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• W2116710692 abstract "Transforming growth factor-β1 (TGF-β1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-β1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-β1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-β1 induced apoptosis. The proapoptotic effects of TGF-β1 occur via a p53-independent pathway, since p53null FDCP-Mix demonstrated the same responses to TGF-β1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-xL expression. In FDCP-Mix cells, neither IL-3 nor TGF-β1 induced any change in Bcl-xL protein levels or the proapoptotic proteins Bad or Bax. However, TGF-β1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-β1-induced apoptosis but was incapable of inhibiting TGF-β1-mediated growth arrest. We conclude that TGF-β1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-xL. The significance of these results for stem cell survival in bone marrow are discussed. Transforming growth factor-β1 (TGF-β1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-β1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-β1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-β1 induced apoptosis. The proapoptotic effects of TGF-β1 occur via a p53-independent pathway, since p53null FDCP-Mix demonstrated the same responses to TGF-β1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-xL expression. In FDCP-Mix cells, neither IL-3 nor TGF-β1 induced any change in Bcl-xL protein levels or the proapoptotic proteins Bad or Bax. However, TGF-β1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-β1-induced apoptosis but was incapable of inhibiting TGF-β1-mediated growth arrest. We conclude that TGF-β1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-xL. The significance of these results for stem cell survival in bone marrow are discussed. transforming growth factor macrophage inflammatory protein interleukin long term repopulating cell Iscove's modified Dulbecco's medium horse serum Proliferation of the hematopoietic stem cell compartment is tightly regulated to ensure that appropriate numbers of stem cells are recruited into the cell cycle to meet demands for mature blood cell production. Despite a high turnover of mature blood cells, hematopoietic stem cells are normally quiescent. This is achieved, at least in part, through the action of hematopoietic growth inhibitors (1Broxmeyer H.E. Whetton A.D. Gordon J. Blood Cell Biochemistry. Plenum Press, New York1996: 121-150Google Scholar). Transforming growth factor-β1 (TGF-β1),1 the prototypic member of a large family of soluble growth and differentiation factors, has been shown to possess potent antiproliferative activity for many cell types including epithelial and hematopoietic cells (2Jetten A.M. Shirley J.E. Stoner G. Exp. Cell Res. 1986; 167: 539-549Crossref PubMed Scopus (127) Google Scholar, 3Kurokowa M. Lynch K. Podolsky D.K. Biochem. Cell Biol. Commun. 1987; 142: 775-782Google Scholar, 4Keller J.R. Mantel C. Sing G.K. Ellingsworth L.R. Ruscetti S.K. Ruscetti F.W. J. Exp. Med. 1988; 168: 737-750Crossref PubMed Scopus (174) Google Scholar, 5Bonnet D. Lemoine F.M. Najman A. Guigon M. Exp. Hematol. 1995; 23: 551-556PubMed Google Scholar, 6Sitnicka E. Ruscetti F.W. Priestley G.V. Wolf N.S. Bartelmez S.H. Blood. 1996; 88: 82-88Crossref PubMed Google Scholar). The growth-suppressive actions of TGF-β1 on hematopoietic cells appear to be specific for primitive hematopoietic cell populations, with more mature, lineage-restricted progenitors being resistant to these effects (4Keller J.R. Mantel C. Sing G.K. Ellingsworth L.R. Ruscetti S.K. Ruscetti F.W. J. Exp. Med. 1988; 168: 737-750Crossref PubMed Scopus (174) Google Scholar, 7Ottmann O.G. Pelus L.M. J. Immunol. 1988; 140: 2661-2665PubMed Google Scholar). Indeed, TGF-β1 has been reported to behave as a bidirectional regulator of hematopoietic cell growth, its effects being dependent on both differentiation status and the presence of other cytokines (8Jacobsen S.E. Keller J.R. Ruscetti F.W. Kondaiah P. Roberts A.B. Falk L.A. Blood. 