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Insulin-like Growth Factor-I Extends in VitroReplicative Life Span of Skeletal Muscle Satellite Cells by Enhancing G1/S Cell Cycle Progression via the Activation of Phosphatidylinositol 3′-Kinase/Akt Signaling Pathway*

Open AccessPublished:November 17, 2000DOI:https://doi.org/10.1074/jbc.M005832200
      Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3′-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G1/S cell cycle progression via a down-regulation of p27 Kip1 . Adenovirally mediated ectopic overexpression of p27 Kip1 in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27 Kip1 in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.
      IGF-I
      insulin-like growth factor I
      PI 3-kinase
      phosphatidylinositol 3-kinase
      BrdUrd
      bromodeoxyuridine
      PD
      population doubling
      FACS
      fluorescence-activated cell sorter
      MAPK
      mitogen-activated protein kinase
      PAGE
      polyacrylamide gel electrophoresis
      WT
      wild type
      MEK
      mitogen-activated extracellular signal-regulate kinase
      Repair or growth of skeletal muscle requires satellite cells for new myonuclei because myonuclei are postmitotic (
      • Stockdale F.E.
      • Holtzer H.
      ). Satellite cells are a population of mononuclear myogenic precursor cells wedged between the basal lamina and muscle fiber sarcolemma (
      • Bischoff R.
      ). Satellite cells differ from immortalized myogenic cell lines in that, like other normal diploid cells, they have a limited capacity to divide, and after a finite number of cell divisions enter a state of irreversible growth arrest, termed replicative senescence (
      • Hayflick L.
      • Moorhead P.S.
      ). Indeed, proliferative potential of these cells has been shown to decrease in muscular dystrophies that are characterized by multiple rounds of regeneration (
      • Wright W.E.
      ), ultimately resulting in their cellular senescence. Therefore, when satellite cells exhaust their finite proliferative reserves, the effectiveness of regeneration, hypertrophy, and myoblast-mediated gene therapy for the reconstruction of muscle is constrained.
      Given the previously documented effects of insulin-like growth factor-I (IGF-I)1 to stimulate myoblast proliferation, myogenic differentiation, and myotube hypertrophy in cultured immortalized myogenic cell lines (
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ), and its ability to induce skeletal muscle hypertrophy in both young and old rodents (
      • Coleman M.E.
      • DeMayo F.
      • Yin K.C.
      • Lee H.M.
      • Geske R.
      • Montgomery C.
      • Schwartz R.J.
      ,
      • Barton-Davis E.R.
      • Shoturma D.I.
      • Musaro A.
      • Rosenthal N.
      • Sweeney H.L.
      ,
      • Adams G.R.
      • McCue S.A.
      ,
      • Chakravarthy M.V.
      • Davis B.S.
      • Booth F.W.
      ), we asked if IGF-I might enhance the proliferative potential of the resident satellite cells within animal skeletal muscles. Such understanding, which is presently lacking in non-immortalized primary satellite cells, could be used to counteract many intractable muscle-wasting conditions.
      A mouse lacking the igf-1 gene in a liver-specific manner demonstrated that IGF-Is action on growth and development is largely autocrine/paracrine (
      • Liu J-L.
      • Yakar S.
      • LeRoith D.
      ). Therefore, a transgenic mouse expressing an IGF-I transgene driven by a skeletal α-actin promoter was selected (
      • Coleman M.E.
      • DeMayo F.
      • Yin K.C.
      • Lee H.M.
      • Geske R.
      • Montgomery C.
      • Schwartz R.J.
      ) to allow the local overexpression of IGF-I in skeletal muscles in order to mimic its autocrine effect in the resident satellite cells. We hypothesized that satellite cells from the IGF-I transgenic (IGF-I Tg) skeletal muscles exposed to continuous high levels of IGF-I might possess a prolonged ability to proliferate in culture, reflecting an increased proliferative potential, relative to those cells isolated from their FVB wild type (WT) littermates.
      We next asked how the IGF-I Tg satellite cells were able to have an enhanced replicative life span, when the corresponding WT satellite cells were growth-arrested? Based upon studies in other cell types (
      • Rittling S.R.
      • Brooks K.M.
      • Cristofalo V.J.
      • Baserga R.
      ,
      • Dulic V.
