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Cyclin D Expression Is Controlled Post-transcriptionally via a Phosphatidylinositol 3-Kinase/Akt-dependent Pathway*

Open AccessPublished:November 06, 1998DOI:https://doi.org/10.1074/jbc.273.45.29864
      Cyclin D expression is regulated by growth factors and is necessary for the induction of mitogenesis. Herbimycin A, a drug that binds to Hsp90, induces the destruction of tyrosine kinases and causes the down-regulation of cyclin D and an Rb-dependent growth arrest in the G1phase of the cell cycle. We find that the induction of D-cyclin expression by serum and its repression by herbimycin A are regulated at the level of mRNA translation. Induction of cyclin D by serum occurs prior to the induction of its mRNA and does not require transcription. Herbimycin A repression is characterized by a decrease in the synthetic rate of D-cyclins prior to changes in mRNA expression and in the absence of changes in the half-life of the protein. This effect on D-cyclin translation is mediated via a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. PI 3-kinase inhibitors such as wortmannin and LY294002, and rapamycin, an inhibitor of FRAP/TOR, cause a decline in the level of D-cyclins, whereas inhibitors of mitogen-activated protein kinase kinase and farnesyltransferase do not. Cells expressing the activated, myristoylated form of Akt kinase, a target of PI 3-kinase, are refractory to the effects of herbimycin A or serum starvation on D-cyclin expression. These data suggest that serum induction of cyclin D expression results from enhanced translation of its mRNA and that this results from activation of a pathway that is dependent upon PI 3-kinase and Akt kinase.
      Cdk
      cyclin-dependent kinase
      PI 3-kinase
      phosphatidylinositol 3-kinase
      DMEM
      Dulbecco's modified Eagle's medium
      MAP
      mitogen-activated protein
      MEK
      MAP kinase kinase
      bp
      base pair(s)
      EGF
      epidermal growth factor
      HA
      hemagglutinin
      PAGe
      polyacrylamide gel electrophoresis.
      Growth factors elicit their biological effects by activating a complex network of receptors and signaling pathways. Activation of transmembrane tyrosine kinases by serum or polypeptide growth factors results in the transit of cells through the G1 phase of the cell cycle into S-phase. Several lines of evidence suggest that the D-type cyclins and their associated kinases (Cdks)1 are among the targets of these growth signals (
      • Sherr C.J.
      ). The D-type cyclins, D1, D2 and D3, are closely related proteins whose expression is induced by mitogens and growth factors (
      • Lanahan A.
      • Williams J.B.
      • Snaders L.K.
      • Nathans D.
      ,
      • Matsushime H.
      • Roussel M.F.
      • Ashmun R.A.
      • Sherr C.J.
      ,
      • Musgrove E.A.
      • Hamilton J.A.
      • Lee C.S.L.
      • Seeney K.L.E.
      • Watts C.K.W.
      • Sutherland R.L.
      ,
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ,
      • Altucci L.
      • Addeo R.
      • Cicatiello L.
      • Dauvois S.
      • Parker M.G.
      • Truss M.
      • Beato M.
      • Sica V.
      • Bresciani F.
      • Weisz A.
      ) and down-regulated by growth factor deprivation or by antimitogens (
      • Watts C.K.W.
      • Sweeney K.J.E.
      • Walters A.
      • Musgrove E.A.
      • Sutherland R.L.
      ,
      • Miyatake S.
      • Nakano H.
      • Park S.Y.
      • Yamazaki T.
      • Tadase K.
      • Matsushime H.
      • Kato A.
      • Saito T.
      ). The D-type cyclins associate with cyclin-dependent protein kinase Cdk4 or Cdk6 to form an active complex that phosphorylates and inactivates the retinoblastoma protein, pRb (
      • Matakeyama M.
      • Brill J.A.
      • Fink G.R.
      • Weinberg R.A.
      ,
      • Resnitzky D.
      • Reed S.I.
      ). Inhibition of cyclin D1 expression either by antisense methodology or antibody microinjection lengthens the duration of the G1 phase and causes a reduction in proliferation (
      • Liu J.
      • Chao J.
      • Jiang M.
      • Ng S.
      • Yen J.J.