1991; 78: 2239-2247Crossref PubMed Google Scholar, 9Keller J.R. Jacobsen S.E. Sill K.T. Ellingsworth L.R. Ruscetti F.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7190-7194Crossref PubMed Scopus (99) Google Scholar, 10Celada A. Maki R.A. J. Immunol. 1992; 148: 1102-1105PubMed Google Scholar, 11Keller J.R. Bartelmez S.H. Sitnicka E. Ruscetti F.W. Ortiz M. Gooya J.M. Jacobsen S.E. Blood. 1994; 84: 2175-2181Crossref PubMed Google Scholar). These bidirectional effects have also been observed in vivo in normal mice injected with TGF-β1 either alone or in combination with other growth factors such as granulocyte-macrophage colony-stimulating factor (12Goey H. Keller J.R. Back T. Longo D.L. Ruscetti F.W. Wiltrout R.H. J. Immunol. 1989; 143: 877-880PubMed Google Scholar, 13Migdalska A. Molineux G. Demuynck H. Evans G.S. Ruscetti F. Dexter T.M. Growth Factors. 1991; 4: 239-245Crossref PubMed Scopus (65) Google Scholar, 14Bursuker I. Neddermann K.M. Petty B.A. Schacter B. Spitalny G.L. Tepper M.A. Pasternak R.D. Exp. Hematol. 1992; 20: 431-435PubMed Google Scholar, 15Hestdal K. Jacobsen S.E. Ruscetti F.W. Longo D.L. Boone T.C. Keller J.R. Exp. Hematol. 1993; 21: 799-805PubMed Google Scholar). In contrast to other growth suppressor molecules such as macrophage inflammatory protein-1α (MIP-1α), the growth-inhibitory effects of TGF-β1 are apparent throughout the whole stem cell hierarchy including the very primitive long term repopulating cells (LTRCs) (6Sitnicka E. Ruscetti F.W. Priestley G.V. Wolf N.S. Bartelmez S.H. Blood. 1996; 88: 82-88Crossref PubMed Google Scholar, 11Keller J.R. Bartelmez S.H. Sitnicka E. Ruscetti F.W. Ortiz M. Gooya J.M. Jacobsen S.E. Blood. 1994; 84: 2175-2181Crossref PubMed Google Scholar, 16Soma T., Yu, J.M. Dunbar C.E. Blood. 1996; 87: 4561-4567Crossref PubMed Google Scholar). The demonstration that neutralizing antibodies to TGF-β1 but not MIP-1α could induce normally quiescent progenitors into the cell cycle in long term bone marrow cultures has suggested that autocrine/paracrine production of TGF-β1 is likely to be an important mechanism of maintaining stem cell quiescence in vivo (17Eaves C.J. Cashman J.D. Kay R.J. Dougherty G.J. Otsuka T. Gaboury L.A. Hogge D.E. Landsdorp P.M. Eaves A.C. Humphries R.K. Blood. 1991; 78: 110-117Crossref PubMed Google Scholar, 18Cashman J.D. Eaves C.J. Sarris A.H. Eaves A.C. Blood. 1998; 92: 2338-2344Crossref PubMed Google Scholar). The precise role of TGF-β1 in hematopoiesis has been further complicated by the demonstration that TGF-β1 can also induce apoptosis of both normal and leukemic progenitors (19Lotem J. Sachs L. Blood. 1992; 80: 1750-1757Crossref PubMed Google Scholar, 20Taetle R. Payne C. Dos Santos B. Russell M. Segarini P. Cancer Res. 1993; 53: 3386-3393PubMed Google Scholar, 21Jacobsen F.W. Stokke T. Jacobsen S.E. Blood. 1995; 86: 2957-2966Crossref PubMed Google Scholar, 22Veiby O.P. Jacobsen F.W. Cui L. Lyman S.D. Jacobsen S.E. J. Immunol. 1996; 157: 2953-2960PubMed Google Scholar). The addition of TGF-β1 to primitive hematopoietic progenitors has been shown to inhibit the survival-promoting effects of several cytokines including stem cell factor and Flt3-L (21Jacobsen F.W. Stokke T. Jacobsen S.E. Blood. 1995; 86: 2957-2966Crossref PubMed Google Scholar, 22Veiby O.P. Jacobsen F.W. Cui L. Lyman S.D. Jacobsen S.E. J. Immunol. 1996; 157: 2953-2960PubMed Google Scholar). These effects could be significant; one study has already indicated that removal of endogenous TGF-β1 from cultures could be of benefit in ex vivoexpansion protocols to improve the number of cells with long term bone marrow reconstitution activity (16Soma T., Yu, J.M. Dunbar C.E. Blood. 