      • Drullinger L.F.
      • Lees E.
      • Reed S.I.
      • Stein G.H.
      ), it seemed likely that this growth arrest in WT satellite cells might be at the G1/S boundary of the cell cycle, a nodal point suggested as one of the biochemical hallmark of replicative senescence. While senescent cells are refractory to mitogens and are terminally non-dividing even under the most optimal growth conditions, quiescent cells can be induced to initiate DNA synthesis by mitogenic stimulation or subcultivation (
      • Smith J.R.
      • Pereira-Smith O.M.
      ). Recent findings have demonstrated an accumulation of cdk-inhibitors such as p16 Ink4, p21 Cip1/Waf1, and p27 Kip1 (
      • Wong H.
      • Riabowol K.
      ,
      • Watanabe Y.
      • Lee S.W.
      • Detmar M.
      • Ajioke I.
      • Dvorak H.F.
      ) in various senescent cell types, further implicating a defect in G1/S transition in cellular senescence. Since IGFs act as progression factors stimulating progression from G1 to S phase of the cell cycle in other cell types (
      • Florini J.R.
      • Ewton D.Z.
      • Coolican S.A.
      ), we reasoned that IGF-I might extend thein vitro replicative life span of satellite cells by modulating cell cycle regulatory molecules at the G1/S boundary. IGF-I exerts its pleiotropic effects by binding to the type I IGF receptor (IGF-IR) (
      • Cohick W.S.
      • Clemmons D.R.
      ), which leads to the activation of both PI 3′-kinase and MAPK pathways (for review, see Ref.
      • Butler A.A.
      • Yakar S.
      • Gewolb I.H.
      • Karas M.
      • Okubu Y.
      • LeRoith D.
      ), in turn producing an increase in cellular proliferation in immortalized myogenic cell lines (
      • Coolican S.A.
      • Samuel D.S.
      • Ewton D.Z.
      • McWade F.J.
      • Florini J.R.
      ,
      • Milasincic D.J.
      • Calera M.R.
      • Farmer S.R.
      • Pilch P.F.
      ) and other tumor cell lines (
      • 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.
      ). Therefore, we hypothesized that the IGF-I induced enhancement of cell cycle progression via modulation of G1/S regulatory molecules in the IGF-I Tg satellite cells might be signaled by either one, or both, of these pathways.
      Here we report that IGF-I Tg satellite cells have an enhanced in vitro replicative life span, and continued to maintain cell cycle progression via activation of the PI 3′-kinase/Akt pathway, which in turn mediated the down-regulation of p27 Kip1 even in late-passage (the point at which WT cells were senesced). Adenovirally mediated ectopic overexpression of p27 Kip1 in growing late-passage IGF-I Tg satellite cells induced a G1 arrest, and mimicked the biochemical events seen in senesced late-passage WT satellite cells, indicating that p27 Kip1 has a critical role in the regulation of satellite cell senescence.

      DISCUSSION

      After 76 days in culture, satellite cells from 1-month-old IGF-I Tg mice continued to proliferate. In contrast, satellite cells from WT littermates at this time point had exhausted their proliferative reserves, were refractive to exogenous LR3-IGF-I, and demonstrated negligible amounts of sarcomeric myosin staining, suggesting that this loss in proliferative capacity was likely due to cellular senescence. Thus, IGF-I overexpression in satellite cells for a period of ∼4 months (∼1-month in the animal + 2.5-months in culture) delayed cellular senescence by extending their in vitro replicative life span. The cumulative number of in vitro population doublings attained by IGF-I Tg satellite cells was ∼5 additional rounds of divisions (i.e. on the average each satellite cell could replicate to 32 more cells), above the maximum attained by corresponding late-passage WT satellite cells derived from FVB littermates. This dramatic increase in proliferation potential seen in IGF-I Tg satellite cells was IGF-I-dependent since the IGF-I receptor antibody blocked this proliferation. These observations raised the critical question of how IGF-I Tg satellite cells were able to sustain their proliferative ability even in late-passages to ultimately result in the observed extension of their in vitro replicative life span.
