Glucocorticoids Inhibit Apoptosis during Fibrosarcoma Development by Transcriptionally Activating Bcl-xL
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m301812200
ISSN1083-351X
AutoresDuncan M. Gascoyne, Robert Kypta, María dM Vivanco,
Tópico(s)ATP Synthase and ATPases Research
ResumoGlucocorticoids influence many physiological processes, and in particular apoptosis, often with opposite effects depending on the cell type examined. We found that during fibrosarcoma development there is a strong increase in apoptosis at the tumor stage, which is repressed by dexamethasone to levels observed in normal fibroblasts. The anti-apoptotic Bcl-2 family protein Bcl-xL is induced by dexamethasone at the transcriptional level at all stages of fibrosarcoma development. The ligand-activated glucocorticoid receptor (GR) activates the Bcl-x promoter in transient transfection experiments, and GR binds to specific Bcl-x promoter sequences in vitro and in vivo. Furthermore, a GR antagonist abolishes this effect, indicating that Bcl-xL induction is mediated by GR. Importantly, exogenous Bcl-xL inhibits apoptosis and caspase-3 activity in fibrosarcoma cells to levels found in dexamethasone-treated fibrosarcoma cells. We conclude that Bcl-xL is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis. Glucocorticoids influence many physiological processes, and in particular apoptosis, often with opposite effects depending on the cell type examined. We found that during fibrosarcoma development there is a strong increase in apoptosis at the tumor stage, which is repressed by dexamethasone to levels observed in normal fibroblasts. The anti-apoptotic Bcl-2 family protein Bcl-xL is induced by dexamethasone at the transcriptional level at all stages of fibrosarcoma development. The ligand-activated glucocorticoid receptor (GR) activates the Bcl-x promoter in transient transfection experiments, and GR binds to specific Bcl-x promoter sequences in vitro and in vivo. Furthermore, a GR antagonist abolishes this effect, indicating that Bcl-xL induction is mediated by GR. Importantly, exogenous Bcl-xL inhibits apoptosis and caspase-3 activity in fibrosarcoma cells to levels found in dexamethasone-treated fibrosarcoma cells. We conclude that Bcl-xL is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis. glucocorticoid receptor chromatin immunoprecipitation normal fibroblast mild fibromatosis aggressive fibromatosis fibrosarcoma glucocorticoid response element 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid basic fibroblast growth factor thymidine kinase Glucocorticoids exert different effects on apoptosis and cell growth depending on the tissues examined. In some cell types, for example thymocytes and some leukemia cell lines, treatment with glucocorticoids induces apoptosis (1Planey S.L. Litwack G. Biochem. Biophys. Res. Commun. 2000; 279: 307-312Crossref PubMed Scopus (126) Google Scholar). This has led to their common use as chemotherapeutic agents in lymphomas and leukemias (2Smets L.A. Salomons G. van den Berg J. Adv. Exp. Med. Biol. 1999; 457: 607-614Crossref PubMed Scopus (42) Google Scholar). In contrast, glucocorticoids have been reported to inhibit apoptosis in a number of other cell types, including glioma and astrocytoma cell lines (3Gorman A.M. Hirt U.A. Orrenius S. Ceccatelli S. Neuroscience. 2000; 96: 417-425Crossref PubMed Scopus (77) Google Scholar), fibroblasts (4Pagliacci M.C. Migliorati G. Smacchia M. Grignani F. Riccardi C. Nicoletti I. J. Endocrinol. Invest. 1993; 16: 591-599Crossref PubMed Scopus (40) Google Scholar), hepatoma cells (5Evans-Storms R.B. Cidlowski J.A. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (83) Google Scholar), gastric cancer cell lines (6Chang T.C. Hung M.W. Jiang S.Y. Chu J.T. Chu L.L. Tsai L.C. FEBS Lett. 1997; 415: 11-15Crossref PubMed Scopus (59) Google Scholar), and mammary epithelial cells (7Feng Z. Marti A. Jehn B. Altermatt H.J. Chicaiza G. Jaggi R. J. Cell Biol. 1995; 131: 1095-1103Crossref PubMed Scopus (224) Google Scholar, 8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar).The glucocorticoid receptor (GR)1 belongs to a superfamily of transcription factors that includes receptors for steroid and thyroid hormones, retinoic acid, and vitamin D3(9Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1630) Google Scholar). GR is normally localized in the cytoplasm in a non-active state in a complex that includes Hsp90. Upon hormone binding, GR changes conformation and migrates to the nucleus, where it induces or represses transcription by binding to specific DNA sequences on target genes (10Cole T.J. Blendy J.A. Monaghan A.P. Schmid W. Aguzzi A. Schutz G. Steroids. 1995; 60: 93-96Crossref PubMed Scopus (64) Google Scholar).Apoptosis, triggered by a variety of intra- and extracellular signals, is important for normal development, to maintain tissue homeostasis, and as a defense strategy against the emergence of cancer (11Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar, 12Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2488) Google Scholar). The apoptotic program is executed by a family of cysteine proteases called caspases, which are activated by proteolytic cleavage (13Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 14Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2428) Google Scholar). Once activated, effector caspases cleave a variety of cellular substrates including structural components, regulatory proteins, and other caspases, resulting in the orchestrated collapse of the cell characteristic to apoptosis.Bcl-2 family proteins play critical roles in the control of apoptosis. Two major groups of Bcl-2 family proteins exist; the pro-survival members, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, etc., and the pro-apoptotic members, including Bax, Bak, Bok, etc. (see Ref. 15Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholarfor review). The ratio between these two groups of family members determines whether a cell will live or die. Downstream of this checkpoint lie the caspase pathway and mitochondria dysfunction, major execution events that lead to irreversible cell death (16Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). Alterations in the expression of anti-apoptotic members such as Bcl-2 and Bcl-xL have been implicated in tumorigenesis in both clinical cases and transgenic models (17Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar). In addition, Bcl-2 members are also important determinants of anticancer drug sensitivity (18Amundson S.A. Myers T.G. Scudiero D. Kitada S. Reed J.C. Fornace Jr., A.J. Cancer Res. 2000; 60: 6101-6110PubMed Google Scholar).The conversion of a normal cell to a neoplastic one occurs in multiple steps (19Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21908) Google Scholar), and one approach to studying this process has been to employ transgenic mice (20Hanahan D. Annu. Rev. Genet. 1988; 22: 479-519Crossref PubMed Scopus (166) Google Scholar). Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a process that involves distinct proliferative stages. These are the normal fibroblasts (NF), and then two histological grades of hyperplasia, mild fibromatosis (MF) and aggressive fibromatosis (AF). Finally, at lower frequency, dermal fibrosarcomas (FS) develop (21Sippola-Thiele M. Hanahan D. Howley P.M. Mol. Cell. Biol. 1989; 9: 925-934Crossref PubMed Google Scholar). The first molecular distinction between the AF and the FS cells to be identified was a dramatic increase in ligand-dependent GR transcriptional activity in FS cells (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar). This increase does not result from changes in the intracellular levels of GR, hormone-dependent nuclear translocation, or specific DNA binding activity, all of which are unaltered throughout the progression. Moreover, analysis of the tumors formed in mice upon inoculation of AF or FS cells indicates a direct correlation between GR transcriptional activity and tumorigenic potential (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar).To understand cancer progression it is important to determine the mechanisms by which signaling proteins influence proliferation and apoptosis at different stages of the tumorigenic process. Here we have examined the effects of dexamethasone on apoptosis in the multistep tumorigenic pathway of fibrosarcoma development. Our observations point to Bcl-xL as a key GR target mediating the inhibition of apoptosis during fibrosarcoma progression.DISCUSSIONApoptosis has been found to be widespread in many tumors (36Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12723) Google Scholar, 37Arends M.J. McGregor A.H. Wyllie A.H. Am. J. Pathol. 1994; 144: 1045-1057PubMed Google Scholar) and yet limited in others (38Kaufmann S.H. Gores G.J. Bioessays. 2000; 22: 1007-1017Crossref PubMed Scopus (180) Google Scholar). GR can display either pro-apoptotic or anti-apoptotic activity depending on cell context (39Tolosa E. Ashwell J.D. Neuroimmunomodulation. 1999; 6: 90-96Crossref PubMed Scopus (17) Google Scholar, 40Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1068) Google Scholar). We show here that in the absence of glucocorticoids apoptosis increases during fibrosarcoma development, whereas in the presence of dexamethasone it is reduced to those levels found in the earlier stages of the tumorigenic pathway.Few studies of caspase activity during tumor development have been reported. Caspase-3-like activity has been observed to increase during the development of colorectal carcinoma (41Leonardos L. Butler L.M. Hewett P.J. Zalewski P.D. Cowled P.A. Cancer Lett. 1999; 143: 29-35Crossref PubMed Scopus (19) Google Scholar), thus representing a situation similar to that found in the absence of glucocorticoids during fibrosarcoma development. Although involved in some forms of apoptosis, caspase-1 also promotes inflammatory responses by regulating cytokine signaling (42Watson R.W. Rotstein O.D. Parodo J. Bitar R. Marshall J.C. William R. Watson G. J. Immunol. 1998; 161: 957-962PubMed Google Scholar). Increasing caspase-1 activity during fibrosarcoma development may therefore have implications not only for apoptosis but also for inflammation.Our results demonstrate that decreasing Bcl-xL expression results primarily from decreasing Bcl-x transcription. This correlates inversely with increasing transcription of the BPV-1 genome and E5, E6, and E7 oncogene expression in MF, AF, and FS cells (21Sippola-Thiele M. Hanahan D. Howley P.M. Mol. Cell. Biol. 1989; 9: 925-934Crossref PubMed Google Scholar,53Roca R. Kypta R.M. Vivanco M.d.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3113-3118Crossref PubMed Scopus (8) Google Scholar). Both BPV-1 E6 and E7 oncoproteins sensitize cells to apoptosis induced by tumor necrosis factor-α (43Liu Z. Liu Y. Hong Y. Rapp L. Androphy E.J. Chen J.J. Virology. 2002; 295: 230-237Crossref PubMed Scopus (9) Google Scholar); thus it can be speculated that these proteins may inhibit Bcl-x transcription. Notably, the angiogenic growth factor bFGF increases Bcl-x expression in epithelial cells (44Bryckaert M. Guillonneau X. Hecquet C. Courtois Y. Mascarelli F. Oncogene. 