1996; 87: 4561-4567Crossref PubMed Google Scholar). Although there are now substantial data on the actions of TGF-β1 on primitive hematopoietic cells, the molecular mechanisms that mediate these effects have remained elusive. While some groups have correlated the growth-regulatory effects of TGF-β1 with changes in receptor expression for survival/growth-stimulatory factors such as the colony-stimulating factors (23Jacobsen S.E. Ruscetti F.W. Dubois C.M. Lee J. Boone T.C. Keller J.R. Blood. 1991; 77: 1706-1716Crossref PubMed Google Scholar, 24Jacobsen S.E. Ruscetti F.W. Roberts A.B. Keller J.R. J. Immunol. 1993; 151: 4534-4544PubMed Google Scholar, 25Dubois C.M. Ruscetti F.W. Stankova J. Keller J.R. Blood. 1994; 83: 3138-3145Crossref PubMed Google Scholar), others have shown that this mechanism cannot always account for the activities of this cytokine (26Fan K. Ruan Q. Sensenbrenner L. Chen B. Blood. 1992; 79: 1679-1685Crossref PubMed Google Scholar, 27Chen A.R. Rohrschneider L.R. Blood. 1993; 81: 2539-2546Crossref PubMed Google Scholar). Postreceptor mechanisms must therefore also be involved in mediating the effects of TGF-β1 on hematopoietic cells. It is becoming increasingly clear that many survival- and death-inducing stimuli mediate their effects, at least in part, through the modulation of Bcl-2 family members (28Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395-419Crossref PubMed Scopus (1528) Google Scholar). This family comprises proteins that can antagonize or promote cell survival. The ratio of homodimers and heterodimers within the Bcl-2 family has been suggested to determine cell survival in many cell systems. For example, homodimers of the proapoptotic family member Bax can induce apoptosis, while the formation of Bax/Bcl-2 heterodimers promote survival (29Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar, 30Yin X.M. Oltval Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar). Positive regulators of cell survival such as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor have been shown to exert their effects by up-regulating the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL, respectively (31Rinaudo M.S. Su K. Falk L.A. Halder S. Mufson R.A. Blood. 1995; 86: 80-88Crossref PubMed Google Scholar, 32Dibbert B. Daigle I. Braun D. Schranz C. Weber M. Blaser K. Zangemaster-Wittke U. Akbar A.N. Simon H. Blood. 1998; 92: 778-783Crossref PubMed Google Scholar, 33Dumon S. Santos S.C. Debierre-Grockiego F. Gouilleux-Gruart V. Cocault L. Boucheron C. Mollat P. Gisselbrecht S. Gouilleux F. Oncogene. 1999; 18: 4191-4199Crossref PubMed Scopus (137) Google Scholar). There is now evidence that modulation of Bcl-2 members can also play a part in the proapoptotic activities of TGF-β1 in a variety of different cell types including some leukemic cells (34Tsukada T. Eguchi K. Migita K. Kawabe Y. Kawakami A. Matsuoka N. Takashima H. Mizokami A. Nagataki S. Biochem. Cell Biol. Commun. 1995; 210: 1076-1082Google Scholar, 35Naas S.J. Li M. Amundadottir L.T. Furth P.A. Dickson R.B. Biochem. Cell Biol. Commun. 1996; 227: 248-256Google Scholar, 36Teramoto T. Kiss A. Thorgeirsson S.S. Biochem. Cell Biol. Commun. 1998; 251: 56-60Google Scholar, 37Selvakumaran M. Lin H.K. Sjin R.T. Reed J.C. Liebermann D.A. Hoffman B. Mol. Cell. Biol. 1994; 14: 2352-2360Crossref PubMed Google Scholar, 38Motyl T. Grzelkowska K. Zimowska W. Skierski J. Wareski P. Proszaj T. Trzeciak L. Eur. J. Cell Biol. 1998; 75: 367-374Crossref PubMed Scopus (56) Google Scholar, 39Saltzman A. Munro R. Searfoss G. Franks C. Jaye M. Ivashchenko Y. Exp. Cell Res. 1998; 242: 244-254Crossref PubMed Scopus (87) Google Scholar). In the present study, the IL-3-dependent and multipotent FDCP-Mix cell line was utilized as a model to study the growth-inhibitory and proapoptotic effects of TGF-β1 on normal, primitive hematopoietic progenitors. FDCP-Mix cells are karyotypically normal and have previously been shown to be responsive to the growth inhibitory effects of MIP-1α (40Heyworth C.M. Pearson M.A. Dexter T.M. Wark G. Owen-Lynch P.J. Whetton A.D. Growth Factors. 