      Analysis of the molecular profile of late-passage IGF-I Tg satellite cells demonstrated characteristics of cycling cells, whereas that of the corresponding WT cells was consistent with a G1/S arrest characteristic of senescent cells. Upon release from serum starvation, the IGF-I Tg cells demonstrated a time-dependent accumulation of S-phase cells (coincident with the induction of cyclin D1 and E proteins), phosphorylated pRb, and up-regulated cyclin A protein. In addition, IGF-I overexpression in late-passage satellite cells down-regulated p27 Kip1 protein levels, leading to an associated increase in the cyclin E/cdk2 kinase activity, relative to WT cells (Fig. 8). Cdk2 is one of the critical kinases required to phosphorylate pRb (
      • Akiyama T.
      • Ohuchi T.
      • Sumida S.
      • Matsumoto K.
      • Toyoshima K.
      ), which in turn has been suggested to be a rate-limiting step controlling cell cycle progression past the R-point to synthesize late-G1 genes necessary for S phase entry (
      • Pardee A.B.
      ,
      • Nevins J.R.
      ). Polyaket al. (
      • Polyak K.
      • Lee M-H.
      • Erdjument-Bromage H.
      • Koff A.
      • Roberts J.M.
      • Tempst P.
      • Massague J.
      ) suggested that p27 Kip1 could affect both the activation and kinase activity of cdk2, since the binding of p27 Kip1 may interfere with phosphorylation of cdk2s activating site (Thr-160). Others have suggested that p27 Kip1 could act as a threshold device controlling cdk2 kinase activity, increased cyclin D1/cdk4 accumulation sequesters free p27 Kip1, decreasing p27 Kip1 -cdk2 association, and consequently allowing for cdk2 activation (
      • Toyoshima H.
      • Hunter T.
      ). Consistent with this notion, we demonstrated an increased abundance of the cyclin D1·cdk4 complex in late-passage IGF-I Tg cells, which we speculate may also help sequester p27 Kip1, effectively decreasing the p27 Kip1 protein that is available to bind and inactivate cdk2.
      Figure thumbnail gr8
      Figure 8Potential model for IGF-I-mediated enhancement of cell cycle progression resulting in an extension of thein vitro replicative life span of satellite cells from IGF-I Tg skeletal muscle. Signaling events utilized by IGF-I to modulate critical cell cycle regulatory molecules at the G1/S boundary. Directionality of the arrows is relative to senesced late-passage WT satellite cells. Placement of the phosphorylation of pRb at the R point is based on the model proposed by Dulic et al. (
      • Dulic V.
      • Drullinger L.F.
      • Lees E.
      • Reed S.I.
      • Stein G.H.
      ) as well as the fact that pRb gets phosphorylated several hours before S phase (
      • Goodrich D.W.
      • Wang N.P.
      • Qian Y-W.
      • Lee E.Y-H.P.
      • Lee W-H.
      ). While the results presented in this study uniquely support the notion that p27 Kip1 is a downstream target of the PI 3′-kinase/Akt pathway in skeletal muscle satellite cells, it remains to be determined if it is directly downstream of Akt or if other molecules activated by PI 3′-kinase, independent of Akt directly modulate its down-regulation. Other processes known to be mediated by activated Akt, but not measured in this study are indicated in parentheses.
      Remarkably, adenoviral-mediated ectopic overexpression of p27 Kip1 in exponentially growing cultures of late-passage IGF-I Tg satellite cells down-regulated cdk2 kinase activity, hypophosphorylated pRb, decreased cyclin A protein, and in vitro cell proliferation, increased cell doubling time by ∼2.5-fold, and induced a G1 arrest, thereby mimicking the changes observed in senesced cultures of late-passage WT satellite cells. Previous studies have shown that p27 Kip1 overexpression (
      • Chen D.
      • Krasinski K.
      • Chen D.
      • Sylvester A.
      • Chen J.
      • Nisen P.D.
      • Andres V.
      ,
      • Schulze A.
      • Zerfass-Thome K.
      • Berges J.
      • Middendorp S.
      • Jansen-Durr P.
      • Henglein B.
      ) repressed transcription from the cyclin A promoter regions that contain an E2F-binding site required for cell cycle-regulated cyclin A gene expression, indicating that p27 Kip1 can regulate transcription of late G1 genes. Furthermore, recent findings of Busse et al. (
      • Busse D.
      • Doughty R.S.
      • Ramsey T.T.