1999; 18: 7584-7593Crossref PubMed Scopus (56) Google Scholar). Because the expression of bFGF becomes increasingly extracellular at the AF stage of fibrosarcoma development (45Kandel J. Bossy-Wetzel E. Radvanyi F. Klagsbrun M. Folkman J. Hanahan D. Cell. 1991; 66: 1095-1104Abstract Full Text PDF PubMed Scopus (476) Google Scholar), it is possible that decreasing levels of intracellular bFGF contribute to decreasing Bcl-xL expression in AF and FS cells. In addition, the transcription factor AP-1 has been proposed to inhibit Bcl-xL expression (46Jacobs-Helber S.M. Wickrema A. Birrer M.J. Sawyer S.T. Mol. Cell. Biol. 1998; 18: 3699-3707Crossref PubMed Scopus (91) Google Scholar), and therefore the increased expression and activity of the AP-1 component proteins c-Jun and JunB in AF and FS cells (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar, 47Bossy-Wetzel E. Bravo R. Hanahan D. Genes Dev. 1992; 6: 2340-2351Crossref PubMed Scopus (68) Google Scholar) may also reduce Bcl-xLexpression.Increased Bcl-xL mRNA expression following glucocorticoid treatment has been observed in human gastric cancer cells (6Chang T.C. Hung M.W. Jiang S.Y. Chu J.T. Chu L.L. Tsai L.C. FEBS Lett. 1997; 415: 11-15Crossref PubMed Scopus (59) Google Scholar), where enhanced Bcl-xL mRNA stability was found to represent a part of the mechanism underlying the protective effect of dexamethasone. However, dexamethasone does not affect the stability of Bcl-xL mRNA in fibrosarcoma cells (53Roca R. Kypta R.M. Vivanco M.d.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3113-3118Crossref PubMed Scopus (8) Google Scholar). The reported increased expression of Bcl-xL mRNA in human myeloid leukemic cells required 24 h of hormone treatment (48Lotem J. Sachs L. Cell Growth & Differ. 1995; 6: 647-653PubMed Google Scholar), whereas we clearly detected up-regulation of Bcl-xLafter 2 h of dexamethasone treatment. Subsequent studies by other groups have also demonstrated the importance of increasing Bcl-xL expression for the inhibition of apoptosis by glucocorticoids in a variety of cell types (3Gorman A.M. Hirt U.A. Orrenius S. Ceccatelli S. Neuroscience. 2000; 96: 417-425Crossref PubMed Scopus (77) Google Scholar, 8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar, 31Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 49Messmer U.K. Winkel G. Briner V.A. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 1673-1683Crossref PubMed Scopus (36) Google Scholar). However, none of these reports identified the mechanism by which GR might control Bcl-x expression.Further extending the initial observations of these studies, we have demonstrated that the up-regulation of Bcl-xL expression by dexamethasone occurs at the transcriptional level and is increased during fibrosarcoma development. The increased expression of Bcl-xL is most likely achieved by specific binding of GR to the identified GRE-like sequences within the Bcl-x promoter. In fact, these sequences also confer hormone responsiveness to a heterologous promoter in COS-7 cells, indicating that the presence of ligand-activated GR is sufficient to induce Bcl-x transcription in other cell types. Furthermore, specific activation of the Bcl-x promoter by dexamethasone requires the cooperation of the various GREs identified, because deletion or mutation of these elements results in a reduction in responsiveness. This observation supports the prediction made by Nordeen and colleagues (35Nordeen S.K. Suh B.J. Kuhnel B. Hutchison C.D. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar) that most natural response elements are suboptimal elements and that cooperativity among these individually weak sites contributes to the inducibility of the promoter. Because Bcl-x expression is higher in untreated NF than in untreated FS cells, one possibility is that the Bcl-x promoter is repressed in FS cells and that dexamethasone treatment results in de-repression. Although this may be the case, the reduced expression of Bcl-x in FS cells is unlikely to involve GR binding to GREs in the Bcl-x promoter, because their mutation does not affect transcription in the absence of hormone.Importantly, Bcl-xL is responsible for the ability of dexamethasone to inhibit apoptosis in fibrosarcoma cells, as shown by ectopic expression of Bcl-xL. As expression of Bcl-xL decreases during fibrosarcoma development in the absence of glucocorticoids, increasing up-regulation of Bcl-xL expression by dexamethasone serves to maintain Bcl-xL levels during tumor development, and thus apoptosis remains approximately constant. In effect, Bcl-xLexpression becomes increasingly dependent on glucocorticoids during fibrosarcoma development. Increased Bcl-xL expression and reduced apoptosis correlate in FS cells with decreased caspase-3, but not caspase-1, activity in response to glucocorticoids. Such a correlation has also been reported by Messmer et al. (49Messmer U.K. Winkel G. Briner V.A. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 1673-1683Crossref PubMed Scopus (36) Google Scholar). Furthermore, levels of apoptosis in hormone-treated FS cells are comparable with those in earlier cell types, despite the presence of higher caspase-1 activity. These data indicate that caspase-3 is likely to play a more important role than caspase-1 in determining apoptosis in FS cells.It may be noteworthy that we also observed dexamethasone regulation of Bcl-x expression in both normal and transformed human mammary epithelial cells. Apoptosis of immortalized mouse mammary epithelial cells can be decreased by dexamethasone treatment, associated with increased Bcl-x mRNA expression, within 2 h of hormone treatment (8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar). Intriguingly, an increase in Bcl-xL protein expression following dexamethasone treatment was readily detectable in transformed SK-BR-3 breast cancer cells but was lower in normal epithelial cells (data not shown). This represents a parallel situation to that observed during fibrosarcoma progression, where the ability of hormone to induce Bcl-xL expression increases during tumor development; it may reflect the ability of normal cells to limit, or of tumor cells to enhance, regulation of gene expression by specific steroid hormone receptors as has been suggested previously (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar). Such a phenomenon may be a result of increased expression of steroid hormone receptor co-factors during tumor development, and indeed co-activators such as E6-AP and AIB1 are overexpressed in certain tumor types (50Sivaraman L. Nawaz Z. Medina D. Conneely O.M. O'Malley B.W. Breast Cancer Res. Treat. 