1995; 12: 165-172Crossref PubMed Scopus (7) Google Scholar). We report that TGF-β1 is able to reversibly inhibit the growth of FDCP-Mix cells cultured in high concentrations IL-3 that promote proliferation, while in lower IL-3 concentrations TGF-β1 induces apoptosis independent of p53, and this is inhibited by Bcl-2 expression. p53null FDCP-Mix cells were generated using long term marrow cultures derived from p53null mice as described previously (41Spooncer E. Lord B.I. Dexter T.M. Nature. 1985; 316: 62-64Crossref PubMed Scopus (34) Google Scholar). FDCP-Mix cells expressing Bcl-2 were generated as described by Fairbairn et al. (42Fairbairn L.J. Cowling G.J. Reipert B.M. Dexter T.M. Cell. 1993; 74: 823-832Abstract Full Text PDF PubMed Scopus (348) Google Scholar). The multipotent FDCP-Mix, Bcl-2-transfected, and p53nullFDCP-Mix cells were maintained in Fischer's medium supplemented with preselected batches of horse serum (HS; 20%, v/v) and 5% IL-3-conditioned medium (43Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-104Crossref PubMed Scopus (1081) Google Scholar). Puromycin (1 mg/ml) was also included in the Bcl-2 FDCP-Mix cell cultures. In these culture conditions, the FDCP-Mix cultures maintain a primitive blast cell phenotype. The cells were subcultured twice a week to a cell concentration of 6–8 × 104 cells/ml and maintained at 37 °C in 5% CO2 in air. Cells in logarithmic phase were washed free of growth factors and plated out (2 × 103cells/plate) in triplicate in Iscove's modified Dulbecco's medium (IMDM), 20% (v/v) HS, 10% (v/v) bovine serum albumin, 5% (v/v) IL-3 conditioned medium, 0.33% (v/v) agar, and the appropriate concentration of TGF-β1 added at 1% (v/v). Plates were incubated at 37 °C in a humidified incubator with 5% CO2. After 7 days, plates were removed, and the number of colonies was scored. Colonies were defined as aggregates of more than 50 cells. The rate of cell proliferation was determined by measuring DNA synthesis using the incorporation of [3H]thymidine into chromosomal DNA as described previously (44Pierce A. Whetton A.D. Owen-Lynch P.J. Tavernier J. Spooncer E. Dexter T.M. Heyworth C.M. J. Cell Sci. 1998; 111: 815-823Crossref PubMed Google Scholar). Cells were washed free of cytokines and seeded (1.5 × 105cells/ml) in IMDM supplemented with 10% (v/v) HS, 10 ng/ml recombinant murine IL-3 (R&D Systems, Abingdon, UK), and the appropriate concentration of TGF-β1 (R&D Systems). Cells were incubated for 48 h at 37 °C in a humidified incubator with 5% CO2, washed thoroughly, and then plated out in soft gel colony-forming assays. Cells were washed and resuspended (1.5 × 105 cells/ml) in IMDM supplemented with 10% (v/v) HS and the appropriate concentration of recombinant murine IL-3 and TGF-β1. After 24, 48, and 72 h, samples were taken, and viability was determined by trypan blue exclusion. (Our previous work has shown that trypan blue exclusion gives similar results to acridine orange staining.) Apoptotic assays were performed using the annexin V, propidium iodide-based assay (R&D Systems) and analyzed using a FacsVantage flow cytometer (Becton Dickinson Co., Mountain View, CA). Western blotting was carried out as described previously (45Spooncer E. Fairbairn L. Cowling G.J. Dexter T.M. Whetton A.D. Owen-Lynch P.J. Leukemia. 1994; 8: 620-630PubMed Google Scholar). Correct protein loading is ensured by measurement of protein concentration, and even transfer was assessed by Ponceau S staining of filters. Antibodies used were polyclonal murine Bcl-2, murine Bax, and human Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Polyclonal antibodies for murine Bad were obtained from Transduction Laboratories, Inc. (Lexington, KY). We have previously shown that FDCP-Mix cells are susceptible to growth suppression by the growth inhibitor MIP-1α (40Heyworth C.M. Pearson M.A. Dexter T.M. Wark G. Owen-Lynch P.J. Whetton A.D. Growth Factors. 