdition of fresh medium, the culture may grow with some evidence of synchrony for one or more divisions. Not all cells enter the cell cycle after being starved, and the ones that do may not do it at the same time, so a low level of synchrony is obtained and it decays rapidly as the cells progress into S phase.

Some of the oldest induction techniques consist of a series of temperature shocks or light changes. These techniques do not produce high degrees of synchrony except in the case of temperature sensitive mutants, which are effectively aligned at specific stages in the cell cycle using temperature changes from the non-permissive to the permissive temperature.

Induction techniques can cause different levels of disruption which can even be lethal to the cell. These inhibitors target specific function in the cell such as DNA synthesis, but other processes such as RNA and protein synthesis are not inhibited, thereby causing unbalanced growth. For instance, cells synchronized at the G1-S phase transition with excess thymidine, mimosine, or aphidicolin showed about 40% increase in the amount of proteins and 2- to 5-fold higher levels of cyclin E, cyclin B1, and cyclin A compared to the equivalent unperturbed cells. As a consequence, any results obtained with these techniques may be impaired by the synchronization technique itself. The side effects of the induction techniques have led to the development of other techniques that do not require the addition of harmful drugs to the cells. These techniques are classified as selection techniques.


Selection techniques. Selection techniques are not as disturbing to the cell as the induction techniques. One example is the mitotic shake-off technique which is used to obtain synchronous cultures from mammalian cells that grow and spread on a solid surface and then round up during mitosis, thereby loosening their attachment to the surface. In this manner it is possible to remove the mitotic cells from the culture by washing off the loosely-attached cells, which then grow as a synchronous culture. The disadvantage of this technique is that the yield of synchronous cells is low because only a few percent of the population is in mitosis and thus washed off.

Another selection technique uses gradient separation. Cells are layered at the top of a sucrose gradient and then centrifuged. After centrifugation, cells will have spread down through the gradient to form a layer with small cells representing the early stages in the cell cycle at the top and large cells representing the late stages of the cell cycle at the bottom. Cells can be removed from any gradient layer and grown as a synchronous culture. However the process of gradient selection may have an adverse effect on the cells. In another technique, called centrifugal elutriation, sedimentation of cells in a centrifugal field is opposed by a counter flow of medium. Cells can be separated by changes in the centrifugal field or the flow rate. The separation is based on size and density. However, artifacts caused by the elutriation procedure have been reported.