The restriction point (R) is a point in G1 of the animal cell cycle at which the cell becomes “committed” to the cell cycle and after which extracellular proliferation stimulants are no longer required.
History of the restriction point
Originally, Temin showed that chicken cells reach a point at which they are committed to replicate their DNA and are not dependent on extracellular signals. About 20 years later, in 1973, Arthur Pardee demonstrated that a single restriction point exists in G1. Previously, G1 had been defined simply as the time between mitosis and S phase. No molecular or morphological place-markers for a cell's position in G1 were known. Pardee used a double-block method in which he shifted cells from one cell cycle block (such as critical amino acid withdrawal or serum withdrawal) to another and compared each block’s efficiency at preventing progression to S phase. He found that both blocks in all cases examined were equally efficient at blocking S phase progression, indicating that they must all act at the same point in G1, which he termed the “restriction point,” or R-point.
In 1985, Zetterberg and Larsson discovered that, in all stages of the cell cycle, serum deprivation results in inhibition of protein synthesis. Only in postmitotic cells (i.e. cells in early G1) did serum withdrawal force cells into quiescence (G0). In fact, Zetterberg found that virtually all of the variability in cell cycle length can be accounted for in the time it takes the cell to move from the restriction point to S phase.
Extracellular Signals and the Restriction Point
Except for early embryonic development, most cells in multicellular organisms persist in a quiescent state known as G0, where proliferation does not occur, and cells are typically terminally differentiated; other specialized cells continue to divide into adulthood. For both of these groups of cells, a decision has been made to either exit the cell cycle and become quiescent (G0), or to reenter G1.
A cell’s decision to enter, or reenter, the cell cycle is made before S-phase in G1 at what is known as the restriction point, and is determined by the combination of promotional and inhibitory extracellular signals that are received and processed. Before the R-point, a cell requires these extracellular stimulants to begin progressing through the first three sub-phases of G1 (competence, entry G1a, progression G1b). After the R-point has been passed in G1b, however, extracellular signals are no longer required, and the cell is irreversibly committed to preparing for DNA duplication. Further progression is regulated by intracellular mechanisms. Removal of stimulants before the cell reaches the R-point may result in the cell’s reversion to quiescence. Under these conditions, cells are actually set back in the cell cycle, and will require additional time (about 8 hours more than the withdrawal time in culture) after passing the restriction point to enter S phase.
Signals from extracellular growth factors are transduced in a typical manner. Growth factor binds to receptors on the cell surface, and a variety of phosphorylation cascades result in Ca2+ uptake and protein phosphorylation. Phosphoprotein levels are counterbalanced by phosphatases. Ultimately, transcriptional activation of certain target genes occurs. Extracellular signaling must be maintained, and the cell must also have access to sufficient nutrient supplies to support rapid protein synthesis. Accumulation of cyclin D's are essential.
Cyclin D-bound cdk’s 4 and 6 are activated by cdk-activating kinase and drive the cell towards the restriction point. Cyclin D, however has a high turnover rate (t1/2<25 min). It is because of this quick turnover rate that the cell is extremely sensitive to mitogenic signaling levels, which not only stimulate cycin D production, but also help to stabilize cyclin D within the cell. In this way, cyclin D acts as a mitogenic signal sensor. Cdk inhibitors (CKI), such as the Ink4 proteins and p21, help to prevent improper cyclin-cdk activity.
Active cyclin D-cdk complexes phosphorylate retinoblastoma protein (pRb) in the nucleus. Unphosphorylated Rb acts as an inhibitor of G1 by preventing E2F-mediated transcription. Once phosphorylated, E2F activates the transcription of cyclins E and A. Active cyclin E-cdk begins to accumulate and completes pRb phosphorylation, as shown in the figure.
A paper published by the Lingchong You and Joe Nevins groups at Duke University in 2008 demonstrated that the a bistable hysteric E2F switch underlies the restriction point. E2F promotes its own activation, and also promotes the inhibition of its own inhibitor (pRb), forming two feedback loops (among others) that are important in establishing bistable systems. The authors of this study used a destabilized GFP-system under the control of the E2F promoter as a readout of E2F activity. Serum-starved cells were stimulated with varying serum concentrations, and the GFP readout was recorded at a single-cell level. They found that the GFP reporter was either on or off, indicating that E2F was either completely activated or deactivated at all of the different serum levels analyzed. Further experiments, in which they analyzed the history-dependence of the E2F system confirmed that it operates as a hysteretic bistable switch.
The restriction point in cancer
Cancer can be seen as a disruption of normal restriction point function, as cells continually and inappropriately reenter the cell cycle, and do not enter G0. Mutations at many steps in the pathway towards the restriction point can result in cancerous growth of cells. Some of the genes most commonly mutated in cancer include Cdk’s and CKI’s; overactive Cdk’s or underactive CKI’s lower the stringency of the restriction point, allowing more cells to bypass senescence.
The restriction point is an important consideration in the development of new drug therapies. Under normal physiological conditions, all cell proliferation is regulated by the restriction point. This can be exploited and used as a way to protect non-cancerous cells from chemotherapy treatments. Chemotherapy drugs typically attack cells that are proliferating rapidly. By using drugs that inhibit completion of the restriction point, such as growth factor receptor inhibitors, normal cells are prevented from proliferating, and are thus protected from chemotherapy treatments.
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