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. Mitogen Signaling Growth factors (e.g., PDGF, FGF, and EGF) regulate entry of cells into the cell cycle and progression to the restriction point. After passing this switch-like “point of no return,” cell cycle completion is no longer dependent on the presence of mitogens. Sustained mitogen signaling promotes cell cycle entry largely through regulation of the G1 cyclins (cyclin D1-3) and their assembly with Cdk4/6, which may be mediated in parallel through both MAPK and PI3K pathways. MAPK Signaling Cascade The binding of extracellular growth factors to their receptor tyrosine kinases (RTK) triggers a conformational change and promotes dimerization and autophosphorylation of tyrosine residues on the cytoplasmic tail of the RTKs. These phosphorylated tyrosine residues facilitate the docking of proteins containing an SH2-domain (e.g., Grb2), which can subsequently recruit other signaling proteins to the plasma membrane and trigger signaling kinase cascades. RTK-associated Grb2 binds Sos, which is a guanine nucleotide exchange factor that converts membrane-bound Ras to its active form (Ras-GDP Ras-GTP). Active Ras activates the MAP kinase cascade, binding and activating Raf, which phosphorylates and activates MEK, which phosphorylates and activates ERK (also known as MAPK, see also MAPK/ERK pathway). Active ERK then translocates into the nucleus where it activates multiple targets, such as the transcription factor serum-response factor (SRF), resulting in expression of immediate early genes—notably the transcription factors Fos and Myc. Fos/Jun dimers comprise the transcription factor complex AP-1 and activate delayed response genes, including the major G1 cyclin, cyclin D1. Myc also regulates expression of a wide variety of pro-proliferative and pro-growth genes, including some induction of cyclin