1995; 12: 165-172Crossref PubMed Scopus (7) Google Scholar). To establish whether FDCP-Mix cells were a good model system for studying TGF-β1 actions on primitive hematopoietic cells, the responses of this cell line to TGF-β1 were assessed. We first examined the effects of TGF-β1 on IL-3-stimulated colony formation. TGF-β1 alone was unable to induce colony formation (data not shown). Concentrations of TGF-β1 of 50 pm or higher were shown to cause a major reduction in colony number compared with control cells treated with IL-3 alone (Fig. 1 A). Maximal inhibition of colony formation occurred at TGF-β1 concentrations above 75 pm. At this dose, TGF-β1 decreased colony number by approximately 70%. As colony-forming assays were performed over a 7-day period, to assess the kinetics of TGF-β1 growth inhibition we cultured FDCP-Mix cells in IL-3 for 24, 48, and 72 h in the presence or absence of TGF-β1 and then performed [3H]thymidine incorporation assays. TGF-β1 had relatively little effect in the presence of IL-3 (10 ng/ml) after 24 h, but beyond 48 h there was a marked inhibition of proliferation at TGF-β1 concentrations above 15 pm (Fig. 1 B). Flow cytometric analysis of cell cycle status revealed that TGF-β1 did not induce growth arrest at any phase of the cell cycle. The proportion of cells in each phase of the cell cycle was similar for cells cultured in the presence or absence of TGF-β1 even after 72 h (percentage of cells in S/G2/M without TGF-β1 was 21.9 ± 1.4%; with TGF-β1, it was 20.8 ± 2.1%). TGF-β1 therefore appears to be slowing down progression of cells through all phases of the cell cycle. We next investigated the reversibility of TGF-β1-induced growth suppression. Following 48-h treatment with IL-3 (10 ng/ml) and various concentrations of TGF-β1, cells were washed free of cytokines and then plated out in soft gel colony-forming assays (Fig. 1 C). Cells pretreated with TGF-β1 were able to form similar numbers of colonies as controls. TGF-β1 does not therefore affect the viability of clonogenic cells. While the viable cells remaining after TGF-β1 treatment are still clonogenic, it still remains possible that TGF-β1 can selectively kill a subset of nonclonogenic cells present in FDCP-Mix cultures. We tested this possibility using first viable cell counts and then the apoptotic-specific annexin V assay. TGF-β1 induced no loss of cells in the presence of 10 ng/ml IL-3 (Fig. 2 D); similarly, there was no increase in the percentage of cells staining for the apoptotic and dead cell-specific markers annexin V and propidium iodide, respectively (Table I). Thus, in the concentration of IL-3 employed in colony forming and [3H]thymidine incorporation assays, TGF-β1-induced growth inhibition was not a result of reduced viability.Table IEffect of TGF-β1 on the viability of FDCP-Mix cellsTimeIL-3TGF-β1Viable, no stainingPI-negative and annexin-positiveAnnexin- and PI-positivehng/mlpm%%%240.01074.3 ± 3.717.7 ± 3.17.9 ± 0.610060.3 ± 4.227.0 ± 3.412.5 ± 0.910094.4 ± 0.64.2 ± 0.41.3 ± 0.110094.6 ± 0.73.9 ± 0.51.3 ± 0.2480.01053.9 ± 3.432.9 ± 3.512.9 ± 2.410020.0 ± 6.658.9 ± 6.720.8 ± 4.510093.2 ± 0.54.4 ± 0.12.3 ± 0.410092.9 ± 1.34.9 ± 0.82.1 ± 0.4720.01043.7 ± 4.641.3 ± 1.314.7 ± 5.41006.8 ± 2.669.7 ± 5.122.9 ± 6.810087.9 ± 2.56.6 ± 1.85.2 ± 0.910092.8 ± 0.85.0 ± 0.91.9 ± 0.2FDCP-Mix A4 cells were washed and incubated in IMDM containing 10% HS (v/v) and the appropriate concentration of IL-3 ± 100 pm TGF-β1. The number of viable, apoptotic, and dead cells was determined using the annexin V, propidium iodide (PI)-based assay and analyzed using a FacsVantage flow cytometer after 24, 48, and 72 h. The data presented are the mean of four individual experiments ± S.E. Open table in a new tab FDCP-Mix A4 cells were washed and incubated in IMDM containing 10% HS (v/v) and the appropriate concentration of IL-3 ± 100 pm TGF-β1. The number of viable, apoptotic, and dead cells was determined using the annexin V, propidium iodide (PI)-based assay and analyzed using a FacsVantage flow cytometer after 24, 48, and 72 h. The data presented are the mean of four individual experiments ± S.E. Previous studies on primitive hematopoietic progenitors have shown that TGF-β1 can antagonize the survival-promoting effects of cytokines such as stem cell factor and Flt3-L (21Jacobsen F.W. Stokke T. Jacobsen S.E. Blood. 