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.
Highlights
The epidermal growth factor receptor (EGFR1; ErbB-1 or HER1) is a 170-kDa member of the ErbB family of transmembrane receptor tyrosine kinases that plays a central role in proliferation, development, differentiation, migration, and oncogenesis of several cell lineages [1,2,3]
Using small molecule quinazoline inhibitors of the EGFR tyrosine kinase [21, 22] as experimental tools, we have studied the cell cycle effects that follow interruption of EGFR function in receptor-overexpressing tumor cells focusing on molecules involved in the G1/S transition
Quinazolines Block Basal and TGF-␣-induced EGFR Phosphorylation on Tyrosine—We initially studied the inhibitory potency of AG-1517 on basal and ligand-induced EGFR phosphorylation in intact A431 and MDA-468 cells
Summary
Vol 275, No 10, Issue of March 10, pp. 6987–6995, 2000 Printed in U.S.A. Reversible G1 Arrest Induced by Inhibition of the Epidermal Growth Factor Receptor Tyrosine Kinase Requires Up-regulation of p27KIP1 Independent of MAPK Activity*. Using small molecule quinazoline inhibitors of the EGFR tyrosine kinase [21, 22] as experimental tools, we have studied the cell cycle effects that follow interruption of EGFR function in receptor-overexpressing tumor cells focusing on molecules involved in the G1/S transition Both of the quinazolines used inhibit the EGFR kinase activity at submicromolar concentrations by reversibly binding to the receptor’s ATP site and inducing the formation of inactive EGFR homodimers [23], thereby preventing the phosphorylation of downstream cellular substrates. These reversible biochemical responses result in G1 arrest of tumor cells expressing high levels of autoactivated EGFR, allowing examination of the temporal correlation between EGFR signal transduction and cell cycle progression
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