Key points are not available for this paper at this time.
Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G1 cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27Kip1 was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27Kip1. The up-regulation of p27Kip1 correlated with increased association of p27Kip1 with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G1 cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27Kip1. Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G1 cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27Kip1 was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27Kip1. The up-regulation of p27Kip1 correlated with increased association of p27Kip1 with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G1 cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27Kip1. mitogen-activated protein kinase extracellular signal-regulated kinase MAP kinase/ERK kinase cyclin-dependent kinase retinoblastoma protein polyacrylamide gel electrophoresis MAP kinase phosphatase 4′,6′-diamidino-2-phenylindole bromodeoxyuridine kilobase pair 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 (1Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 2Widmann C. Gibson S. Jarpe B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2274) Google Scholar). 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 (1Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 2Widmann C. Gibson S. Jarpe B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2274) Google Scholar). Aberrant activation of signal transducing proteins has been linked with cancer. For example, constitutively active mutants of Ras (3Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3778) Google Scholar) and Raf-1 (4Nakatsu Y. Nomoto S. Oh-uchida M. Shimizu K. Sekiguchi M. Cold Spring Harbor Symp. Quant. Biol. 1985; 51: 1001-1008Crossref Google Scholar) have been observed in several human tumors, and constitutively active mutants of MEK-1 have been shown to transform mammalian cells (5Mansour S.J. Mattern W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar, 6Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar). 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 (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar, 8Oka H. Chatani Y. Hoshino R. Ogawa O. Kakehi Y. Terachi T. Okada Y. Kawaichi M. Kohno M. Yoshida O. Cancer Res. 1995; 55: 4182-4187PubMed Google Scholar). Activation of the ERKs is also associated with prostate cancer progression (9Gioeli D. Mandell J.W. Petroni G.R. Frierson Jr., H.F. Weber M.J. Cancer Res. 1999; 59: 279-284PubMed Google Scholar). 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 (10Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar) and U0126 (11Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar), which specifically inhibit MEK activity. Our results demonstrate that these MEK inhibitors induce a remarkable G1 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 G1-arrested tumor cells. Human cell lines A-172 (glioblastoma), TGW (neuroblastoma), GOTO (neuroblastoma), HT1080 (fibrosarcoma), RPMI-SE (renal cell carcinoma), Colo320 (colon adenocarcinoma), and TIG-3 (diploid fibroblasts) (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The polyclonal anti-ERK antibody has been described previously (12Chatani Y. Tanaka E. Tobe K. Hattori A. Sato M. Tamemoto H. Nishizawa N. Nomoto H. Takeya T. Kadowaki T. Kasuga M. Kohno M. J. Biol. Chem. 1992; 267: 9911-9916Abstract Full Text PDF PubMed Google Scholar, 13Chatani Y. Tanimura S. Miyoshi N. Hattori A. Sato M. Kohno M. J. Biol. Chem. 1995; 270: 30686-30692Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Antibodies against p16INK4a (SC-1661), p19INK4d (SC-1063), p21Cip1 (SC-6246), p27Kip1 (SC-528), p57Kip2 (SC-1040), cyclin A (SC-239), cyclin D1 (SC-6281), cyclin E (SC-247), and pRb (SC-102) were obtained from Santa Cruz Biotechnology. Anti-cyclin B1 antibody (CC-03) was from Calbiochem. 2-(2-Amino-3-methoxyphenyl) chromone, which is identical to the published compound PD98059 (10Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar), was synthesized as described previously (14Tanimura S. Chatani Y. Hoshino R. Sato M. Watanabe S. Kataoka T. Nakamura T. Kohno M. Oncogene. 