The 41-/43-kDa mitogen-activated protein (MAP) 1 kinase pathway, also called the extracellular signal-regulated kinase (ERK) pathway, is activated in a variety of cell types by diverse extracellular stimuli and is among the most thoroughly studied of signaling pathways that connect different membrane receptors to the nucleus (1, 2). Activation of the ERK pathway involves the activation of Ras at the plasma membrane, and the sequential activation of a series of protein kinases. Initially, Ras interacts with and activates Raf-1, which in turn activates MAP kinase/ERK kinase (MEK)-1 and -2 by serine phosphorylation. MEK-1/2 then catalyze the phosphorylation of 41-and 43-kDa MAP kinases (ERK2 and ERK1, respectively) on tyrosine and threonine residues, and these activated MAP kinases can phosphorylate cytoplasmic and nuclear targets. The ERK pathway participates in a wide range of cellular programs including proliferation, differentiation, and movement (1, 2).Aberrant activation of signal transducing proteins has been linked with cancer. For example, constitutively active mutants of Ras (3) and Raf-1 (4) have been observed in several human tumors, and constitutively active mutants of MEK-1 have been shown to transform mammalian cells (5, 6). We recently examined whether constitutive activation of the ERK pathway is associated with the neoplastic phenotype of human tumor cells. Constitutive activation of ERKs and MEK was observed in a relatively large number of tumors; tumor cells derived from pancreas, colon, lung, ovary, and kidney tissues showed especially high frequencies (30 -50%) and a high degree of kinase activation (7,8). Activation of the ERKs is also associated with prostate cancer progression (9). The precise cause of constitutive activation of the ERK pathway in many of these tumor cells remains unclear. However, such high frequencies of ERK/ MEK activation in human tumors indicate that specific inhibitors might be developed against these protein kinases for cancer therapy, especially for treatment of tumors showing constitutive activation of the ERK pathway.In the present study, we have examined the effect of blockade of the ERK pathway on the proliferation of human tumor cells. We utilized small molecule inhibitors of this pathway, PD98059 (10) and U0126 (11), which specifically inhibit MEK activity. Our results demonstrate that these MEK inhibitors induce a remarkable G 1 cell cycle arrest, followed by a modest apoptotic response, in tumor cells in which the ERK pathway is constitutively activated. Up-regulation of the CDK inhibitor p27Kip1 was observed in these G 1 -arrested tumor cells.

Hepatocyte growth factor (HGF) markedly induced the spreading, dissociation and scattering of Madin ± Darby canine kidney epithelial cells (MDCK) and human stomach adenocarcinoma cells (TMK1). Scattering of MDCK and TMK1 cells was induced by 12-Otetradecanoyl-phorbol-13-acetate (PMA) and epidermal growth factor (EGF), respectively. In all these agentstimulated cells, rapid activation of Raf-1, MAP kinase/ ERK kinase (MEK), 41/43 kDa MAP kinases and p90 rsk was commonly observed. In contrast, PMA neither induced the scattering nor activation of all these kinases in TMK1 cells. Pretreatment of MDCK and TMK1 cells with 2-(2-amino-3-methoxyphenyl) choromone (AMPC), a speci®c inhibitor of MEK, selectively inhibited the HGF-, PMA-and EGF-stimulated activities of MEK, 41/43 kDa MAP kinases and p90 rsk in a dose dependent manner. AMPC-pretreatment, however, did not aect HGF-, PMA-or EGF-induced activation of Raf-1, nor HGF-induced activation of phosphatidylinositol 3-kinase in these cells. Importantly, HGF-, PMA-and EGFinduced scattering of MDCK and TMK1 cells was inhibited at doses of AMPC similar to those that gave comparable levels of inhibition of the activities of MEK, 41/43 kDa MAP kinases and p90 rsk . These results suggest that activation of the 41/43 kDa MAP kinase signaling pathway is required for the motility response of MDCK and TMK1 cells induced by agents such as HGF, PMA and EGF.

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