2000; 62: 185-195Crossref PubMed Scopus (19) Google Scholar,51Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1422) Google Scholar).It is interesting to consider that the discovery of regulation of Bcl-xL expression by glucocorticoids during tumorigenesis may have clinical relevance. Glucocorticoids such as dexamethasone are used as anti-emetics in the treatment of several cancers. One study has identified a strong negative correlation between Bcl-xLexpression and sensitivity to a wide variety of cytotoxic agents in 60 cancer cell lines (18Amundson S.A. Myers T.G. Scudiero D. Kitada S. Reed J.C. Fornace Jr., A.J. Cancer Res. 2000; 60: 6101-6110PubMed Google Scholar). Our results suggest that Bcl-xLexpression (and thus cell survival) may be promoted by dexamethasone. Therefore, glucocorticoid treatment of tumors may reduce the cytotoxic effects of chemotherapy on tumor cells and may be contraindicated in those tumors with highly inducible Bcl-xL expression. A similar situation may exist in malignant gliomas, where glucocorticoids have been reported to interfere with chemotherapy response (52Weller M. Schmidt C. Roth W. Dichgans J. Neurology. 1997; 48: 1704-1709Crossref PubMed Scopus (91) Google Scholar). Importantly, indications that expression of Bcl-xL may be more glucocorticoid-dependent in tumor cells than in normal cells suggest that glucocorticoid therapy could have a certain degree of tumor specificity.The enhanced expression of Bcl-xL by GR during fibrosarcoma development reflects the unexpected transition in the transcriptional activity of GR and reveals an alternative mode of regulation for steroid hormone receptors. In summary, increasing GR transcriptional activity is likely to exert a protective effect by enabling the reduction of apoptosis at the tumor stage of fibrosarcoma development and, therefore, prolonging tumor cell survival. Glucocorticoids exert different effects on apoptosis and cell growth depending on the tissues examined. In some cell types, for example thymocytes and some leukemia cell lines, treatment with glucocorticoids induces apoptosis (1Planey S.L. Litwack G. Biochem. Biophys. Res. Commun. 2000; 279: 307-312Crossref PubMed Scopus (126) Google Scholar). This has led to their common use as chemotherapeutic agents in lymphomas and leukemias (2Smets L.A. Salomons G. van den Berg J. Adv. Exp. Med. Biol. 1999; 457: 607-614Crossref PubMed Scopus (42) Google Scholar). In contrast, glucocorticoids have been reported to inhibit apoptosis in a number of other cell types, including glioma and astrocytoma cell lines (3Gorman A.M. Hirt U.A. Orrenius S. Ceccatelli S. Neuroscience. 2000; 96: 417-425Crossref PubMed Scopus (77) Google Scholar), fibroblasts (4Pagliacci M.C. Migliorati G. Smacchia M. Grignani F. Riccardi C. Nicoletti I. J. Endocrinol. Invest. 1993; 16: 591-599Crossref PubMed Scopus (40) Google Scholar), hepatoma cells (5Evans-Storms R.B. Cidlowski J.A. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (83) Google Scholar), gastric cancer cell lines (6Chang T.C. Hung M.W. Jiang S.Y. Chu J.T. Chu L.L. Tsai L.C. FEBS Lett. 1997; 415: 11-15Crossref PubMed Scopus (59) Google Scholar), and mammary epithelial cells (7Feng Z. Marti A. Jehn B. Altermatt H.J. Chicaiza G. Jaggi R. J. Cell Biol. 1995; 131: 1095-1103Crossref PubMed Scopus (224) Google Scholar, 8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar). The glucocorticoid receptor (GR)1 belongs to a superfamily of transcription factors that includes receptors for steroid and thyroid hormones, retinoic acid, and vitamin D3(9Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1630) Google Scholar). GR is normally localized in the cytoplasm in a non-active state in a complex that includes Hsp90. Upon hormone binding, GR changes conformation and migrates to the nucleus, where it induces or represses transcription by binding to specific DNA sequences on target genes (10Cole T.J. Blendy J.A. Monaghan A.P. Schmid W. Aguzzi A. Schutz G. Steroids. 1995; 60: 93-96Crossref PubMed Scopus (64) Google Scholar). Apoptosis, triggered by a variety of intra- and extracellular signals, is important for normal development, to maintain tissue homeostasis, and as a defense strategy against the emergence of cancer (11Reed J.C. J. Clin. Oncol. 1999; 17: 2941-2953Crossref PubMed Google Scholar, 12Raff M.C. Nature. 1992; 356: 397-400Crossref PubMed Scopus (2488) Google Scholar). The apoptotic program is executed by a family of cysteine proteases called caspases, which are activated by proteolytic cleavage (13Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 14Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2428) Google Scholar). Once activated, effector caspases cleave a variety of cellular substrates including structural components, regulatory proteins, and other caspases, resulting in the orchestrated collapse of the cell characteristic to apoptosis. Bcl-2 family proteins play critical roles in the control of apoptosis. Two major groups of Bcl-2 family proteins exist; the pro-survival members, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, etc., and the pro-apoptotic members, including Bax, Bak, Bok, etc. (see Ref. 15Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholarfor review). The ratio between these two groups of family members determines whether a cell will live or die. Downstream of this checkpoint lie the caspase pathway and mitochondria dysfunction, major execution events that lead to irreversible cell death (16Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3238) Google Scholar). Alterations in the expression of anti-apoptotic members such as Bcl-2 and Bcl-xL have been implicated in tumorigenesis in both clinical cases and transgenic models (17Evan G. Littlewood T. Science. 