title: Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity.
abstract: We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: D
authors.initials: D
authors.lastname: Busse
authors.forename: R S
authors.initials: RS
authors.lastname: Doughty
authors.forename: T T
authors.initials: TT
authors.lastname: Ramsey
authors.forename: W E
authors.initials: WE
authors.lastname: Russell
authors.forename: J O
authors.initials: JO
authors.lastname: Price
authors.forename: W M
authors.initials: WM
authors.lastname: Flanagan
authors.forename: L K
authors.initials: LK
authors.lastname: Shawver
authors.forename: C L
authors.initials: CL
authors.lastname: Arteaga
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins; D019938:Cyclin D1 / Q000032:analysis; D050760:Cyclin-Dependent Kinase Inhibitor p27; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005260:Female; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011799:Quinazolines / Q000494:pharmacology; D016160:Retinoblastoma Protein / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D020032:Tyrphostins / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: C108081:AG 1517; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011799:Quinazolines; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D020032:Tyrphostins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; C101044:RTKI cpd; D066246:ErbB Receptors; D020929:Mitogen-Activated Protein Kinase Kinases