      • Yang-Yen H.
      ,
      • Filmus J.
      • Robels A.L.
      • Shi W.
      • Wong M.J.
      • Colombo L.L.
      • Conti C.J.
      ). Aberrant overexpression of D-type cyclins resulting from upstream growth factor receptor activation, gene amplification or rearrangement, or an increase in mRNA stability seems to be a common feature of a number of human cancers and may reduce the cell's dependence on physiologic growth stimuli (
      • Buckley M.F.
      • Sweeney K.J.E.
      • Hamilton J.A.
      • Sini R.L.
      • Manning D.L.
      • Nicholson R.J.
      • deFrazio A.
      • Watts C.K.W.
      • Musgrove E.A.
      • Sutherland R.L.
      ,
      • Jiang W.
      • Kahn S.M.
      • Zhou P.
      • Zhang Y.
      • Cacace A.M.
      • Infante A.S.
      • Doi S.
      • Santella R.M.
      • Weinstein I.B.
      ,
      • Bartkova J.
      • Lukas J.
      • Strauss M.
      • Bartek J.
      ,
      • Lebwohl D.E.
      • Muise-Helmericks R.C.
      • Sepp-Lorenzino L.
      • Serve S.
      • Timaul M.
      • Bol R.
      • Borgen P.
      • Rosen N.
      ).
      Changes in cyclin D expression integrate the proliferative effects of an array of extracellular factors, including cytokines, polypeptide growth factors, and steroid hormones (
      • Lanahan A.
      • Williams J.B.
      • Snaders L.K.
      • Nathans D.
      ,
      • Matsushime H.
      • Roussel M.F.
      • Ashmun R.A.
      • Sherr C.J.
      ,
      • Musgrove E.A.
      • Hamilton J.A.
      • Lee C.S.L.
      • Seeney K.L.E.
      • Watts C.K.W.
      • Sutherland R.L.
      ,
      • Watts C.K.W.
      • Sweeney K.J.E.
      • Walters A.
      • Musgrove E.A.
      • Sutherland R.L.
      ). Cellular stress results in the loss of cyclin D1 expression, with a concomitant arrest in the G1 phase of the cell cycle (
      • Miyatake S.
      • Nakano H.
      • Park S.Y.
      • Yamazaki T.
      • Tadase K.
      • Matsushime H.
      • Kato A.
      • Saito T.
      ,

      Tomida, A., Suzuki, H., Kim, H., and Tsuruo, T. (1997)Oncogene 2699–2705

      ). The networks of pathways responsible for the transduction of these signals are complex and not completely understood. There is some evidence suggesting that a Ras- and MAP kinase-dependent signaling pathway is involved. Expression of activated Ras is associated with the increased expression of cyclin D1 in both epithelial cells (
      • Filmus J.
      • Robels A.L.
      • Shi W.
      • Wong M.J.
      • Colombo L.L.
      • Conti C.J.
      ) and fibroblasts (
      • Liu J.
      • Chao J.
      • Jiang M.
      • Ng S.
      • Yen J.J.
      • Yang-Yen H.
      ). Moreover, in the absence of growth factors, activation of the Raf1 → MEK → MAP kinase pathway has been shown to be sufficient to induce cyclin D1 transcription (
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ). Herbimycin A is a natural product that binds to a specific site in Hsp90 and causes the degradation of transmembrane tyrosine protein kinases, Raf1, and steroid hormone receptors (
      • Sepp-Lorenzino L.
      • Ma Z.
      • Lebwohl D.E.
      • Vinitsky A.
      • Rosen N.
      ,
      • Stebbins C.E.
      • Russo A.A.
      • Schneider C.
      • Rosen N.
      • Hartl F.U.
      • Pavletich N.P.
      ,
      • Xu Y.
      • Lindquist S.
      ,
      • Schulte T.W.
      • Blagosklonny M.V.
      • Ingui C.
      • Neckers L.
      ,
      • Stancato L.F.
      • Chow Y.H.
      • Hutchison K.A.
      • Perdew G.H.
      • Jove R.
      • Pratt W.B.
      ,
      • Schneider C.
      • Sepp-Lorenzino L.
      • Nimmesgern E.
