Mitogenic Signaling of Insulin-like Growth Factor I in MCF-7 Human Breast Cancer Cells Requires Phosphatidylinositol 3-Kinase and Is Independent of Mitogen-activated Protein Kinase

Abstract

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21WAF1/CIP1, an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells. Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21WAF1/CIP1, an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells. Insulin-like growth factor I (IGF-I) 1The abbreviations used are: IGF-I, insulin-like growth factor I; cdk, cyclin-dependent kinase; IRS-1, insulin receptor substrate 1; MAPK, mitogen-activated protein kinase; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; TPA, 12-O-tetradecanoylphorbol-13-acetate; CKIs, cyclin-dependent kinase inhibitors; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FCS, fetal calf serum; DTT, dithiothreitol; pRb, retinoblastoma protein; FACS, fluorescence-activated cell sorter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEK, MAPK/ERK kinase; ERK, extracellular response kinase. is a well established mitogen to many different cell types in vitro. The components of the pathway transducing the mitogenic stimulus of IGF-I to the cell cycle machinery have been identified in part. The first step of IGF-I signaling involves its binding to the type 1 IGF surface receptor, consisting of two extracellular α-subunits and two membrane-spanning β-subunits (1Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.-C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1526) Google Scholar). The activated receptor functions as a tyrosine kinase. IRS-1, a 185-kDa protein, is an important substrate of this kinase and becomes extensively phosphorylated (2Izumi T. White M.F. Kadowaki T. Takaku F. Akanuma Y. Kasuga M. J. Biol. Chem. 1987; 262: 1282-1287Abstract Full Text PDF PubMed Google Scholar, 3Shemer J. Adamo M. Wilson G.L. Heffez D. Zick Y. LeRoith D. J. Biol. Chem. 1987; 262: 15476-15482Abstract Full Text PDF PubMed Google Scholar). Activated IRS-1 then serves as a multisite docking protein by binding through its phosphorylated residues to numerous SH2 domain-containing proteins. These include the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) and the guanine-nucleotide exchange factor Grb2/SOS (4Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (386) Google Scholar, 5Backer J.M. Myers Jr., M.G. Sun X.J. Chin D.J. Shoelson S.E. Miralpeix M. White M.F. J. Biol. Chem. 1993; 268: 8204-8212Abstract Full Text PDF PubMed Google Scholar, 6Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J.K. Chawla A. Banerjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (232) Google Scholar, 7Skolnik E.Y. Batzer A. Li N. Lee C.-H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (505) Google Scholar, 8Skolnik E.Y. Lee C.-H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar). Putative downstream effectors of PI3-K are the ribosomal protein kinase p70S6K (9Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1002) Google Scholar), the Rho family polypeptides Rac and Cdc42 (10Chou M.M. Blenis J. Cell. 1996; 85: 573-583Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), and the serine/threonine protein kinase PKB/Akt (11Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar). Grb2/SOS binding subsequently results in activation of the proto-oncogenes p21ras and raf and the mitogen-activated protein kinase (MAPK)/extracellular response kinase (ERK) kinase MEK1, which activates the MAP kinases. Whether and how these pathways ultimately connect the surface receptor to the cell cycle components in the nucleus is still largely unknown. The effectiveness of IGF-I as a mitogen of the human breast cancer-derived MCF-7 cell line is well documented (12Karey K.P. Sirbashu D.A. Cancer Res. 1988; 48: 4083-4092PubMed Google Scholar, 13Huff K.K. Kaufman D. Gabbay K.H. Spencer E.M. Lippman M.E. Dickson R.B. Cancer Res. 1986; 46: 4613-4619PubMed Google Scholar, 14Van der Burg B. Rutteman G.R. Blankenstein M.A. de Laat S.W. van Zoelen E.J.J. J. Cell. Physiol. 