      • Russell W.E.
      • Price J.O.
      • Flanagan W.M.
      • Shawver L.K.
      • Arteaga C.L.
      ) showed that antisense p27 Kip1 essentially reversed both p27 Kip1 up-regulation and the G1 arrest in A431 cells treated with the tyrosine kinase inhibitor AG-1478. The role of p27 Kip1 as a critical regulator of cellular proliferation is further illustrated by p27 Kip1 knockout mouse models, which exhibits gigantism, organomegaly, and enhanced spontaneous tumor formation (
      • Nakayama K.
      • Ishida N.
      • Shirane M.
      • Inomata A.
      • Inoue T.
      • Shishido N.
      • Horii I.
      • Loh D.Y.
      • Nakayama K.
      ,
      • Kiyokawa H.
      • Kineman R.D.
      • Manova-Todorova K.O.
      • Soares V.C.
      • Hoffman E.S.
      • Ono M.
      • Khanam D.
      • Hayday A.C.
      • Frohman L.A.
      • Koff A.
      ,
      • Fero M.L.
      • Rivkin M.
      • Tasch M.
      • Porter P.
      • Carow C.E.
      • Firpo E.
      • Polyak K.
      • Tsai L.H.
      • Broudy V.
      • Perlmutter R.M.
      • Kaushansky K.
      • Roberts J.M.
      ). Given that cdk2 kinase activity, phosphorylation of pRb, and up-regulation of cyclin A are critical for cell cycle progression (
      • Dulic V.
      • Drullinger L.F.
      • Lees E.
      • Reed S.I.
      • Stein G.H.
      ,
      • Smith J.R.
      • Pereira-Smith O.M.
      ), and the fact that all of these regulatory factors are inhibited by p27 Kip1 overexpression in growing late-passage IGF-I Tg cells, it suggests that p27 Kip1 is a critical mediator through which IGF-I delays G1/S arrest in these cells. These results also support the possibility that p27 Kip1 could be an initiator of a cascade of G1/S cell cycle events, to consequently regulate replicative senescence (Fig. 8).
      Interruption of PI 3′-kinase activity in IGF-I Tg cells by LY294002 mimicked the molecular events seen in senesced late-passage WT satellite cells, i.e. increased p27 Kip1, decreased pRb phosphorylation and cyclin A protein, and caused a G1/S cell cycle arrest. In contrast, blockade of the activated MAPK by PD098059 resulted in neither an appreciable accumulation of cells in G1, nor an increase in p27 Kip1 protein levels. These observations in IGF-I Tg satellite cells extend the previous findings of Collado et al. (
      • Collado M.
      • Medema R.H.
      • Garcia-Cao I.
      • Dubuisson M.L.L.
      • Barradas M.
      • Glassford J.
      • Rivas C.
      • Burgering B.M.T.
      • Serrano M.
      • Lam E.W-F.
      ), who showed that inhibition of PI 3′-kinase pathway induced a senescent-like arrest that was mediated by p27 Kip1 in mouse embryo fibroblasts. We therefore infer that PI 3′-kinase activation induced by IGF-I overexpression in late-passage IGF-I Tg satellite cells are necessary and sufficient for the down-regulation of p27 Kip1, and consequently for the enhanced G1 to S-phase cell cycle progression, resulting in the increased in vitro doubling potential of these cells (Fig. 8).
      Thus, it is probable that Akt itself or other molecules activated by the PI 3′-kinase signaling pathway is essential for G1 cell cycle progression in skeletal muscle satellite cells from IGF-I Tg mice. Indeed, a requirement of an intact PI 3′-kinase signaling pathway (independent of MAPK activity in many of these cells), for G1 to S cell cycle progression has previously been reported in other cell types (
      • 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.
      ,
      • Li D.
      • Sun H.
      ,
      • Busse D.
      • Doughty R.S.
      • Ramsey T.T.
      • Russell W.E.
      • Price J.O.
      • Flanagan W.M.
      • Shawver L.K.
      • Arteaga C.L.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Klippel A.
      • Escobedo M.A.
      • Wachowicz M.S.
      • Apell G.
      • Brown T.W.
      • Giedlin M.A.
      • Kavanaugh W.M.
      • Williams L.T.
      ,
      • Brennan P.