1998; 17: 57-65Crossref PubMed Scopus (92) Google Scholar). U0126 (11Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar) was purchased from Promega. Other chemicals and reagents were of the purest grade available. For monolayer growth, cells were plated at a density of 1 × 104 cells per 35-mm dish and incubated for 24 h at 37 °C. Cells were then mock-treated or treated with 50 μm PD98059 or 20 μmU0126 for up to 5 days. Cells were harvested by trypsinization, and viable cells which excluded trypan blue were counted using a hemocytometer. For anchorage-independent growth, 1 × 104 cells were suspended in 3 ml of 0.33% Difco agar in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in the presence or absence of 50 μm PD98059, overlaid on a 5-ml layer of 0.5% agar in the respective medium in a 60-mm dish, and cultured for 14 days. Cells were scraped off in cell 2 1 50 2 and and by for were by at × for and protein were using the protein Cell of were by to membrane and with the antibody and were with the (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar, S. M. Watanabe K. Hoshino R. M. Kohno M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). ERK was in kinase as described previously (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar, Y. Tanaka E. Tobe K. Hattori A. Sato M. Tamemoto H. Nishizawa N. Nomoto H. Takeya T. Kadowaki T. Kasuga M. Kohno M. J. Biol. Chem. 1992; 267: 9911-9916Abstract Full Text PDF PubMed Google Scholar, 13Chatani Y. Tanimura S. Miyoshi N. Hattori A. Sato M. Kohno M. J. Biol. Chem. 1995; 270: 30686-30692Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). cell as described of were by for 3 h at with polyclonal anti-ERK antibody to with kinase A 1 and 10% was incubated for at with 20 μm 1 of and of protein in of kinase A. protein was by Cells were in treated with A and with 20 J. F. Cell Biol. 1994; PubMed Scopus Google Scholar). 1 × 104 cells from were for using a of cells in and were using Cells on were treated with 50 μm PD98059 for with apoptotic cells with or were by Cells were in and 0.5% on for were treated with of A for was and by electrophoresis on as described J. 1987; Google Scholar). gel electrophoresis was on as described K. S. M. H. F. S. J. Cell Biol. 1998; PubMed Scopus Google Scholar) using a Cells were in cell 10% 1 50 1 and on for Cell of were incubated with 2 of antibody Santa Cruz or 3 of E antibody for 3 h at °C. were on protein with kinase and 1 and in of kinase supplemented with 2 of μm and of were incubated for at and the of phosphorylation was by and The for M. U. R. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was by and the a kinase-negative form of MEK1 phosphorylation and with was by Van of these RPMI-SE cells × 104 of was using to the after cells were as described A. D. P. S. S. J. J. 1999; 18: PubMed Scopus Google Scholar) using polyclonal antibody Santa Cruz antibody or antibody as the and or as the For 20 μm was to the h after and then cells were incubated for 24 cells were treated with to and then with antibody We have recently that human tumor cells can be with to the activation of the ERK pathway (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar). cells in which constitutive activation of the ERK pathway is as or tumor cells in which the degree of activation of the ERK pathway is especially cells in which constitutive activation of the ERK pathway is as or tumor cells in which the ERK pathway is activated cells with 10% this is identical to that observed in tumor cells with to activation of the ERK pathway, activation of the pathway is cells with 10% serum. We examined the effect of MEK PD98059 and on the activation of ERKs in tumor cells. Activation of ERKs was by different as by a in of the using protein as the and by the of the of which in described previously (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar), (RPMI-SE and and tumor cells a high degree of ERK activation of these tumor cells with U0126 or PD98059 suppressed the activation of ERKs in a U0126 ERK activation PD98059, with complete suppression observed at 20 or 50 a degree of ERK activation was in and and tumor treatment with PD98059 or U0126 also the degree of ERK activation in these cells. the inhibition of ERK activation in RPMI-SE and Colo320 cells. with PD98059 or U0126 the growth of of the tumor cells examined 1 These results the ERK pathway is the cytoplasmic kinase pathway and is activated by stimuli that with a of In activation of the ERK pathway has been shown to be for proliferation (5Mansour S.J. Mattern W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar, 6Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, P. S. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, to these MEK inhibitors showed among cell The growth of tumor cells in which the ERK pathway was constitutively activated was tumor or completely tumor by 50 or 20 μm the growth of TGW tumor cells and TIG-3 to a the inhibition was proliferation of tumor cells to on the activation of the ERK pathway, the growth of these cells. ERK 1 be for the proliferation of tumor inhibition of the ERK by treatment might have in the growth inhibition observed in these cells. Consistent with its antiproliferative effect on monolayer growth, PD98059 completely the anchorage-independent growth of RPMI-SE tumor cells Colo320 tumor cells 1 the the antiproliferative effect of MEK cells were treated with 50 μm PD98059 for with and to of cell cycle Consistent with the marked effect on cell proliferation, PD98059 induced remarkable G1 cell cycle arrest in tumor cells The of G1 was as as h after PD98059 treatment of RPMI-SE and HT1080 and complete G1 cell cycle arrest was observed by 24 at which the of cells in G1 increased from to or to and the of cells in from to or to in RPMI-SE or HT1080 PD98059 treatment induced in these tumor cells a in the of cells with which is a of T. F. 1997; PubMed Scopus Google Scholar). The of such cells h after PD98059 treatment was in RPMI-SE cells and in HT1080 the in mock-treated cells were and PD98059 also induced prominent G1 cell cycle arrest in A-172 tumor cells. However, the of A-172 cells in G1 was that observed in tumor the of A-172 cells in G1 increased from to by at which the of cells in from to In MEK inhibition the cell cycle of TGW tumor cells and TIG-3 to a small PD98059 induced a most in the of these cells in G1 and a decrease in the of cells in modest effect of PD98059 treatment on the cell cycle of these cells to in the growth suppression described 1 PD98059 the cell cycle of Colo320 or GOTO tumor cells. the cell by PD98059, we examined the nuclear of RPMI-SE and HT1080 cells with a treatment with 50 μm PD98059 for of the RPMI-SE cell and of the HT1080 cell and of apoptotic cell such nuclear was in mock-treated cells. PD98059 treatment also induced in HT1080 this was most after PD98059 induced of RPMI-SE of high gel electrophoresis the presence of in RPMI-SE cells after has recently been that the of large to the of and that such large as for the K. S. M. H. F. S. J. Cell Biol. 1998; PubMed Scopus Google F. J.W. C. M. J. PubMed Scopus Google Scholar). to be the of cell in RPMI-SE and HT1080 tumor cells. the of the G1 cell cycle arrest observed in RPMI-SE and HT1080 we examined whether in proteins in these on the retinoblastoma protein Y. Biochem. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar). shown in PD98059 treatment the phosphorylation state of the to by and complete of the was observed h after PD98059 treatment of RPMI-SE and HT1080 cells. In PD98059 treatment the of cyclin A and is with inhibition of and of these is to the ERK pathway has been shown to the of cyclin D1 A. R. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), PD98059 inhibition of the pathway the of either cyclin D1 or cyclin E in these tumor cells. We for on the CDK inhibitors and C.J. J.M. 1999; PubMed Scopus Google Scholar). RPMI-SE and HT1080 cells of these CDK PD98059 treatment induced a marked in p27Kip1 in these tumor which at h and a by the up-regulation of p27Kip1 in the of RPMI-SE cells In PD98059 induce in or p19INK4d in either of the tumor cell lines Up-regulation of p27Kip1 has been shown to its association with such as cyclin in kinase inhibition and to C.J. J.M. 1999; PubMed Scopus Google Scholar). the of p27Kip1 up-regulation in RPMI-SE cell were with against or cyclin and kinase and cyclin kinase were using as a PD98059 treatment of the cells kinase and cyclin kinase activity, with complete inhibition by h of the proteins in p27Kip1 in the and the cyclin E after PD98059 increased of p27Kip1 to cyclin in to PD98059 treatment the of either or cyclin E in the In Colo320 and GOTO tumor PD98059 treatment induce a in the phosphorylation state of or in the of proteins such as cyclin cyclin cyclin or cyclin this was also the in TGW tumor cells and These results were in with the effect of PD98059 on the growth of these tumor cells that specific blockade of the ERK pathway induced up-regulation of RPMI-SE cells were with either kinase-negative form of or is a of the MAP kinase phosphatase and is in that is in the and specifically in to kinase or MAP kinase M. U. R. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). using and that and were in the and completely the activation of up-regulation of p27Kip1 was observed in the of RPMI-SE cells in the absence of PD98059 treatment of or to in the as by the of the In RPMI-SE cells that the kinase-negative form of MEK1 or ERK activation the cells as as the up-regulation of p27Kip1 was observed in these cells after treatment with PD98059 for 24 In the present study, we examined the effect of a specific blockade of the ERK pathway on the growth of human tumor cells in using the specific MEK inhibitors PD98059 and U0126 (10Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar). inhibitors suppressed ERK activation in of the tumor cells examined 