1988; 134: 101-108Crossref PubMed Scopus (224) Google Scholar). These cells arrest in the G0/G1 phase of the cell cycle upon withdrawal of serum, and the proliferative process may be restored by the addition of IGF-I at 10 ng/ml to the medium. In the present study, we have addressed the question whether the activation of the PI3-K and/or MAPK pathways is an essential step in the mitogenic action of IGF-I in MCF-7 cells. The MAPK signal transduction cascade derives its name from findings that suggest a key role for MAPK (i.e. Erk-1 and -2) in the proliferative process. MAPK activity is elevated in response to many different growth factors. Furthermore, a large number of oncogenes share the activation of MAPK as a common pathway (15Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar). The tumor promoting phorbol ester TPA is a strong inducer of MAPK activity and has been shown to be a mitogen of many different cell lines in vitro (16Winkelstein A. Simon P.L. Myers 1986; 14: Google Scholar, L. U. Res. 1987; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). addition of TPA to cell MCF-7 cells, to of activation of MAPK is observed A. N. S. F. M.A. F. Cancer Res. 1987; Google Scholar, Y. J. 1994; PubMed Scopus Google Scholar, J. J. Cell. Physiol. 1992; PubMed Scopus Google Scholar). This observation the question whether MAPK activation a role in growth as well as in growth in MCF-7 cells. the PI3-K activation by many growth and addition of IGF-I to MCF-7 cells may be in the transduction of the mitogenic in mitogenic we have used specific by numerous well documented of PI3-K A. M.P. J. 1993; PubMed Scopus Google and C.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google and of the L. A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). for mitogenic IGF-I signaling and of growth TPA we have the of the cyclin hyperphosphorylation of the retinoblastoma protein pRb, of DNA synthesis, and effects on cell in of MCF-7 cells. of the has been shown to an essential step in of mitogenic signaling to the cell cycle This has been for MCF-7 cells, in which mitogenic from different of were shown to at and to cyclin activity to J. J. J. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). and are not in MCF-7 cells, and cyclin D1 is the essential cyclin M. Y. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). of cyclin D1 has been shown to cell cycle growth serum in MCF-7 cells R. der Burg B. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). the in cyclin D1 and the activity of the cyclin in with the cyclin to hyperphosphorylation of pRb and of the in through the cell cycle then becomes growth and DNA by cell on the have been shown to the synthesis of cyclin-dependent kinase These the activity of pRb from and arrest in the phase of the cell Addition of TPA to MCF-7 cells the of of the CKIs, p21WAF1/CIP1, which may for the growth observed with this Chen J. D.J. P.J. J. 1995; PubMed Scopus Google Scholar). we on the effects of the specific PI3-K and on the IGF-I signal and on the TPA signal to the signal transduction in these in MCF-7 cells. D1 and were from from and were from The cyclin D1 from D. is a the human of cyclin in the of the Y. T. B. D. Cell. Full Text PDF PubMed Scopus Google Scholar). The by R. is a human by of a cell two a and a on the of the human S. Lee D. Mol. 1995; PubMed Scopus Google Scholar). This of a for the of in used P. L. M. S.E. C. P. J.M. Res. 1985; PubMed Scopus Google Scholar). MCF-7 cells were in essential with fetal calf serum, and to a of of the effect of on MCF-7 cells der Burg B. Laat S.W. Zoelen E.J.J. Mol. Cell. PubMed Scopus Google Scholar), the cells were for in fetal calf serum, with bovine serum 10 and the cells in the medium. from from and and were from cells were with IGF-I the cells were with and then from the in for at the cells were in 10 and were by and protein of the were by the protein M.M. PubMed Scopus Google Scholar). of protein were in and to were with to for and blocked with for 1 with the first at in 10 were in and were to for at in the of protein were in at were blocked with in with in for at with in for 1 at were in cells were with IGF-I for with with for 1 stimulation the and of to the cells. were for were on a and to a The blocked with in with the in for 1 at and with the in for at were in were with IGF-I and then with and in 1 1 DTT, 10 10 1 of with of protein by with of protein for 1 at were then for at with of protein to were with with 1 DTT, and then in kinase 10 1 DTT, 10 1 with and to a of The for at which the with of DTT, The and and the on a The and to were in cells were IGF-I of the addition of IGF-I TPA, cells were with in and in by were in cells were with IGF-I the and cells were in phosphate-buffered and with for at