      • Babbage J.W.
      • Burgering B.M.T.
      • Groner B.
      • Reif K.
      • Cantrell D.A.
      ). Our results are also consistent with Milasincic et al. (
      • Milasincic D.J.
      • Calera M.R.
      • Farmer S.R.
      • Pilch P.F.
      ) who had previously shown that the mitogenic response of IGF-I was mediated via the activation of the PI 3′-kinase pathway, independent of MAPK in C2C12 murine cell line, but they had not examined cell cycle markers. However, our results contradict the report in rat L6A1 immortalized myoblasts, which had demonstrated that pharmacological inhibition of MAPK activation attenuated the proliferative response by LR3-IGF-I in these cells (
      • Coolican S.A.
      • Samuel D.S.
      • Ewton D.Z.
      • McWade F.J.
      • Florini J.R.
      ). Thus, it appears that the pluripotent effects of factors such as IGF-I and their consequent signaling may depend on the cell context in which its receptor is activated.
      Clearly, processes as complex as cellular proliferation and replicative senescence involve multiple mediators and cross-talk mechanisms. The activation of PI 3′-kinase/Akt to signal the down-regulation of p27 Kip1 is a crucial phenomenon that leads to an enhanced proliferative potential, resulting in extension of the in vitro replicative life span of IGF-I Tg satellite cells. However, this need not be the exclusive mechanism. IGF-I-evoked modulation of other regulatory pathways such as the inhibition of the pro-apoptotic factor BAD via phosphorylation by Akt/PKB to enhance survival (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ), up-regulation of telomerase activity (
      • Tu W.
      • Zhang D.K.
      • Cheung P.T.
      • Tsao S.W.
      • Lau Y.L.
      ), and/or changes in IGF-I-binding protein concentrations (
      • Goldstein S.
      • Moerman E.J.
      • Baxter R.C.
      ) are some of the mechanisms that need to be further explored.
      While previous studies have shown that the number of replications can be extended in other cell types by other means (
      • Watanabe Y.
      • Lee S.W.
      • Detmar M.
      • Ajioke I.
      • Dvorak H.F.
      ,
      • Smith J.R.
      • Pereira-Smith O.M.
      • Braunschweiger K.I.
      • Roberts T.W.
      • Whitney R.G.
      ,
      • Rheinwald J.G.
      • Green H.
      ,
      • Angello J.C.
      ,
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C.P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ), this is the first demonstration of extending the replicative life span of skeletal muscle stem cells with a growth factor. Given that satellite cells are absolutely required for postnatal muscle growth and repair (
      • Bischoff R.
      ), our data provide novel insights into some of the regulatory events which may serve as attractive tools for potential ex vivomanipulation of stem cell autografts to extend the replicative life span of old satellite cells for gene therapy in muscle wasting conditions.

      Acknowledgments

      We thank Dr. Jeanie McMillin for critically reading the manuscript and helpful suggestions, Louise Barnett for assistance with flow cytometry, Bilal Thair and Sandra Higam for assistance with cell culture. The mouse monoclonal antibodies D3 and MF20 developed by Danto and Fischman (
      • Danto S.I.
      • Fischman D.A.
      ) and Bader et al.(
      • Bader D.
      • Masaki T.
      • Fischman D.A.
      ), respectively, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences.

      REFERENCES

        • Stockdale F.E.
        • Holtzer H.
        Exp. Cell Res. 1961; 24: 508-520
        • Bischoff R.
        Engle A.G. Franzini-Armstrong C. 2nd Ed. Myology. 1. McGraw-Hill, New York1994: 97-118
        • Hayflick L.
        • Moorhead P.S.
        Exp. Cell Res. 1961; 25: 585-621
        • Wright W.E.
        Exp. Cell. Res. 1985; 157: 343-354
        • Florini J.R.
        • Ewton D.Z.
        • Coolican S.A.
        Endocr. Rev. 1996; 17: 481-517
        • Coleman M.E.
        • DeMayo F.
        • Yin K.C.
        • Lee H.M.
        • Geske R.
        • Montgomery C.
        • Schwartz R.J.
        J. Biol. Chem. 1995; 270: 12109-12116
        • Barton-Davis E.R.
        • Shoturma D.I.
        • Musaro A.