1 complete inhibition of the ERK pathway suppressed the growth of tumor cells However, the of cells to the blockade of the ERK pathway showed among cell types and to on the activation state of ERKs (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar). The growth of tumor cells with constitutively high of ERK activation tumor was suppressed by the growth of tumor cells with of ERK activation tumor was suppressed by the MEK inhibitors 1 The different of PD98059 on the anchorage-independent growth of and tumor cells shown in 1 B. These results that the for the ERK pathway in proliferation among human tumor tumor cells on the activation of the ERK pathway for proliferation, proliferation of tumor cells to on the ERK pathway (7Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (611) Google Scholar). Consistent with its prominent antiproliferative effect, PD98059 induced G1 cell cycle arrest in tumor cells in which the ERK pathway is constitutively activated These results with the that activation of the ERK pathway is for cells to the G1 P. S. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). has recently been that the ERK pathway in the also in the from to in mammalian E. R.L. R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). In this of HT1080 cells treated with 50 for 24 h at complete of the cells in was by the cells with and then with a antibody and for Cells at were observed in RPMI-SE or A-172 cell for the ERK pathway in among tumor cells. progression the cell cycle is by several of in turn by CDK inhibitors C.J. J.M. 1999; PubMed Scopus Google Scholar). We that PD98059 induced up-regulation of p27Kip1 in RPMI-SE and HT1080 cells. PD98059 induce a in p27Kip1 in tumor cells in which constitutive activation of the ERK pathway is in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type induced up-regulation of p27Kip1 These results suggest that specific blockade of the ERK pathway marked up-regulation of p27Kip1 in tumor cells in which the pathway is constitutively activated. The of p27Kip1 in CDK and cell cycle progression is C.J. J.M. 1999; PubMed Scopus Google Scholar). p27Kip1 cell cycle arrest in to such as growth serum and p27Kip1 with and the of such as cyclin is for of cells of cyclin E-CDK2 is which from such as to for We that the up-regulation of p27Kip1 observed in RPMI-SE cells correlated with in p27Kip1 associated with cyclin E-CDK2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation of which would culminate in the G1 cell cycle arrest of these cells. results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27Kip1. of p27Kip1 is mediated by and We in p27Kip1 in RPMI-SE cells treated with PD98059 for up to h R. and M. by which p27Kip1 a the that cyclin E-CDK2 p27Kip1 and its by the pathway C.J. J.M. 1999; PubMed Scopus Google Scholar, R. M. M. J. B. 1997; PubMed Scopus Google Scholar), that the of p27Kip1 induced by the ERK pathway is a of of p27Kip1 at the protein PD98059 treatment induced a in the of cells with and in tumor cells in which the ERK pathway is activated PD98059 induced the of in HT1080 cells or in RPMI-SE cells. was observed the cells were with 50 μm a inhibitor Hoshino and M. PD98059 treatment induced a in the in these tumor which was as a These results suggest that specific blockade of the ERK pathway by PD98059 in HT1080 and RPMI-SE cells. In this have that of p27Kip1 to in cancer cells Y. M. P. Cancer Res. 1997; Google Scholar, M. Y. Oncogene. 1997; PubMed Scopus Google Scholar). the of in cells a at in in the of in these tumor cells. blockade of the ERK pathway would cause in HT1080 and RPMI-SE which culminate in the of these tumor cells to on the ERK pathway for growth, and for A. A. Science. 1999; PubMed Scopus Google Scholar). The for the of and of up-regulation of p27Kip1 specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated remains to be of is a for human T. Science. 1998; PubMed Scopus Google Scholar). we have shown that specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated. PD98059 and U0126 the proliferation of human to a degree in the of with after days. proliferation after of the inhibitors results suggest that the ERK pathway is a in a of tumor cells in which the pathway is constitutively activated. is by a showing that a MEK the growth of human tumor in D.T. R. Van K. A. H. Bridges A. S. Saltiel A.R. 1999; PubMed Scopus Google Scholar). In we have in this that specific blockade of the ERK pathway completely the growth of tumor cells in which the pathway is constitutively and we that this prominent growth inhibition is mediated by the up-regulation of p27Kip1. We and Van for for the of p27Kip1 and for of the
Hoshino et al. (Mon,) studied this question.