were with and with for with and then with of for 1 to the at were in cells were IGF-I with with this cells were and then in and in were then in in cells were in of for at The cells were in in PBS, and then in of for at in and in in PBS, cells were in of in and with 10 and were with of in DNA then by from were with and in and to the and in of were in and by in to and to the by The were for at in BSA, and in the to which the specific were The were by were to at were in and in 10 were by with of protein to serum for at were then with of protein to for at were in in 10 and in 10 were then in of kinase 10 1 DTT, with of and of The for at which the with of DTT, The for and and the on a The and to in a the of MCF-7 cells through the phase of the cell cycle addition of IGF-I to the medium. MCF-7 cells were IGF-I and were at different of cyclin D1 and cyclin and of pRb were by Furthermore, the activity of and DNA that the of cyclin D1 protein is the addition of IGF-I and that at a The of protein not upon IGF-I addition not elevated at IGF-I are at and then of pRb first be at IGF-I addition and at least to of cyclin and of the cyclin with is observed from These are in with the results on DNA synthesis to the addition of IGF-I to the and a a in cell number is and the number of cells has The of the cells is on the IGF-I of 10 ng/ml results in a to that observed with fetal calf serum these that addition of IGF-I to the of quiescent MCF-7 cells results in through the of the cell cycle by of D1 and and by of pRb, by the in activity and DNA synthesis, by synthesis and of MCF-7 cells in response to IGF-I and in MCF-7 cells. cells were IGF-I TPA and were the to the 1 DNA as are of cells were and subsequently for to in in 10 ng/ml IGF-I the cell number the as are of cells were for in IGF-I in and 10 this cells were for to in 10 ng/ml TPA Cell were cells were IGF-I TPA a of IGF-I and TPA at 10 of and as are of The phorbol ester TPA activates MAPK and is a inhibitor of growth in MCF-7 cells. this in a growth in which cells were FCS, FCS, in with 10 ng/ml Addition of proliferation. TPA is this stimulation is Addition of IGF-I to quiescent MCF-7 cells results in an in DNA and stimulation in to cells. Addition of TPA this in DNA synthesis the signal transduction by which IGF-I and TPA inhibits cell cycle in MCF-7 cells, we the effects of of these on cell cycle in MCF-7 cells in the of specific of key in the PI3-K and MAPK signal transduction for cell cycle progression, we the of cyclin D1 TPA is to synthesis of this protein Chen J. D.J. P.J. J. 1995; PubMed Scopus Google Scholar), of the In we hyperphosphorylation of pRb and DNA MCF-7 cells, by serum were for 10 with the specific PI3-K inhibitor the p70S6K inhibitor PI3-K activation by IGF-I and the effectiveness of to PI3-K activation in these cells were an for PKB, an established downstream of PI3-K (11Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google of by these IGF-I TPA In of the ng/ml TPA to the cells, and in 10 ng/ml in the effects of TPA at these two have been The of 10 ng/ml is for the growth were and cell were by and The results are shown in is a of the of in the and is a The in cyclin D1 and protein is by addition of results were the PI3-K inhibitor not not is observed the cells are with TPA to cyclin D1 to an to that observed with This in cyclin D1 is by and by at the and protein and This that IGF-I and TPA the synthesis of cyclin D1 and protein in quiescent MCF-7 cells a signal transduction with IGF-I results in a in of the p21WAF1/CIP1, with quiescent cells. This is not by with The in protein is TPA causes a strong in and protein of and This is not by Addition of TPA to cells results in which is not by not This that of by TPA in quiescent and MCF-7 cells is independent of we the of the pathway in the of cyclin D1 and cells were for with IGF-I TPA with a of 1 with PD098059, a specific inhibitor of MAPK/ERK kinase Cell were used for and to the effects on cyclin D1 and and protein The results are shown in a and a of the of independent is shown in B. the IGF-I the in cyclin D1 and protein is by with PD098059, activation of MAPK is by the of used in these and J. R. P.H. van der Burg B. Scholar). In contrast, the strong of and protein upon addition of TPA The results were TPA to MCF-7 cells in not This that in quiescent and MCF-7 cells by TPA is on the MAPK of signal of MAPK on cyclin D1 and and of cyclin D1 