      • Ouerfelli O.
      • Danishefsky S.
      • Rosen N.
      • Hartl F.-U.
      ). We found that treatment of tumor cells with this drug causes a decrease in the expression of D-type cyclins and an Rb-dependent G1block.
      N. Rosen, manuscript in preparation.
      2N. Rosen, manuscript in preparation.
      We report here that the reduction in the level of D-type cyclins induced by herbimycin A is due to inhibition of translation of cyclin D mRNAs. Furthermore, the increase in the level of D-type cyclins in cells treated with serum is due to an increase in the translation of their mRNAs. These effects are due to the regulation of a PI 3-kinase/Akt kinase-dependent, Raf1- and MAP kinase-independent pathway. This pathway is activated by serum and is blocked by the drug herbimycin A.

      DISCUSSION

      The signals induced by extracellular growth factors regulate levels of expression of D-cyclins (
      • Lanahan A.
      • Williams J.B.
      • Snaders L.K.
      • Nathans D.
      ,
      • Matsushime H.
      • Roussel M.F.
      • Ashmun R.A.
      • Sherr C.J.
      ,
      • Musgrove E.A.
      • Hamilton J.A.
      • Lee C.S.L.
      • Seeney K.L.E.
      • Watts C.K.W.
      • Sutherland R.L.
      ,
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ,
      • Altucci L.
      • Addeo R.
      • Cicatiello L.
      • Dauvois S.
      • Parker M.G.
      • Truss M.
      • Beato M.
      • Sica V.
      • Bresciani F.
      • Weisz A.
      ). We have shown that the ansamycin antibiotic herbimycin A induces a specific Rb-dependent G1 block, which is characterized by an early decline in the levels of D-cyclins in epithelial tumor cells. We sought to determine whether this decline is due to a direct effect of ansamycins on the half-life of D-cyclin proteins or to the degradation of a protein that transduces an upstream signal necessary for D-cyclin expression. We show that down-regulation of cyclin D1 and D3 proteins is neither secondary to changes in the levels of the mRNAs that encode the D cyclins nor to an increase in the rate of D-cyclin turnover (Figs. 1 and 3). We conclude that herbimycin A inhibits the translation of D-cyclin mRNA and that this drug may be affecting a member of an upstream signaling pathway.
      Ansamycin antibiotics, which include herbimycin A and geldanamycin, were discovered on the basis of their ability to revert the malignant phenotype of v-src-transformed cells (
      • Uehara Y.
      • Murakami Y.
      • Sugimoto S.
      • Mizuno S.
      ,
      • Uehara Y.
      • Murakami Y.
      • Mizuno S.
      • Kawai S.
      ). The direct target of these drugs seems to be the chaperone Hsp90. This chaperone contains a conserved, deep pocket that binds tightly to ansamycins (
      • Stebbins C.E.
      • Russo A.A.
      • Schneider C.
      • Rosen N.
      • Hartl F.U.
      • Pavletich N.P.
      ). Occupancy of the pocket by the drug inhibits Hsp90-mediated refolding of proteins but not their translation or initial folding. Apparently, this phenomenon is a consequence of inhibition of the ATP-dependent release of the refolded protein from Hsp90 (
      • Schneider C.
      • Sepp-Lorenzino L.
      • Nimmesgern E.
      • Ouerfelli O.
      • Danishefsky S.
      • Rosen N.
      • Hartl F.-U.
      ). The stabilized complex is then degraded. However, ansamycins induce the degradation of only a selected subset of cellular proteins. These include steroid receptors, the Raf1 kinase, v-Src, and certain transmembrane tyrosine kinases, notably members of the HER and IGF receptor families (
      • Sepp-Lorenzino L.
      • Ma Z.
      • Lebwohl D.E.
      • Vinitsky A.
      • Rosen N.
      ,
      • Xu Y.
      • Lindquist S.
      ,
      • Schulte T.W.
      • Blagosklonny M.V.
      • Ingui C.
      • Neckers L.
      ,
      • Stancato L.F.
      • Chow Y.H.
      • Hutchison K.A.
      • Perdew G.H.
      • Jove R.
      • Pratt W.B.
      ).
