G1/S Checkpoint
Cell Cycle

Author: Pietro Garofalo
Date: 25/03/2010

Description

DEFINITION

G1/S is the first checkpoint and it is located at the end of the cell cycle's G1 phase, just before entry into S phase, making the key decision of whether the cell should divide, delay division, or enter a resting stage. Many cells stop at this stage and enter a resting state called G0. Liver cells, for instance, only enter mitosis around once or twice a year (because of mild liver damage as a slight alcoholic intoxication, the damaged cells die, and the space left stimulated ITO cells to produce HGF which induce epatocite proliferation).
The G1 checkpoint is where eukaryotes typically arrest the cell cycle if environmental conditions make cell division impossible or if the cell passes into G0 for an extended period. In animal cells, the G1 phase checkpoint is called the restriction point, and in yeast cells it is called the start point.

G1/S Checkpoint cheks the existence of all conditions (nutrients and enzymes) required for DNA synthesis. Growth factors, hormones etc simply are signals supplying information about the local conditions (aminoacids, glucose, NAPH, O2 etc)

A putative model for the role of mitochondria in the G1–S transition. Throughout most of the cell cycle, mitochondria appear as a combination of either tubular or fragmented morphologies. Surprisingly, at the G1–S transition, the mitochondria coalesce into a giant, single tubular network. This network is electrically coupled and exhibits a hyperpolarized mitochondrial membrane potential (ψm). Because ψm is the ionic gradient used to generate ATP, it is not surprising that this unique morphological and bioenergetic mitochondrial network appears to allow for increased ATP generation. Based on ref. 1, and previous studies in mammalian cells and lower organisms, the absence of this energetic boost may trigger a G1–S checkpoint that involves the sequential activation of AMPK and p53 and ultimately the down-regulation of cyclin E levels. In addition, ref. 1 suggests that increased mitochondrial activity can positively regulate cyclin E levels and trigger S-phase progression. Note, in this putative model, p53 regulates events in several different contexts, including the transcriptional induction of p21, the cell cycle regulator, and SCO2, a factor that has been demonstrated to regulate mitochondrial oxygen consumption.
The Krebs cycle meets the cell cycle: Mitochondria and the G1–S transition 2009

Many different stimuli exert checkpoint control including TGFb, DNA damage, contact inhibition, replicative senescence, and growth factor withdrawal.

INIBITION OF PROLIFERATION: many stimuli involved:

  • TGF-beta1
  • DNA Damage
  • Contact inibition
  • Growfactor withdrawal

TGF-beta1

Transforming growth factor beta (TGF-β) is a protein that controls proliferation, cellular differentiation, and other functions in most cells. It plays a role in immunity, cancer, heart disease, diabetes, and Marfan syndrome. TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. TGF-β is a secreted protein that exists in three isoforms called TGF-β1, TGF-β2 and TGF-β3 ( W )
Transforming growth factor beta 1 prevents phosphorylation of RB scheduled in mid to late G1 and arrests cells in late G1. TGF-beta1 once bound to its receptor activates an intracellular pathway that involves the binding of SMAD3-SMAD4 which activates cyclin-dependent kinase inhibitors (p16, p15, p21, p27). TGFb additionally inhibits the transcription of Cdc25A, a phosphatase that activates the cell cycle kinases. For this reason cell cycle kinases (CDK2, CDK4, CDK6) bound to Cyclin E, and Cyclin D cannot phosphorilate pRb.

DNA Damage

In normal cells p53 is usually inactive, bound to MDM2 protein that inhibits the protein and promotes the degradation of functioning as a ubiquitin ligase. The activation of p53 is induced after the effects of various carcinogens such as UV, oncogenes and drugs or other substances that damage DNA.
The damage to DNA are found in specific "stages" Control of cell cycle proteins that induce various - such as ATM, Chk1 and Chk2 - to phosphorylate p53 sites near or within the region that binds MDM2 (inhibiting the attack). Even oncogenes stimulate the activation of p53 by p14ARF protein. Some other oncogenes, however, stimulate the transcription of a protein that inhibits MDM2. Once activated, p53 activates the transcription of many genes including that for p21, which binds the complex G1-S/CDK and D / CDK (molecules important for the transition from G1 to S phase) by inhibiting their activity ( and avoiding the proliferation of mutated cells).
Another important function of p53 tumor suppression is inhibition of angiogenesis. Recent research has also established a link between the pathways of p53 and RB1 through p14ARF, raising the possibility that the two ways you can adjust each other

