Invest. 1995Invest. . 95:1897Invest. -1905

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

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Epidemiological studies have linked dietary fiber to the prevention of human colorectal cancer and suggest that short chain fatty acids such as butyric acid, which is produced by fermentation of dietary fiber in the large intestine, may be an important mediator of the protective effects of fiber. We investigated the role of Bcl-2 deregulation on the sensitivity of colorectal carcinoma cells to undergo butyrate-induced apoptosis. Here we report an inverse relationship between the levels of Bcl-2 and the sensitivity of colorectal carcinoma cell lines to undergo apoptosis in response to butyrate. Overexpression of Bcl-2 in colorectal carcinoma DiFi cells resulted in suppression of butyrate-induced apoptosis and enhanced cell survival in response to butyrate. Butyrate-induced apoptosis was accompanied by inhibition of expression of a 30 kDa protein (p30, immunorecognized by anti-Bcl-2 mAb) and this cellular effect of butyrate was inhibited by Bcl-2 overexpression. These findings suggest that deregulation of Bcl-2 in human colorectal carcinoma cells confers resistance to induction of apoptosis by butyrate, a dietary micronutrient.

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