1998; 281: 1317-1322Crossref PubMed Scopus (1360) Google Scholar). In addition, Bcl-2 members are also important determinants of anticancer drug sensitivity (18Amundson S.A. Myers T.G. Scudiero D. Kitada S. Reed J.C. Fornace Jr., A.J. Cancer Res. 2000; 60: 6101-6110PubMed Google Scholar). The conversion of a normal cell to a neoplastic one occurs in multiple steps (19Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21908) Google Scholar), and one approach to studying this process has been to employ transgenic mice (20Hanahan D. Annu. Rev. Genet. 1988; 22: 479-519Crossref PubMed Scopus (166) Google Scholar). Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a process that involves distinct proliferative stages. These are the normal fibroblasts (NF), and then two histological grades of hyperplasia, mild fibromatosis (MF) and aggressive fibromatosis (AF). Finally, at lower frequency, dermal fibrosarcomas (FS) develop (21Sippola-Thiele M. Hanahan D. Howley P.M. Mol. Cell. Biol. 1989; 9: 925-934Crossref PubMed Google Scholar). The first molecular distinction between the AF and the FS cells to be identified was a dramatic increase in ligand-dependent GR transcriptional activity in FS cells (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar). This increase does not result from changes in the intracellular levels of GR, hormone-dependent nuclear translocation, or specific DNA binding activity, all of which are unaltered throughout the progression. Moreover, analysis of the tumors formed in mice upon inoculation of AF or FS cells indicates a direct correlation between GR transcriptional activity and tumorigenic potential (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar). To understand cancer progression it is important to determine the mechanisms by which signaling proteins influence proliferation and apoptosis at different stages of the tumorigenic process. Here we have examined the effects of dexamethasone on apoptosis in the multistep tumorigenic pathway of fibrosarcoma development. Our observations point to Bcl-xL as a key GR target mediating the inhibition of apoptosis during fibrosarcoma progression. DISCUSSIONApoptosis has been found to be widespread in many tumors (36Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12723) Google Scholar, 37Arends M.J. McGregor A.H. Wyllie A.H. Am. J. Pathol. 1994; 144: 1045-1057PubMed Google Scholar) and yet limited in others (38Kaufmann S.H. Gores G.J. Bioessays. 2000; 22: 1007-1017Crossref PubMed Scopus (180) Google Scholar). GR can display either pro-apoptotic or anti-apoptotic activity depending on cell context (39Tolosa E. Ashwell J.D. Neuroimmunomodulation. 1999; 6: 90-96Crossref PubMed Scopus (17) Google Scholar, 40Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1068) Google Scholar). We show here that in the absence of glucocorticoids apoptosis increases during fibrosarcoma development, whereas in the presence of dexamethasone it is reduced to those levels found in the earlier stages of the tumorigenic pathway.Few studies of caspase activity during tumor development have been reported. Caspase-3-like activity has been observed to increase during the development of colorectal carcinoma (41Leonardos L. Butler L.M. Hewett P.J. Zalewski P.D. Cowled P.A. Cancer Lett. 1999; 143: 29-35Crossref PubMed Scopus (19) Google Scholar), thus representing a situation similar to that found in the absence of glucocorticoids during fibrosarcoma development. Although involved in some forms of apoptosis, caspase-1 also promotes inflammatory responses by regulating cytokine signaling (42Watson R.W. Rotstein O.D. Parodo J. Bitar R. Marshall J.C. William R. Watson G. J. Immunol. 1998; 161: 957-962PubMed Google Scholar). Increasing caspase-1 activity during fibrosarcoma development may therefore have implications not only for apoptosis but also for inflammation.Our results demonstrate that decreasing Bcl-xL expression results primarily from decreasing Bcl-x transcription. This correlates inversely with increasing transcription of the BPV-1 genome and E5, E6, and E7 oncogene expression in MF, AF, and FS cells (21Sippola-Thiele M. Hanahan D. Howley P.M. Mol. Cell. Biol. 1989; 9: 925-934Crossref PubMed Google Scholar,53Roca R. Kypta R.M. Vivanco M.d.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3113-3118Crossref PubMed Scopus (8) Google Scholar). Both BPV-1 E6 and E7 oncoproteins sensitize cells to apoptosis induced by tumor necrosis factor-α (43Liu Z. Liu Y. Hong Y. Rapp L. Androphy E.J. Chen J.J. Virology. 2002; 295: 230-237Crossref PubMed Scopus (9) Google Scholar); thus it can be speculated that these proteins may inhibit Bcl-x transcription. Notably, the angiogenic growth factor bFGF increases Bcl-x expression in epithelial cells (44Bryckaert M. Guillonneau X. Hecquet C. Courtois Y. Mascarelli F. Oncogene. 