Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition.

Abstract

Abstract. Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21 cipl/wAvl mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21 cipl/wAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21 cipvwAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21 cipl/wAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cipl/wAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cipl/wAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.PIDERMAL growth factor (EGF) ~ is a potent mitogen for most epithelial tissues. Binding of EGF to specific cell surface receptors stimulates formation of noncovalent homodimers (or oligomers) involving two (or more) EGF receptors, which activates receptor tyrosine kinase and results in transphosphorylation of tyrosine residues on the opposite member of the pair. The tyrosine autophosphorylated EGF receptors exhibit increased binding affinities for the Src homology 2 regions of substrate molecules. Substrate binding to receptors is followed by phosphorylation of the substrate molecules on tyrosine residues, which results in a cascade of biochemical steps involved in the mitogenic signal transduction pathways of cells (Carpenter, 1992;Ullrich and Schlessinger, 1990).While EGF has a stimulatory effect on the proliferation

Differential modulation of paclitaxel-mediated apoptosis by p21Waf1 and p27Kip1

“…Cell cycle distribution analysis with propidium iodide was previously described (Fan et al, 1995). For analysis with mithramycin staining, an aliquot of 2610 6 cells was incubated with 2 ml of 0.1% nonidet P-40 in PBS for 5 min, followed by ®xation with the addition of 0.7 ml of 4% formaldehyde in PBS and gentle shaking of the cells at 48C for 15 min.…”

“…Cells were lysed as previously described (Fan et al, 1995). Speci®c signals were visualized by the ECL chemoluminescence detection kit (Amersham, Arlington Heights, IL, USA).…”

“…The kinase assay with Rb or histone H1 as substrates was performed as previously described (Fan et al, 1995;Wu et al, 1996).…”

Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells

“…Low doses of radiation induce expression of p21 Cip-1 via a p42 MAP kinase dependent mechanism High dose irradiation of cells has been shown to induce expression of the cyclin dependent kinase (cdk) inhibitor protein p21 , and other studies have demonstrated that growth factors can induce p21 Cip-1 expression via a p42 MAP kinase dependent mechanism (Pumiglia and Decker, 1997;Whalen et al, 1997;Lloyd et al, 1997;Fan et al, 1995;Missero et al, 1996;Macloed et al, 1995;Liu et al, 1996;Deng et al, 1995;Van den Heuvel and Harlow, 1993;Lees et al, 1993;Brugarolas et al, 1995). However, it has been suggested that radiation and growth factors induce Time Course of inhibition of GSK3 activity after 1 Gy exposure of A431 cells A431 cells (150 mm dishes) were exposed to 1 Gy (1.8 Gy/min) and at speci®ed time points media aspirated and the plates snap frozen.…”

“…Acute activation of the MAP kinase cascade by growth factors has been shown to potentiate proliferation, whereas chronic elevation of MAP kinase activity has been demonstrated to be cytoprotective against irradiation (Canman et al, 1995), and to inhibit DNA synthesis potentially via induction of the cyclin dependent kinase (cdk) inhibitor protein p21 Cip-1 (Pumiglia and Decker, 1997;Wixler et al, 1996;Traverse et al, 1994;Whalen et al, 1997;Lloyd et al, 1997;Fan et al, 1995;Missero et al, 1996;Macloed et al, 1995;Liu et al, 1996;Deng et al, 1995). The cdk inhibitor protein p21 Cip-1 can also be induced in response to radiation exposure of cells, and cultured ®broblasts from p21 Cip-1`k nock-out' mice which cannot express p21 have been demonstrated to be more radiosensitive than wild type cells, demonstrating that expression of this molecule is cytoprotective against radiation (Missero et al, 1996;Macloed et al, 1995;Liu et al, 1996;Deng et al, 1995).…”