1995; 86: 2957-2966Crossref PubMed Google Scholar, 22Veiby O.P. Jacobsen F.W. Cui L. Lyman S.D. Jacobsen S.E. J. Immunol. 1996; 157: 2953-2960PubMed Google Scholar). To determine whether TGF-β1 could also inhibit IL-3-stimulated survival, FDCP-Mix cells were cultured in a range of IL-3 concentrations (0–10 ng/ml) in the presence or absence of TGF-β1 (0–100 pm), and then the cell viability and the number of apoptotic cells were assayed. In concentrations of IL-3 that promoted survival with little detectable proliferation (0.01 and 0.1 ng/ml), TGF-β1 (>50 pm) induced a marked decrease in cell viability (Fig. 2, A andB). These effects became more prominent over time, with the most significant effects on viability seen after 72 h. Annexin V assays performed under the same conditions confirmed that this reduction in viability was a result of TGF-β1-induced apoptosis. After 72 h in the presence of 0.01 ng/ml IL-3, only 7% of cells treated with 100 pm TGF-β1 were still viable compared with 44% of untreated cells (Table I). Having established that the FDCP-Mix cell line is a good model for studying both the antiproliferative and proapoptotic actions of TGF-β1 on primitive progenitors, we next attempted to discern the mechanisms by which these effects occur. Studies on nonhematopoietic cells have implicated the tumor suppressor p53 in the growth-suppressive effects of TGF-β1 (46Reiss M. Brash D.E. Munoz-Antonia T. Simon J.A. Ziegler A. Velluci V.F. Zhou Z.L. Oncology Res. 1992; 4: 349-356PubMed Google Scholar, 47Wyllie F.S. Dawson T. Bond J.A. Goretzki P. Game S. Prime S. Wynford-Thomas D. Mol. Cell. Endocrinol. 1991; 76: 13-21Crossref PubMed Scopus (54) Google Scholar, 48Gerwin B.I. Spillare E. Forrester K. Lehman T.A. Kispert J. Welsh J.A. Pfeifer A.M. Lechner J.F. Baker S.J. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2759-2763Crossref PubMed Scopus (124) Google Scholar, 49Reiss M. Vellucci V.F. Zhou Z.L. Cancer Res. 1993; 53: 899-904PubMed Google Scholar). The detection of a TGF-β1-resistant subpopulation in high proliferative potential colony-forming cells (HPP-CFC) isolated from p53-deficient mice (but not wild type littermates) has suggested that p53 may play a role in TGF-β1 growth-inhibitory pathways in hematopoietic cells (50Sasaki H. Matsuda M. Lu Y. Ikuta K. Matsuyama S. Hirabayashi Y. Mitsui H. Matsumura T. Muramatsu M. Tsukada T. Aizawa S. Inoue T. Leukemia. 1997; 11: 239-244Crossref PubMed Scopus (9) Google Scholar). In order to test this, we utilized a multipotent and IL-3-dependent FDCP-Mix cell line isolated from long term bone marrow cultures derived from p53null mice (p53null FDCP-Mix). TGF-β1 reversibly suppressed IL-3-stimulated proliferation of p53null FDCP-Mix cells in a dose-dependent manner as determined by soft gel colony-forming assays (Fig. 3 A) and [3H]thymidine incorporation assays (Fig. 3 B). Since p53 has been shown to mediate the effects of many apoptotic stimuli in hematopoietic cells, we also examined the effects of TGF-β1 on p53null FDCP-Mix viability. While apoptosis induced by IL-3 withdrawal was delayed in p53null FDCP-Mix cells compared with FDCP-Mix cells expressing wild type p53, no significant differences in their responses to TGF-β1 could be detected. This delay in apoptosis, in p53null cells, has been previously reported in mast cells derived from p53null mice (51Silva A. Wyllie A. Collins M.K.L. Blood. 