and and protein in cells and cells. cells were for in the of 10 ng/ml IGF-I 10 ng/ml TPA with with for 1 by of the to a The of a of the of The in cells were at and the in were cyclin D1 the are of and and were MAPK activation by cells were for with IGF-I with for 1 with of the and phosphorylated of MAPK are The and the in cyclin D1 by IGF-I and The in by TPA is blocked by these are to pRb this cells were with IGF-I TPA with with ng/ml Cell were used for that addition of IGF-I to the cells, pRb has This is by with and by with for 1 not have an Addition of TPA to the cells not hyperphosphorylation of pRb, and with and not this with PD098059, to a of pRb hyperphosphorylation This may be by the of by of the activity of the cyclin The in the of with the of cyclin D1 observed with TPA may in the of a of the of we the effects of and on DNA Addition of IGF-I to MCF-7 cells results in a in DNA of the cells with this the cells are with the DNA synthesis of that observed The that to the of DNA synthesis cyclin D1 and pRb hyperphosphorylation may from in numerous by the MAPK signaling pathway for In the effects of and on cell cycle phase were by were and then in with 10 ng/ml IGF-I for with with for 10 and of the cells, the cells in for the is in the of the cell cycle The results are for cells for the not with IGF-I results in a of G0/G1 cells and a of the number of cells in and with this In contrast, the effect of IGF-I the of the of PI3-K by IGF-I in MCF-7 cells and the effectiveness of in this activation an for the downstream PI3-K (11Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar). cells were with IGF-I with with for 10 for 1 were from the and its kinase activity as a substrate and as The on a and by with IGF-I results in a activation of with quiescent cells. This activation is for at least with the activation of not activation by These that IGF-I activates the pathway in these cells and that this pathway is by In many have been on the effects of insulin and IGF-I and the signal transduction pathways to these The from these is that the PI3-K pathway is with the of insulin and IGF-I, as stimulation of by cells Vlahos C.J. Res. 1994; PubMed Scopus Google Scholar, K. A.J. K. K. Kasuga M. Res. 1995; PubMed Scopus Google Scholar), of P. M. Nature. 1995; PubMed Scopus Google Scholar, R. K. Y. K. S. M. M. J. Akanuma Y. S. Y. Kadowaki T. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and stimulation of protein synthesis D. C. T. J. 1996; PubMed Scopus Google Scholar). p21ras of that the signaling pathway MEK1, and MAPK is essential in the of the of insulin and IGF-I K. J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, B. J.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, T. M. T. Y. T. J.M. M. 1996; PubMed Scopus Google Scholar). results with MCF-7 cells to this In the present we that IGF-I cyclin D1 and protein synthesis in MCF-7 cells. The of the of cyclin are not by IGF-I, and the of the is Furthermore, IGF-I stimulation results in hyperphosphorylation of pRb, DNA and an in cell and not the in cyclin D1 protein to be the of the process by of the cells with LY294002, a specific inhibitor of PI3-K C.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), cyclin D1 Furthermore, inhibits pRb hyperphosphorylation and stimulation of DNA synthesis by of cyclin D1 observed a PI3-K inhibitor to A. M.P. J. 1993; PubMed Scopus Google Scholar), used not that this is in transducing the IGF-I stimulus to cyclin D1 but still of cyclin D1 with a specific inhibitor of p70S6K D.J. J.R. J. Science. 1992; PubMed Scopus Google Scholar). The effect of on pRb hyperphosphorylation to the effect of of hyperphosphorylation these we that activation of the PI3-K pathway is an essential step in proliferative signaling of IGF-I in MCF-7 cells. This is by the finding that the inhibitor not the cyclin D1 the hyperphosphorylation of pRb by IGF-I, that the is not in this process. The effectiveness of the inhibitor is by the that the and MAPK activation observed in MCF-7 cells is by at the used in the and MAPK activation by TPA in these cells is blocked by the inhibitor at this J. R. P.H. van der Burg B. Scholar). The finding that activation of MAPK is not required for mitogenic signaling of IGF-I in these cells is since the of the p21ras signaling pathway in mitogenic stimulation by IGF-I is well p21ras is the of the these K. J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, B. J.