Reversible G1 arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity

Dagmar Busse, Rachel S. Doughty, Timothy T. Ramsey, William E. Russell, James O. Price, W. Michael Flanagan, Laura K. Shawver, Carlos L. Arteaga

Research output: Contribution to journalArticlepeer-review

193 Scopus citations


We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G1 to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1- 10 μM 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 μM AG-1517 abrogated 125I- 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 μM. 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 G1 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 G1 arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G1 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 G1. 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 G1 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.

Original languageEnglish (US)
Pages (from-to)6987-6995
Number of pages9
JournalJournal of Biological Chemistry
Issue number10
StatePublished - Mar 10 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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