Contact Inibition

Recent results obtained on cell cultures show that the loss of contact inhibition of highly malignant cells of thyroid anaplastic carcinoma could be related with the observed reduction of the expression on the cell membrane complex E-caderina/beta-catenina, believed to compromise the cell-cell adhesion and polarity of epithelial cells. A reduction in the levels of E-cadherin would also be a loss of up-regulation of the expression p27 required for negative regulation of cell growth. For this reason contact inition seems to activate the expression of p27 which is a cyclin dependent kinase inibitor and is important to stop cell division in presence of cell contact.
Taken from: http://progettooncologia.cnr.it/strategici/tiroide/03-ti.html

Growth factor withdrawal

Growth factor withdrawal activates GSK3b, which phosphorylates cyclin D, leading to its rapid ubiquitination and proteosomal degradation. Ubiquitination, nuclear export, and degradation are mechanisms commonly used to rapidly reduce the concentration of cell-cycle control proteins.

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ACTIVATION OF PROLIFERATION: many grow factor involved, for example:

  • IGF1 and FGF2
  • PDGF

IGF1 an FGF2

GF-I and FGF-2 coordinately enhance cyclin D1 and cyclin E-cdk2 association and activity to promote G1 progression in oligodendrocyte progenitor cells. Insulin-like growth factor (IGF)-I and fibroblast growth factor (FGF)-2 have known functions individually in development of neural stem cells as well as more restricted neuronal and glial progenitor cells. IGF-I enhanced FGF-2 induction of cyclin D1, activation of G(1) cyclin-cyclin-dependent kinase (cdk) complexes, and hyperphosphorylation of retinoblastoma protein (pRb). Moreover, IGF-I was required for G(2)/M progression. In contrast, FGF-2 decreased levels of the cdk inhibitor p27(Kip1) associated with cyclin E-cdk2.
GF-I and FGF-2 coordinately enhance cyclin D1 and cyclin E-cdk2 association and activity to promote G1 progression in oligodendrocyte progenitor cells

PDGF

Exposure to PDGF, which stimulates cell cycle entry but not progression through GGraphic, induces the formation of cyclin DGraphic-Cdk4 complexes that bind p27Graphic and titrate the pool of Kip1 available to inhibit Cdk2. In addition, PDGF stimulates a moderate transient reduction in the abundance of p27Graphic protein. However, limited expression of cyclin E and cyclin A is observed after PDGF treatment, and in the absence of PPP, p27 levels are sufficient to bind and inactivate existing cyclin-Cdk complexes. Although plasma does not significantly increase the proportion of Kip1 bound to cyclin DGraphic-Cdk4, stimulation of PDGF-treated cells with plasma does overcome the threshold inhibition of p27Graphic by further increasing the expression of cyclins E and A and decreasing the amount of Kip1 over a prolonged time period.
Differential Modulation of Graphic Cyclins and the Cdk Inhibitor p27Graphic by Platelet-derived Growth Factor and Plasma Factors in Density-arrested Fibroblasts

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Every intracellular pathway of this checkpoint ended with the activation or the inactivation of Retinoblastoma Protein which is the pivotal transcriptional factor of G1/S checkpoint.

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pRb (Retinoblastoma Protein)

The retinoblastoma protein is a tumor suppression protein that is dysfunctional in many types of cancer. One highly studied function of pRb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. It is also a recruiter of several chromatin remodelling enzymes such as methylases and acetylases.

