The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-speci®c manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90 rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.

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.

Transforming growth factor ␤1 (TGF-␤1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-␤1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-␤1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41-and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-␤1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-␤1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-␤1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-␤1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.

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Aim : The 41-and 43-kDa mitogen-activated protein kinases (MAPK; ERK2 and ERK1, respectively) play pivotal roles in the mitogenic signal transduction pathway. We previously demonstrated that constitutive activation of the MAPK cascade was related to the carcinogenesis of human tumors. In this study, we examined whether constitutive activation of MAPK was related to the progression to androgen independence of prostate cancer. Methods : MAPK activation was examined by the appearance of phosphorylated forms and an in vitro kinase assay in four human (androgen-dependent and independent) prostate cancer cell lines and rat prostate cancer cell line Dunning (androgen-sensitive G line, and androgen-independent AT-3, AT-6 sublines). In addition, when androgen-dependent mouse Shionogi Carcinoma 115 (SC115) cells were serially cultured without androgen to obtain androgen-independent cells, the time and degree of MAPK activation were examined. Results : One of three human androgen-independent cell lines (DU145) showed constitutive activation of MAPK, while an androgen-dependent cell line (LNCaP) did not show MAPK activation. While MAPK were not activated in an androgen-sensitive Dunning G cell line, MAPK were activated in androgen-independent sublines (AT-3 and AT-6) derived from a G cell line. In addition, when SC115 cells were serially cultured without androgen, the cells 16-24 weeks after androgen removal showed MAPK activation. Furthermore, in subcloned cells, MAPK activation was observed even in the cells maintained for 9 weeks in medium without testosterone.Conclusions : The present findings suggest that constitutive activation of MAPK may be associated with the acquisition of hormone independence in prostate cancer and that clonal selection after androgen removal and hormone-independent growth through the MAPK signal transduction pathway could begin at a relatively early period in the individual cells.

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) So far, two major signal transduction pathways have been shown to be involved in cell growth stimulation: 1) protein-tyrosine phosphorylation, and 2) breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol

The function of hepatocyte growth factor/scatter factor (HGF/SF) is to increase proliferation as well as to stimulate motility and disperse cell colonies of epithelial cells. In this study, we examined the motogenic and mitogenic responses of two human gastric carcinoma cell types, MKN7 and MKN74. Cell motility of both cell lines was markedly stimulated by HGF/SF. In contrast, HGF/SF stimulated cell growth of MKN74 cells, but did not stimulate growth of MKN7 cells. To address the cause of the difference in response of these cells, which may reflect some differences in signaling pathways downstream from the HGF/SF receptor, c‐Met, we investigated the induction of the proto‐oncogene c‐fos. The level of c‐fos mRNA increased and reached a maximum approximately 40 min after HGF/SF stimulation in MKN74 cells, and thereafter its level rapidly decreased. In contrast, the level of c‐fos expression was very low irrespective of the stimulation in MKN7 cells. c‐fos protein was transiently induced only in MKN74 cells 1 h after treatment with HGF/SF, and its levels subsequently decreased. We subsequently examined the activation of mitogen‐activated‐protein kinase, which is a major mediator in the signaling pathway leading to the stimulation of c‐fos transcription, after HGF/SF treatment in both cell lines. Mitogen‐activated‐protein kinase was markedly activated by this treatment in MKN74 cells, but was only slightly activated in MKN7 cells. These results suggest that although mitogen‐activated‐protein kinase activation and c‐fos induction play an essential role in the signaling pathway leading to cell growth, they are not required for the motility response induced by HGF/SF.