      Ansamycins could affect D-cyclin translation via a direct effect on the translational machinery or by interrupting an upstream regulatory pathway. In this regard, we showed that serum induction of D-cyclin protein expression precedes any effect on D-cyclin mRNA and, in addition, is actinomycin D-resistant (Figs. 4 and 5). Thus, it seems that serum activates and herbimycin A suppresses a pathway(s) required for D-cyclin translation. Activation of receptor tyrosine kinases has been shown to up-regulate D-cyclin expression, and this effect is thought to involve engagement of the Ras → Raf1 → MAP kinase cascade (
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ). As both receptor tyrosine kinases and Raf1 kinase are direct targets of herbimycin A action, it seemed likely that degradation of these targets was responsible for the action of the herbimycin A. However, whereas PI 3-kinase inhibitors and rapamycin were found to decrease D-cyclin expression, MEK inhibitors do not (Fig. 6). Furthermore, neither reversion of Ha-ras-transformed cells by farnesyltransferase inhibitors nor destruction of Raf1 by herbimycin A in cells expressing constitutively active Akt kinase is associated with a decrease in D-cyclin levels. These data suggest that, in these cells, D-cyclin expression is under the control of a PI 3-kinase-dependent pathway and is regulated independently of Raf1 and MAP kinase.
      To confirm these results, we tested the effects of herbimycin A in cells in which an activated form of the Akt kinase is either induced or expressed constitutively. Akt kinase is a serine kinase that lies downstream of PI 3-kinase and is activated by phosphatidylinositol 3′-phosphates (
      • Franke T.F.
      • Yang S.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Datta K.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Kippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). Herbimycin A does not affect D-cyclins but still causes Raf1 degradation in cells expressing activated Akt kinase (Fig. 7). These results confirm that cyclin D translation is regulated by a PI 3-kinase/Akt-dependent pathway. Serum and growth factors stimulate this pathway, in part by engaging receptor tyrosine kinases that may activate PI 3-kinase via Ras-dependent and -independent mechanisms (
      • Franke T.F.
      • Yang S.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaiesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Klinghoffer R.A.
      • Duckworth B.
      • Valius M.
      • Cantley L.
      • Kazlauskas A.
      ). Herbimycin A does not affect PI 3-kinase levels but may down-regulate this pathway in at least two ways. First, it causes the degradation of several receptor tyrosine kinases (
      • Sepp-Lorenzino L.
      • Ma Z.
      • Lebwohl D.E.
      • Vinitsky A.
      • Rosen N.
      ). In addition, we have observed that the expression of Akt is reduced in MCF7 cells treated with herbimycin A (Fig. 7). Whether this is a direct effect of herbimycin A on Akt or due to down-regulation of upstream signals is under investigation. These data suggest that D-cyclin translation is regulated by serum and ansamycins, in opposite directions, via a PI 3-kinase/Akt kinase-dependent pathway.
      Others have reported that growth factor activation of receptor tyrosine kinases leads to increased transcription of D-cyclin genes and decreased turnover of D-cyclin proteins (
      • Matsushime H.
      • Roussel M.F.
      • Ashmun R.A.
      • Sherr C.J.
      ,
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ,
      • Altucci L.
      • Addeo R.
      • Cicatiello L.
      • Dauvois S.
      • Parker M.G.
      • Truss M.
      • Beato M.
      • Sica V.
      • Bresciani F.
      • Weisz A.
      ,
      • Daksis J.I.
      • Lu R.Y.
      • Facchini L.M.
      • Marhin W.W.
      • Penn L.J.Z.
      ,
      • Diehl J.A.
      • Zindy F.
      • Sherr C.J.
      ). These data are not inconsistent with the observations reported here, as they were obtained in different systems. We also observed changes in D-cyclin mRNA levels after exposure of cells to herbimycin A, but these changes occur after the changes in protein expression. Others have suggested a role for the post-transcriptional control of cyclin D expression. Overexpression of cyclin D1 mRNA, ectopically or by amplification, does not necessarily lead to an increased protein expression (
      • Buckley M.F.
      • Sweeney K.J.E.
      • Hamilton J.A.
      • Sini R.L.
      • Manning D.L.
      • Nicholson R.J.