1999; 18: 7584-7593Crossref PubMed Scopus (56) Google Scholar). Because the expression of bFGF becomes increasingly extracellular at the AF stage of fibrosarcoma development (45Kandel J. Bossy-Wetzel E. Radvanyi F. Klagsbrun M. Folkman J. Hanahan D. Cell. 1991; 66: 1095-1104Abstract Full Text PDF PubMed Scopus (476) Google Scholar), it is possible that decreasing levels of intracellular bFGF contribute to decreasing Bcl-xL expression in AF and FS cells. In addition, the transcription factor AP-1 has been proposed to inhibit Bcl-xL expression (46Jacobs-Helber S.M. Wickrema A. Birrer M.J. Sawyer S.T. Mol. Cell. Biol. 1998; 18: 3699-3707Crossref PubMed Scopus (91) Google Scholar), and therefore the increased expression and activity of the AP-1 component proteins c-Jun and JunB in AF and FS cells (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar, 47Bossy-Wetzel E. Bravo R. Hanahan D. Genes Dev. 1992; 6: 2340-2351Crossref PubMed Scopus (68) Google Scholar) may also reduce Bcl-xLexpression.Increased Bcl-xL mRNA expression following glucocorticoid treatment has been observed in human gastric cancer cells (6Chang T.C. Hung M.W. Jiang S.Y. Chu J.T. Chu L.L. Tsai L.C. FEBS Lett. 1997; 415: 11-15Crossref PubMed Scopus (59) Google Scholar), where enhanced Bcl-xL mRNA stability was found to represent a part of the mechanism underlying the protective effect of dexamethasone. However, dexamethasone does not affect the stability of Bcl-xL mRNA in fibrosarcoma cells (53Roca R. Kypta R.M. Vivanco M.d.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3113-3118Crossref PubMed Scopus (8) Google Scholar). The reported increased expression of Bcl-xL mRNA in human myeloid leukemic cells required 24 h of hormone treatment (48Lotem J. Sachs L. Cell Growth & Differ. 1995; 6: 647-653PubMed Google Scholar), whereas we clearly detected up-regulation of Bcl-xLafter 2 h of dexamethasone treatment. Subsequent studies by other groups have also demonstrated the importance of increasing Bcl-xL expression for the inhibition of apoptosis by glucocorticoids in a variety of cell types (3Gorman A.M. Hirt U.A. Orrenius S. Ceccatelli S. Neuroscience. 2000; 96: 417-425Crossref PubMed Scopus (77) Google Scholar, 8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar, 31Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 49Messmer U.K. Winkel G. Briner V.A. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 1673-1683Crossref PubMed Scopus (36) Google Scholar). However, none of these reports identified the mechanism by which GR might control Bcl-x expression.Further extending the initial observations of these studies, we have demonstrated that the up-regulation of Bcl-xL expression by dexamethasone occurs at the transcriptional level and is increased during fibrosarcoma development. The increased expression of Bcl-xL is most likely achieved by specific binding of GR to the identified GRE-like sequences within the Bcl-x promoter. In fact, these sequences also confer hormone responsiveness to a heterologous promoter in COS-7 cells, indicating that the presence of ligand-activated GR is sufficient to induce Bcl-x transcription in other cell types. Furthermore, specific activation of the Bcl-x promoter by dexamethasone requires the cooperation of the various GREs identified, because deletion or mutation of these elements results in a reduction in responsiveness. This observation supports the prediction made by Nordeen and colleagues (35Nordeen S.K. Suh B.J. Kuhnel B. Hutchison C.D. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar) that most natural response elements are suboptimal elements and that cooperativity among these individually weak sites contributes to the inducibility of the promoter. Because Bcl-x expression is higher in untreated NF than in untreated FS cells, one possibility is that the Bcl-x promoter is repressed in FS cells and that dexamethasone treatment results in de-repression. Although this may be the case, the reduced expression of Bcl-x in FS cells is unlikely to involve GR binding to GREs in the Bcl-x promoter, because their mutation does not affect transcription in the absence of hormone.Importantly, Bcl-xL is responsible for the ability of dexamethasone to inhibit apoptosis in fibrosarcoma cells, as shown by ectopic expression of Bcl-xL. As expression of Bcl-xL decreases during fibrosarcoma development in the absence of glucocorticoids, increasing up-regulation of Bcl-xL expression by dexamethasone serves to maintain Bcl-xL levels during tumor development, and thus apoptosis remains approximately constant. In effect, Bcl-xLexpression becomes increasingly dependent on glucocorticoids during fibrosarcoma development. Increased Bcl-xL expression and reduced apoptosis correlate in FS cells with decreased caspase-3, but not caspase-1, activity in response to glucocorticoids. Such a correlation has also been reported by Messmer et al. (49Messmer U.K. Winkel G. Briner V.A. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 1673-1683Crossref PubMed Scopus (36) Google Scholar). Furthermore, levels of apoptosis in hormone-treated FS cells are comparable with those in earlier cell types, despite the presence of higher caspase-1 activity. These data indicate that caspase-3 is likely to play a more important role than caspase-1 in determining apoptosis in FS cells.It may be noteworthy that we also observed dexamethasone regulation of Bcl-x expression in both normal and transformed human mammary epithelial cells. Apoptosis of immortalized mouse mammary epithelial cells can be decreased by dexamethasone treatment, associated with increased Bcl-x mRNA expression, within 2 h of hormone treatment (8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar). Intriguingly, an increase in Bcl-xL protein expression following dexamethasone treatment was readily detectable in transformed SK-BR-3 breast cancer cells but was lower in normal epithelial cells (data not shown). This represents a parallel situation to that observed during fibrosarcoma progression, where the ability of hormone to induce Bcl-xL expression increases during tumor development; it may reflect the ability of normal cells to limit, or of tumor cells to enhance, regulation of gene expression by specific steroid hormone receptors as has been suggested previously (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar). Such a phenomenon may be a result of increased expression of steroid hormone receptor co-factors during tumor development, and indeed co-activators such as E6-AP and AIB1 are overexpressed in certain tumor types (50Sivaraman L. Nawaz Z. Medina D. Conneely O.M. O'Malley B.W. Breast Cancer Res. Treat. 