pRB role and mechanism of activation

pRB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorilation during the G1/S transition. pRB plays an essential role in the G1 arrest induced by a variety of growth inhibitory signals. For example pRb prevents the cell from replicating damaged DNA by preventing its progression along the cell cycle through G1 (first gap phase) into S (synthesis phase).
The antiproliferative activity of RB is mediated by its ability to inhibit the transcription of genes that are required for cell cycle progression, e.g., cyclin A.
pRb binds and inhibits transcription factors of the E2F family, which are composed of dimers of an E2F protein and a DP protein. The transcription activating complexes of E2 promoter-binding–protein-dimerization partners (E2F-DP) can push a cell into S phase. As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. When pRb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. The pRb-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin. Because histone deacetylase modifies chromatin to a closed state through deacetylation, transcription is repressed.
Additionally, it has recently been reported that the RB-mediated repression of specific cell cycle genes (e.g., the cyclin A gene) is dependent on association with SWI/SNF chromatin remodelling activity. The mechanism through which the SWI/SNF complex mediates RB-dependent transcriptional repression is not clearly understood. However, loss of SWI/SNF activity disrupts RB-mediated repression of specific cell cycle targets and renders cells resistant to RB-mediated cell cycle arrest. Lastly, RB may interact with specific components of the basal transcription machinery (e.g., TFII250) to regulate their activities. Through these collective mechanisms of transcriptional regulation, RB exerts its antiproliferative action. In general, RB activity is induced in response to environmental signals which favor halting the cell cycle. For example, the antimitogenic activity of TGFb requires RB activation.

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ACTIVATION and INACTIVATION of pRB: In the hypophosphorylated state, pRb is active and carries out its role as tumor suppressor by inhibiting cell cycle progression. Phosphorylation inactivates pRb, during the M-to-G1 transition; pRb is progressively dephosphorylated by PP1, returning to its growth-suppressive hypophosphorylated state.
The ability of RB to inhibit cellular proliferation is counterbalanced by the action of cyclin-dependent kinases (Cdks). In response to proliferative signals, Cdks are activated by their
cyclin regulatory subunits to phosphorylate RB and thereby inactivate its protein binding function.
Specifically, when quiescent cells are stimulated to enter the cell cycle, Cdk4/6-cyclin D complexes become active in mid-G1 and initiate the phosphorylation of RB. Later in G1, RB becomes hyperphosphorylated through the combined actions of Cdk4-cyclin D, Cdk2-cyclin E, and Cdk2-cyclin A. The activities of Cdk2-cyclin E and Cdk2-cyclin A are both rate limiting and required for entry into S phase. Phosphorylation of RB is maintained throughout S and G2, until RB is finally dephosphorylated by a phosphatase at the M/G1 transition. The E2F binding function of RB can be inactivated by the phosphorylation of several Cdk phosphorylation sites. The binding of RB to c-Abl tyrosine kinase is inactivated by the phosphorylation of two specific
serine sites, and binding to viral oncoproteins or histone deacetylase is inactivated by two specific threonine sites. These data show that phosphorylation of RB is a highly regulated process and that specific phosphorylation events result in distinct outcomes.
Phosphorylation of pRb allows E2F-DP to dissociate from pRb and become active. When E2F is free it activates factors like cyclins (e.g. Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA.

The importance of RB phosphorylation is underscored by the prevalence of mutations in cancer that result in deregulation of RB phosphorylation. For example, amplification or overexpression of cyclin D and Cdk4/6, or loss of the Cdk4/6 inhibitor p16ink4a, occurs with high frequency in human tumors. Each of these types of mutations results in increased RB phosphorylation and inactivation of RB function. Accordingly, RB mutant proteins that lack the Cdk phosphorylation sites which regulate E2F binding are potent inhibitors of the cell cycle. These phosphorylation site-mutated RB proteins (PSM-RB) cause a cell cycle arrest in G1, which can be overridden by the increased expression of cyclin E. Interestingly, however, overproduction of cyclin E does not rescue cell cycle inhibition imposed by PSM-RB (constitutively active RB), as these cells entered but could not progress through S phase. The S-phase inhibitory action of RB cannot be mimicked by Cdk inhibitors such as p16ink4a, p21Cip1, or p27Kip1. The observation that PSM-RB could inhibit S-phase progression was consistent with the continued phosphorylation of RB throughout S phase and suggested that RB might become dephosphorylated under specific conditions, resulting in the inhibition of DNA replication.

Knudsen et al, University of Cincinnati, in a study of 2000 stated that pRB is not only involved in the regulation of the G1/S Checkpoint but it is also required for an intra-S-phase response to DNA damage.

VHL

Gene array of VHL mutation and hypoxia shows novel hypoxia-induced genes and that cyclin D1 is a VHL target gene. 2004

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