1997; 89: 2717-2722Crossref PubMed Google Scholar) and with results obtained with a dominant negative p53 in a hematopoietic cell line (52Gottlieb E. Haffner R. von-Ruden T. Wagner E.F. Oren M. EMBO J. 1994; 13: 1368-1374Crossref PubMed Scopus (169) Google Scholar). TGF-β1 induced a dose-dependent decrease in p53nullFDCP-Mix cell viability in the presence of IL-3 concentrations that promoted survival alone (Fig. 4,A and B). These proapoptotic effects of TGF-β1, however, were completely overcome by higher concentrations of IL-3 as seen in FDCP-Mix cells (Fig. 4, C and D). Retroviral transfection of the p53null FDCP-Mix cells with p53 gave similar results (data not shown). We therefore conclude that p53 is not absolutely required for TGF-β1-induced growth suppression or apoptosis in primitive myeloid progenitors.Figure 4The effect of TGF -β1 on p53null FDCP-Mix cell death. Cells were washed free of cytokines and cultured in the presence of 0.01 (A), 0.1 (B), 1 (C), or 10 ng/ml IL-3 (D) and a range of TGF-β1 concentrations. The percentage of dead cells after 24, 48, and 72 h was determined using trypan blue exclusion. Data are the mean values ± S.E. of representative experiments performed three times, with triplicates in each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Members of the Bcl-2 family are known to be major regulators of apoptosis in many cell types including hematopoietic cells. To investigate whether TGF-β1-induced apoptosis of FDCP-Mix cells is mediated through the modulation of these proteins, we analyzed the expression of both proapoptotic and antiapoptotic Bcl-2 family members. We first examined the expression of the antiapoptotic member Bcl-xL, which has been shown to be up-regulated by a variety of survival stimuli in hematopoietic cells including cytokines (31Rinaudo M.S. Su K. Falk L.A. Halder S. Mufson R.A. Blood. 1995; 86: 80-88Crossref PubMed Google Scholar, 32Dibbert B. Daigle I. Braun D. Schranz C. Weber M. Blaser K. Zangemaster-Wittke U. Akbar A.N. Simon H. Blood. 1998; 92: 778-783Crossref PubMed Google Scholar, 33Dumon S. Santos S.C. Debierre-Grockiego F. Gouilleux-Gruart V. Cocault L. Boucheron C. Mollat P. Gisselbrecht S. Gouilleux F. Oncogene. 1999; 18: 4191-4199Crossref PubMed Scopus (137) Google Scholar, 53Chen Q. Turner J. Watson A.J.M. Dive C. Oncogene. 1997; 15: 2249-2254Crossref PubMed Scopus (23) Google Scholar). Protein levels of Bcl-xL were unaltered by IL-3 removal or by the addition of TGF-β1 to FDCP-Mix cells (Fig. 5 A). Similarly, the levels of the proapoptotic proteins Bax and Bad were unaffected by the reduction of IL-3 levels or TGF-β1 treatment (Fig. 5, B andC). However, in contrast, the expression of Bcl-2 was dramatically reduced by TGF-β1. Bcl-2 protein levels were decreased by 2-fold 24 h after TGF-β1 treatment, and this decrease was still maintained after 48 h (Fig. 5 D). While others have shown that IL-3 can increase Bcl-2 expression in some hematopoietic cells (31Rinaudo M.S. Su K. Falk L.A. Halder S. Mufson R.A. Blood. 1995; 86: 80-88Crossref PubMed Google Scholar), we detected no significant differences in Bcl-2 expression in FDCP-Mix cells cultured in low or high concentrations of IL-3 (Fig. 5 D). Furthermore, we found that TGF-β1-induced decreases in Bcl-2 levels occurred in high concentrations of IL-3 that can abolish the proapoptotic actions of this cytokine. These data therefore demonstrate that high levels of Bcl-2 are not absolutely necessary for FDCP-Mix survival. To determine whether TGF-β1-induced apoptosis is mediated by down-regulation of Bcl-2, we used retroviral technology to generate clonal cell populations of FDCP-Mix that express human Bcl-2 (Fig. 6 A). These FDCP-Mix Bcl-2 cell lines were able to undergo normal myeloid differentiation when cultured in appropriate conditions (42Fairbairn L.J. Cowling G.J. Reipert B.M. Dexter T.M. Cell. 1993; 74: 823-832Abstract Full Text PDF PubMed Scopus (348) Google Scholar). Overexpression of Bcl-2 did not affect the sensitivity of FDCP-Mix cells to the growth-inhibitory effects of TGF-β1. IL-3-stimulated colony formation (Fig. 6 B) and [3H]thymidine incorporation (Fig. 6 C) of FDCP-Mix Bcl-2 cells were reversibly suppressed by