2000; 62: 185-195Crossref PubMed Scopus (19) Google Scholar,51Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1422) Google Scholar).It is interesting to consider that the discovery of regulation of Bcl-xL expression by glucocorticoids during tumorigenesis may have clinical relevance. Glucocorticoids such as dexamethasone are used as anti-emetics in the treatment of several cancers. One study has identified a strong negative correlation between Bcl-xLexpression and sensitivity to a wide variety of cytotoxic agents in 60 cancer cell lines (18Amundson S.A. Myers T.G. Scudiero D. Kitada S. Reed J.C. Fornace Jr., A.J. Cancer Res. 2000; 60: 6101-6110PubMed Google Scholar). Our results suggest that Bcl-xLexpression (and thus cell survival) may be promoted by dexamethasone. Therefore, glucocorticoid treatment of tumors may reduce the cytotoxic effects of chemotherapy on tumor cells and may be contraindicated in those tumors with highly inducible Bcl-xL expression. A similar situation may exist in malignant gliomas, where glucocorticoids have been reported to interfere with chemotherapy response (52Weller M. Schmidt C. Roth W. Dichgans J. Neurology. 1997; 48: 1704-1709Crossref PubMed Scopus (91) Google Scholar). Importantly, indications that expression of Bcl-xL may be more glucocorticoid-dependent in tumor cells than in normal cells suggest that glucocorticoid therapy could have a certain degree of tumor specificity.The enhanced expression of Bcl-xL by GR during fibrosarcoma development reflects the unexpected transition in the transcriptional activity of GR and reveals an alternative mode of regulation for steroid hormone receptors. In summary, increasing GR transcriptional activity is likely to exert a protective effect by enabling the reduction of apoptosis at the tumor stage of fibrosarcoma development and, therefore, prolonging tumor cell survival. Apoptosis has been found to be widespread in many tumors (36Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12723) Google Scholar, 37Arends M.J. McGregor A.H. Wyllie A.H. Am. J. Pathol. 1994; 144: 1045-1057PubMed Google Scholar) and yet limited in others (38Kaufmann S.H. Gores G.J. Bioessays. 2000; 22: 1007-1017Crossref PubMed Scopus (180) Google Scholar). GR can display either pro-apoptotic or anti-apoptotic activity depending on cell context (39Tolosa E. Ashwell J.D. Neuroimmunomodulation. 1999; 6: 90-96Crossref PubMed Scopus (17) Google Scholar, 40Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1068) Google Scholar). We show here that in the absence of glucocorticoids apoptosis increases during fibrosarcoma development, whereas in the presence of dexamethasone it is reduced to those levels found in the earlier stages of the tumorigenic pathway. Few studies of caspase activity during tumor development have been reported. Caspase-3-like activity has been observed to increase during the development of colorectal carcinoma (41Leonardos L. Butler L.M. Hewett P.J. Zalewski P.D. Cowled P.A. Cancer Lett. 1999; 143: 29-35Crossref PubMed Scopus (19) Google Scholar), thus representing a situation similar to that found in the absence of glucocorticoids during fibrosarcoma development. Although involved in some forms of apoptosis, caspase-1 also promotes inflammatory responses by regulating cytokine signaling (42Watson R.W. Rotstein O.D. Parodo J. Bitar R. Marshall J.C. William R. Watson G. J. Immunol. 1998; 161: 957-962PubMed Google Scholar). Increasing caspase-1 activity during fibrosarcoma development may therefore have implications not only for apoptosis but also for inflammation. Our results demonstrate that decreasing Bcl-xL expression results primarily from decreasing Bcl-x transcription. This correlates inversely with increasing transcription of the BPV-1 genome and E5, E6, and E7 oncogene expression in MF, AF, and FS cells (21Sippola-Thiele M. Hanahan D. Howley P.M. Mol. Cell. Biol. 1989; 9: 925-934Crossref PubMed Google Scholar,53Roca R. Kypta R.M. Vivanco M.d.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3113-3118Crossref PubMed Scopus (8) Google Scholar). Both BPV-1 E6 and E7 oncoproteins sensitize cells to apoptosis induced by tumor necrosis factor-α (43Liu Z. Liu Y. Hong Y. Rapp L. Androphy E.J. Chen J.J. Virology. 2002; 295: 230-237Crossref PubMed Scopus (9) Google Scholar); thus it can be speculated that these proteins may inhibit Bcl-x transcription. Notably, the angiogenic growth factor bFGF increases Bcl-x expression in epithelial cells (44Bryckaert M. Guillonneau X. Hecquet C. Courtois Y. Mascarelli F. Oncogene. 