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, T. M. T. Y. T. J.M. M. 1996; PubMed Scopus Google have that this pathway is important in proliferation. in addition to activated by the IGF-I receptor, PI3-K has been shown to be a downstream of p21ras P. P.H. R. B. M.J. J. Nature. 1994; PubMed Scopus Google Scholar). we the of an IGF-I signaling the type 1 receptor, activation of and activation of PI3-K, and these are not C. J. N. D. A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google have that of cyclin D1 is by of MAPK and is by the of the in stimulation of cyclin D1 and cell and present results are in with these The and of MAPK have been in and results may be of essential in IGF-I signaling in on and MCF-7 breast cells on the growth factor of many different cell types but are not mitogenic in MCF-7 cell der Burg B. Rutteman G.R. Blankenstein M.A. de Laat S.W. van Zoelen E.J.J. J. Cell. Physiol. 1988; 134: 101-108Crossref PubMed Scopus (224) Google Scholar). these activation of MAPK upon addition to MCF-7 cells, to the of activation observed with IGF-I C. R. J. 1996; Scholar). The substrate of the insulin and type I IGF receptor, IRS-1, has been shown to be to PI3-K by binding to its p85 subunit J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun Miralpeix M. Hu P. Margolis B. E.Y. Schlessinger J. White M.F. EMBO J. 1992; PubMed Scopus Google Scholar). IRS-1 is not phosphorylated by the growth factor and growth factor This observation to the important role of PI3-K in the of by IGF-I in MCF-7 cells. on the signal transduction pathways of IGF-I in cell types be to this In of findings in MCF-7 cells, IGF-I has been to PI3-K in IGF-I a mitogenic response in these cells that MAPK activity IGF-I PI3-K activation not blocked by and the suggest a signaling of PI3-K with IRS-1 D.J. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). The PI3-K inhibitor not used in these Furthermore, to the mitogenic signal transduction pathway of IGF-I in of human suggest that PI3-K activity MAPK activity with Y. K. Kadowaki D. Y. Y. Akanuma Y. Kadowaki T. PubMed Scopus Google Scholar). have been M.J. Cooper M.E. Science. PubMed Scopus Google Scholar, A. P. E. C. Coffer P. J. Nature. PubMed Scopus Google Scholar, M. LeRoith D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in which the effect of IGF-I in cell types is shown to be by the PI3-K signaling In addition to the effects of the mitogen IGF-I, we have the effects of the growth inhibitor TPA on the of cyclin and and on hyperphosphorylation of The growth of TPA in MCF-7 cells have been A. N. S. F. M.A. F. Cancer Res. 1987; Google Scholar, M.J. Cancer Res. 1992; Google Scholar). and of by TPA have been documented in cell J. J. Cell. Physiol. 1992; PubMed Scopus Google Scholar, G.R. J. Cell. Physiol. 1994; PubMed Scopus Google and cells Y. J. 1994; PubMed Scopus Google Scholar, P. C. L. Cell. Res. 1995; PubMed Scopus Google Scholar). In contrast, TPA has been shown to the growth of cell (16Winkelstein A. Simon P.L. Myers 1986; 14: Google Scholar), cells L. U. Res. 1987; PubMed Scopus Google Scholar), and PubMed Scopus Google Scholar). that TPA cyclin D1 and protein in MCF-7 cells to an to the observed with IGF-I, TPA not Furthermore, TPA of and protein, and hyperphosphorylation of pRb is observed TPA addition to MCF-7 cells. TPA is to MCF-7 cells. this we the of cyclin TPA has growth in these cells of its strong stimulatory effect on This results in of the cyclin and pRb D1 by TPA is by to a by not stimulation of cyclin D1 these we that PI3-K is a for the of cyclin D1 by TPA, the is not have been on signaling the and effect of the PI3-K inhibitor on cyclin D1 by TPA that TPA is to PI3-K in the MCF-7 cell TPA of is to and This that by TPA is the pathway and that the PI3-K is not of by TPA has been in human in cell and in MCF-7 cells Chen J. D.J. P.J. J. 1995; PubMed Scopus Google Y. M. M. Res. 1995; PubMed Scopus Google Scholar, 1996; 12: Google Scholar). Furthermore, for cells has been shown that the pathway is in the activation of Y. M. Cancer Res. 1996; Google Scholar). that the is for MCF-7 cells. In to the In human MCF-7 breast cells, PI3-K is for the of the growth stimulatory IGF-I the is not in mitogenic of the growth signal of TPA, in of p21WAF1/CIP1, not PI3-K but involves the for the cyclin D1 for the of the and for on the for on the pRb and and de for with the kinase