TGF-β1 to a similar degree as parental cells. Trypan blue and annexin V assays demonstrated that these Bcl-2-expressing cell lines were more resistant than parental cells to apoptosis induced by IL-3 deprivation. When we expressed human Bcl-2 in FDCP-Mix cells, we observed a delay in apoptosis of the cells cultured in the absence or in low concentrations of IL-3 (Fig. 7 A, TableII). However, when we examined the effects of TGF-β1 on FDCP-Mix Bcl-2 cells cultured in low concentrations of IL-3, there was a complete abrogation of TGF-β1-induced apoptosis (Fig. 7, A and B). TGF-β1-treated FDCP-Mix Bcl-2 cells underwent apoptosis at a similar rate as untreated cells in all concentrations of IL-3 tested (Fig. 7,A–D). Thus, it appears that TGF-β1-mediated effects on primitive myeloid cell survival can be inhibited by ectopic expression of Bcl-2 protein. This effect, in the presence of low concentrations of IL-3, has a major impact on cellular survival.Figure 7TGF -β1-induced apoptosis is blocked by high levels of Bcl-2. The effect of Bcl-2 on TGF-β1-mediated cell death of FDCP-Mix cells was assessed using the FDCP-Mix Bcl-2 (clone 15) cell line" @default.
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• W2116710692 title "Transforming Growth Factor-β1 Induces Apoptosis Independently of p53 and Selectively Reduces Expression of Bcl-2 in Multipotent Hematopoietic Cells" @default.
• W2116710692 cites W1489642554 @default.
• W2116710692 cites W1501273614 @default.
• W2116710692 cites W1527321128 @default.
• W2116710692 cites W1529680890 @default.
• W2116710692 cites W1535779673 @default.
• W2116710692 cites W1558535920 @default.
• W2116710692 cites W1574447385 @default.
• W2116710692 cites W1575606714 @default.
• W2116710692 cites W1749033347 @default.
• W2116710692 cites W1865619950 @default.
• W2116710692 cites W1921728185 @default.
• W2116710692 cites W1966507279 @default.
• W2116710692 cites W1970834072 @default.
• W2116710692 cites W1975988004 @default.
• W2116710692 cites W1981221225 @default.
• W2116710692 cites W1982102756 @default.
• W2116710692 cites W1982872895 @default.
• W2116710692 cites W1987297184 @default.
• W2116710692 cites W1989867073 @default.
• W2116710692 cites W1990162889 @default.
• W2116710692 cites W1993112793 @default.
• W2116710692 cites W1996522179 @default.
• W2116710692 cites W1997257161 @default.
• W2116710692 cites W2000739442 @default.
• W2116710692 cites W2001917467 @default.
• W2116710692 cites W2005794507 @default.
• W2116710692 cites W2005836348 @default.
• W2116710692 cites W2006897943 @default.
• W2116710692 cites W2019714026 @default.
• W2116710692 cites W2024196729 @default.
• W2116710692 cites W2030573603 @default.
• W2116710692 cites W2033847504 @default.
• W2116710692 cites W2056395300 @default.
• W2116710692 cites W2062455027 @default.
• W2116710692 cites W2064645060 @default.
• W2116710692 cites W2081773727 @default.
• W2116710692 cites W2093500426 @default.
• W2116710692 cites W2093628776 @default.
• W2116710692 cites W2116039073 @default.
• W2116710692 cites W2119836992 @default.
• W2116710692 cites W2124221273 @default.
• W2116710692 cites W2128045505 @default.
• W2116710692 cites W2134979684 @default.
• W2116710692 cites W2137055826 @default.
• W2116710692 cites W2137986638 @default.
• W2116710692 cites W2156471525 @default.
• W2116710692 cites W2266660623 @default.
• W2116710692 cites W2314160756 @default.
• W2116710692 cites W2318698183 @default.
• W2116710692 cites W2338263125 @default.
• W2116710692 cites W2408968820 @default.
• W2116710692 cites W2415581815 @default.
• W2116710692 cites W2417569905 @default.
• W2116710692 cites W2417869212 @default.
• W2116710692 cites W2431051467 @default.
• W2116710692 cites W347889507 @default.
• W2116710692 cites W4236025207 @default.
• W2116710692 cites W4256409628 @default.
• W2116710692 cites W72367593 @default.
• W2116710692 cites W950059845 @default.
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