      • deFrazio A.
      • Watts C.K.W.
      • Musgrove E.A.
      • Sutherland R.L.
      ,
      • Tam S.W.
      • Theodoras A.M.
      • Shay J.W.
      • Draetta G.F.
      • Pagano M.
      ). In MCF10A cells inhibition of EGF signaling, using a blocking antibody against the receptor, leads to the down-regulation of cyclin D1. Forced expression of cyclin D in these cells does not abrogate this effect, excluding a promoter effect.
      J. Chow and J. Mendelsohn, personal communication.
      Additionally, Zhuet al. (
      • Zhu X.
      • Ohtsubo M.
      • Bohmer R.M.
      • Roberts J.M.
      • Assoian R.K.
      ) have shown that adherence is required for cyclin D1 expression in fibroblasts and that a component of this effect is translational. In the systems we have employed, neither herbimycin A nor growth factor-induced changes in D-cyclin expression are associated with obvious changes in cellular adherence.
      The Ras → MAP kinase pathway has been shown in several systems to be responsible for the induction of D-cyclin transcription (
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ,
      • Liu J.
      • Chao J.
      • Jiang M.
      • Ng S.
      • Yen J.J.
      • Yang-Yen H.
      ,
      • Filmus J.
      • Robels A.L.
      • Shi W.
      • Wong M.J.
      • Colombo L.L.
      • Conti C.J.
      ) and also for assembly of the active cyclin D-Cdk4 protein kinase complex (
      • Cheng M.
      • Sexl V.
      • Sherr C.J.
      • Roussel M.F.
      ). In the epithelial cell lines we examined, inhibition of Ras processing, MEK kinase activity, or Raf1 expression did not affect D-cyclin levels. In these systems, the Raf1 → MEK → MAP kinase pathway is not required for cyclin D-expression (Fig. 6). Whether this is generally true in epithelial cells or reflects activation of an alternative pathway in these carcinoma cells is unknown. However, a potential link between cyclin D expression and PI 3-kinase has been demonstrated in epithelial cells, as inhibition of PI 3-kinase by LY294002 or by inostamycin, a phosphatidylinositol synthesis inhibitor, leads to a decrease in cyclin D levels (
      • Alblas J.
      • Slager-Davidov R.
      • Steenbergh P.H.
      • Sussenbach J.S.
      • van der Burg B.
      ,
      • Deguchi A.
      • Imoto M.
      • Umezawa K.
      ,
      • Dufourny B.
      • Alblas J.
      • van Teeffelen H.A.A.M.
      • van Schaik F.M.A.
      • van der Burg B.
      • Steenbergh P.H.
      • Sussenbach J.S.
      ). We show a direct link between PI 3-kinase/Akt kinase activation and cyclin D expression, with a combination of inhibitors and by expression of an activated form of Akt (Figs. 6 and 7). The role of Ras is less clear. Farnesyltransferase inhibitors efficiently prevent the processing of Ha-Ras but not N- or Ki-Ras. Even so, reversion of Ha-ras-transformed cells by the drug is not associated with repression of D-cyclin expression.
      The mechanism whereby Akt stimulates D-cyclin translation is not completely understood. It could directly regulate the translational apparatus, resulting in an increase in the efficiency of translation of D-cyclin message, or cause changes in the compartmentalization of D-cyclin mRNA. Mitogenic stimulation is associated with a generalized 2–3-fold increase in translation, while a group of transcripts are translated at much higher rates. The explanation of how Akt affects D-cyclin translation is likely to be more complex since G1 progression is also associated with decreased translation of certain inhibitors such as p27kip1 (
      • Hengst L.
      • Reed S.I.
      ). The PI 3-kinase/Akt kinase pathway has been implicated in the growth factor-dependent phosphorylation of a number of key regulators of translation such as p70S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ) and PHAS-1 (4EBP-1) (
      • Mendez R.
      • Myers M.G.
      • White M.F.
      • Rhoads R.E.
      ,
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence Jr., J.C.
      • Roth R.A.
      ), an inhibitor of the mRNA cap binding protein eIF4E (
      • Pause A.
      • Belsham G.J.
      • Gingras A.
      • Donze O.