        • Rosenthal N.
        • Sweeney H.L.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15603-15607
        • Adams G.R.
        • McCue S.A.
        J. Appl. Physiol. 1998; 84: 1716-1722
        • Chakravarthy M.V.
        • Davis B.S.
        • Booth F.W.
        J. Appl. Physiol. 2000; 89: 1365-1379
        • Liu J-L.
        • Yakar S.
        • LeRoith D.
        Proc. Soc. Exp. Biol. Med. 2000; 223: 344-351
        • Rittling S.R.
        • Brooks K.M.
        • Cristofalo V.J.
        • Baserga R.
        Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3316-3320
        • Dulic V.
        • Drullinger L.F.
        • Lees E.
        • Reed S.I.
        • Stein G.H.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11034-11038
        • Smith J.R.
        • Pereira-Smith O.M.
        Science. 1996; 273: 63-67
        • Wong H.
        • Riabowol K.
        Exp. Gerontol. 1996; 31: 311-325
        • Watanabe Y.
        • Lee S.W.
        • Detmar M.
        • Ajioke I.
        • Dvorak H.F.
        Oncogene. 1997; 14: 2025-2032
        • Cohick W.S.
        • Clemmons D.R.
        Annu. Rev. Physiol. 1993; 55: 131-153
        • Butler A.A.
        • Yakar S.
        • Gewolb I.H.
        • Karas M.
        • Okubu Y.
        • LeRoith D.
        Comp. Biochem. Physiol. Part B. 1998; 121: 19-26
        • Coolican S.A.
        • Samuel D.S.
        • Ewton D.Z.
        • McWade F.J.
        • Florini J.R.
        J. Biol. Chem. 1997; 272: 6653-6662
        • Milasincic D.J.
        • Calera M.R.
        • Farmer S.R.
        • Pilch P.F.
        Mol. Cell. Biol. 1996; 16: 5964-5973
        • 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.
        J. Biol. Chem. 1997; 272: 31163-31171
        • Kaufman S.J.
        • Foster R.F.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9606-9610
        • Decary S.
        • Mouly V.
        • Hamida C.B.
        • Sautet A.
        • Barbet J.P.
        • Butler-Browne G.S.
        Hum. Gene Ther. 1997; 8: 1429-1438
        • Smith J.R.
        • Pereira-Smith O.M.
        • Braunschweiger K.I.
        • Roberts T.W.
        • Whitney R.G.
        Mech. Ageing Dev. 1980; 12: 355-365
        • Li D.
        • Sun H.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15406-15411
        • Busse D.
        • Doughty R.S.
        • Ramsey T.T.
        • Russell W.E.
        • Price J.O.
        • Flanagan W.M.
        • Shawver L.K.
        • Arteaga C.L.
        J. Biol. Chem. 2000; 275: 6987-6995
        • Chen J.
        • Willingham T.
        • Shuford M.
        • Nisen P.D.
        J. Clin. Invest. 1996; 97: 1983-1988
        • Chen D.
        • Krasinski K.
        • Chen D.
        • Sylvester A.
        • Chen J.
        • Nisen P.D.
        • Andres V.
        J. Clin. Invest. 1997; 99: 2334-2341
        • Criswell D.S.
        • Booth F.W.
        • DeMayo F.
        • Schwartz R.J.
        • Gordon S.E.
        • Fiorotto M.L.
        Am. J. Physiol. 1998; 275: E373-E379
        • Pardee A.B.
        Science. 1989; 246: 603-608
        • Goodrich D.W.
        • Wang N.P.
        • Qian Y-W.
        • Lee E.Y-H.P.
        • Lee W-H.
        Cell. 1991; 67: 293-302
        • Nevins J.R.
        Science. 1992; 258: 424-429
        • Ohtsubo M.
        • Roberts J.M.
        Science. 1993; 259: 1908-1912
        • Stein G.H.
        • Beeson M.
        • Gordon L.
        Science. 1990; 249: 666-669
        • Lees E.
        Curr. Opin. Cell Biol. 1995; 7: 773-780
        • Sherr C.J.
        • Roberts J.M.
        Genes Dev. 1995; 9: 1149-1163
        • Kandel E.S.
        • Hay N.