      • Lin T.
      • Lawrence J.C.
      • Sonenberg N.
      ). Both eIF4E and p70S6 kinase are activities that correlate with the increase in protein synthesis caused by growth factors such as insulin (
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ,
      • Mendez R.
      • Myers M.G.
      • White M.F.
      • Rhoads R.E.
      ,
      • Pause A.
      • Belsham G.J.
      • Gingras A.
      • Donze O.
      • Lin T.
      • Lawrence J.C.
      • Sonenberg N.
      ). Phosphorylation of PHAS-1 and p70S6 kinase are also rapamycin sensitive, implicating an additional need for the TOR/FRAP pathway in the activation of these two regulators of translation (
      • Beretta L.
      • Gingras A.
      • Svitkin Y.V.
      • Hall M.N.
      • Sonenberg N.
      ,
      • Phillip A.
      • Schneider A.
      • Vasrik I.
      • Finke K.
      • Xiong Y.
      • Beach D.
      • Alitalo K.
      • Eilers M.
      ,
      • Han E.
      • Sgambato A.
      • Jiang W.
      • Zhang Y.
      • Santella R.M.
      • Doki Y.
      • Cacace A.M.
      • Schieren I.
      • Weinstein I.B.
      ). Importantly, forced overexpression of eIF4E has been shown to cause an increase in cyclin D1 levels by increasing the nucleocytoplasmic export of cyclin D1 mRNA (
      • Rosenwald I.B.
      • Lazaris-Karatzas A.
      • Sonenberg N.
      • Schmidt E.V.
      ,
      • Rousseau D.
      • Kaspar R.
      • Rosenxald I.
      • Gehrke L.
      • Sonenberg N.
      ).
      Akt kinase has been shown to be a potent regulator of cellular proliferation. v-akt encodes a mutant activated kinase and is an oncogene (
      • Bellacosa A.
      • Franke T.F.
      • Gonzalez-Portal M.E.
      • Datta K.
      • Taguchi T.
      • Gardener J.
      • Cheng J.Q.
      • Testa J.R.
      • Tsichlis P.N.
      ). Expression of activated Akt rescues G1arrest, stimulating cell cycle progression in the absence of growth factors, in part by affecting the expression of c-Myc and Bcl-2 (
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ). It has also been reported that Akt activation leads to Rb phosphorylation (
      • Brennan P.
      • Babbage J.W.
      • Boudewijin M.T.
      • Groner B.
      • Reif K.
      • Cantrell D.A.
      ). These results, together with the data presented here, suggest a crucial connection between PI 3-kinase/Akt kinase and the cell cycle machinery. The herbimycin A-induced G1arrest is not, however, alleviated by expression of activated Akt (data not shown). This result is not unexpected since this drug, in addition to its effect on cyclin D, also affects other important regulators of cell growth. Inhibition of apoptosis by growth factors such as interleukin-2 and IGF1, is also mediated by Akt kinase (
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ) possibly through the phosphorylation of BAD, a member of the Bcl-2 family (
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ). Activation of PI 3-kinase and Akt by tyrosine kinases in tumor cells can therefore lead to both activation of cell cycle progression and a decrease in apoptosis via stimulation of PI 3-kinase and Akt kinase. Activation of PI 3-kinase by mutated Ras would also feed into this pathway. This is perhaps one explanation for the high frequency of mutational activation of Ras and tyrosine kinases, but not Raf1 and MAP kinase in human cancers. A variety of environmental stresses also lead to a specific down-regulation of D cyclins and G1 block (

      Tomida, A., Suzuki, H., Kim, H., and Tsuruo, T. (1997)Oncogene 2699–2705

      ). It is tempting to speculate that this results from engagement of an Hsp90-regulated pathway and is mimicked by herbimycin A binding to Hsp90. In many tumor cell lines, the ansamycins cause a striking reversion of transformation associated with G1 arrest and apoptosis. This phenomenon may reflect down-regulation of a key pathway for the maintenance of the malignant phenotype and could be the basis of a new set of therapeutic strategies.

      ACKNOWLEDGEMENT

      We thank Laura Sepp-Lorenzino for helpful advice and for the critical reading of the manuscript.

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