        Exp. Cell Res. 1999; 253: 210-229
        • Dudley D.T.
        • Pang L.
        • Decker S.J.
        • Bridges A.J.
        • Saltiel A.R.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689
        • Vlahos C.J.
        • Matter W.F.
        • Hui K.Y.
        • Brown R.F.
        J. Biol. Chem. 1994; 269: 5241-5248
        • Akiyama T.
        • Ohuchi T.
        • Sumida S.
        • Matsumoto K.
        • Toyoshima K.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7900-7904
        • Polyak K.
        • Lee M-H.
        • Erdjument-Bromage H.
        • Koff A.
        • Roberts J.M.
        • Tempst P.
        • Massague J.
        Cell. 1994; 78: 59-66
        • Toyoshima H.
        • Hunter T.
        Cell. 1994; 78: 67-74
        • Schulze A.
        • Zerfass-Thome K.
        • Berges J.
        • Middendorp S.
        • Jansen-Durr P.
        • Henglein B.
        Mol. Cell. Biol. 1996; 16: 4632-4638
        • Nakayama K.
        • Ishida N.
        • Shirane M.
        • Inomata A.
        • Inoue T.
        • Shishido N.
        • Horii I.
        • Loh D.Y.
        • Nakayama K.
        Cell. 1996; 85: 707-720
        • Kiyokawa H.
        • Kineman R.D.
        • Manova-Todorova K.O.
        • Soares V.C.
        • Hoffman E.S.
        • Ono M.
        • Khanam D.
        • Hayday A.C.
        • Frohman L.A.
        • Koff A.
        Cell. 1996; 85: 721-732
        • Fero M.L.
        • Rivkin M.
        • Tasch M.
        • Porter P.
        • Carow C.E.
        • Firpo E.
        • Polyak K.
        • Tsai L.H.
        • Broudy V.
        • Perlmutter R.M.
        • Kaushansky K.
        • Roberts J.M.
        Cell. 1996; 85: 733-744
        • Collado M.
        • Medema R.H.
        • Garcia-Cao I.
        • Dubuisson M.L.L.
        • Barradas M.
        • Glassford J.
        • Rivas C.
        • Burgering B.M.T.
        • Serrano M.
        • Lam E.W-F.
        J. Biol. Chem. 2000; 275: 21960-21968
        • Roche S.
        • Koegl M.
        • Courtneidge S.A.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9185-9189
        • Ahmed N.N.
        • Grimes H.L.
        • Bellacosa A.
        • Chan T.O.
        • Tsichlis P.N.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632
        • Klippel A.
        • Escobedo M.A.
        • Wachowicz M.S.
        • Apell G.
        • Brown T.W.
        • Giedlin M.A.
        • Kavanaugh W.M.
        • Williams L.T.
        Mol. Cell. Biol. 1998; 18: 5699-5711
        • Brennan P.
        • Babbage J.W.
        • Burgering B.M.T.
        • Groner B.
        • Reif K.
        • Cantrell D.A.
        Immunity. 1997; 7: 679-689
        • Datta S.R.
        • Dudek H.
        • Tao X.
        • Masters S.
        • Fu H.
        • Gotoh Y.
        • Greenberg M.E.
        Cell. 1997; 91: 231-241
        • Tu W.
        • Zhang D.K.
        • Cheung P.T.
        • Tsao S.W.
        • Lau Y.L.
        Br. J. Haematol. 1999; 104: 785-794
        • Goldstein S.
        • Moerman E.J.
        • Baxter R.C.
        J. Cell. Physiol. 1993; 156: 294-302
        • Rheinwald J.G.
        • Green H.
        Nature. 1977; 265: 421-424
        • Angello J.C.
        Mech. Ageing Dev. 1992; 62: 1-12
        • Bodnar A.G.
        • Ouellette M.
        • Frolkis M.
        • Holt S.E.
        • Chiu C.P.
        • Morin G.B.
        • Harley C.B.
        • Shay J.W.
        • Lichtsteiner S.
        • Wright W.E.
        Science. 1998; 279: 349-352
        • Danto S.I.
        • Fischman D.A.
        J. Cell Biol. 1984; 98: 2179-2191
        • Bader D.
        • Masaki T.
        • Fischman D.A.
        J. Cell Biol. 1982; 95: 763-770