1999; 18: 7584-7593Crossref PubMed Scopus (56) Google Scholar). Because the expression of bFGF becomes increasingly extracellular at the AF stage of fibrosarcoma development (45Kandel J. Bossy-Wetzel E. Radvanyi F. Klagsbrun M. Folkman J. Hanahan D. Cell. 1991; 66: 1095-1104Abstract Full Text PDF PubMed Scopus (476) Google Scholar), it is possible that decreasing levels of intracellular bFGF contribute to decreasing Bcl-xL expression in AF and FS cells. In addition, the transcription factor AP-1 has been proposed to inhibit Bcl-xL expression (46Jacobs-Helber S.M. Wickrema A. Birrer M.J. Sawyer S.T. Mol. Cell. Biol. 1998; 18: 3699-3707Crossref PubMed Scopus (91) Google Scholar), and therefore the increased expression and activity of the AP-1 component proteins c-Jun and JunB in AF and FS cells (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar, 47Bossy-Wetzel E. Bravo R. Hanahan D. Genes Dev. 1992; 6: 2340-2351Crossref PubMed Scopus (68) Google Scholar) may also reduce Bcl-xLexpression. Increased Bcl-xL mRNA expression following glucocorticoid treatment has been observed in human gastric cancer cells (6Chang T.C. Hung M.W. Jiang S.Y. Chu J.T. Chu L.L. Tsai L.C. FEBS Lett. 1997; 415: 11-15Crossref PubMed Scopus (59) Google Scholar), where enhanced Bcl-xL mRNA stability was found to represent a part of the mechanism underlying the protective effect of dexamethasone. However, dexamethasone does not affect the stability of Bcl-xL mRNA in fibrosarcoma cells (53Roca R. Kypta R.M. Vivanco M.d.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3113-3118Crossref PubMed Scopus (8) Google Scholar). The reported increased expression of Bcl-xL mRNA in human myeloid leukemic cells required 24 h of hormone treatment (48Lotem J. Sachs L. Cell Growth & Differ. 1995; 6: 647-653PubMed Google Scholar), whereas we clearly detected up-regulation of Bcl-xLafter 2 h of dexamethasone treatment. Subsequent studies by other groups have also demonstrated the importance of increasing Bcl-xL expression for the inhibition of apoptosis by glucocorticoids in a variety of cell types (3Gorman A.M. Hirt U.A. Orrenius S. Ceccatelli S. Neuroscience. 2000; 96: 417-425Crossref PubMed Scopus (77) Google Scholar, 8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar, 31Pecci A. Scholz A. Pelster D. Beato M. J. Biol. Chem. 1997; 272: 11791-11798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 49Messmer U.K. Winkel G. Briner V.A. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 1673-1683Crossref PubMed Scopus (36) Google Scholar). However, none of these reports identified the mechanism by which GR might control Bcl-x expression. Further extending the initial observations of these studies, we have demonstrated that the up-regulation of Bcl-xL expression by dexamethasone occurs at the transcriptional level and is increased during fibrosarcoma development. The increased expression of Bcl-xL is most likely achieved by specific binding of GR to the identified GRE-like sequences within the Bcl-x promoter. In fact, these sequences also confer hormone responsiveness to a heterologous promoter in COS-7 cells, indicating that the presence of ligand-activated GR is sufficient to induce Bcl-x transcription in other cell types. Furthermore, specific activation of the Bcl-x promoter by dexamethasone requires the cooperation of the various GREs identified, because deletion or mutation of these elements results in a reduction in responsiveness. This observation supports the prediction made by Nordeen and colleagues (35Nordeen S.K. Suh B.J. Kuhnel B. Hutchison C.D. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar) that most natural response elements are suboptimal elements and that cooperativity among these individually weak sites contributes to the inducibility of the promoter. Because Bcl-x expression is higher in untreated NF than in untreated FS cells, one possibility is that the Bcl-x promoter is repressed in FS cells and that dexamethasone treatment results in de-repression. Although this may be the case, the reduced expression of Bcl-x in FS cells is unlikely to involve GR binding to GREs in the Bcl-x promoter, because their mutation does not affect transcription in the absence of hormone. Importantly, Bcl-xL is responsible for the ability of dexamethasone to inhibit apoptosis in fibrosarcoma cells, as shown by ectopic expression of Bcl-xL. As expression of Bcl-xL decreases during fibrosarcoma development in the absence of glucocorticoids, increasing up-regulation of Bcl-xL expression by dexamethasone serves to maintain Bcl-xL levels during tumor development, and thus apoptosis remains approximately constant. In effect, Bcl-xLexpression becomes increasingly dependent on glucocorticoids during fibrosarcoma development. Increased Bcl-xL expression and reduced apoptosis correlate in FS cells with decreased caspase-3, but not caspase-1, activity in response to glucocorticoids. Such a correlation has also been reported by Messmer et al. (49Messmer U.K. Winkel G. Briner V.A. Pfeilschifter J. Br. J. Pharmacol. 2000; 129: 1673-1683Crossref PubMed Scopus (36) Google Scholar). Furthermore, levels of apoptosis in hormone-treated FS cells are comparable with those in earlier cell types, despite the presence of higher caspase-1 activity. These data indicate that caspase-3 is likely to play a more important role than caspase-1 in determining apoptosis in FS cells. It may be noteworthy that we also observed dexamethasone regulation of Bcl-x expression in both normal and transformed human mammary epithelial cells. Apoptosis of immortalized mouse mammary epithelial cells can be decreased by dexamethasone treatment, associated with increased Bcl-x mRNA expression, within 2 h of hormone treatment (8Schorr K. Furth P.A. Cancer Res. 2000; 60: 5950-5953PubMed Google Scholar). Intriguingly, an increase in Bcl-xL protein expression following dexamethasone treatment was readily detectable in transformed SK-BR-3 breast cancer cells but was lower in normal epithelial cells (data not shown). This represents a parallel situation to that observed during fibrosarcoma progression, where the ability of hormone to induce Bcl-xL expression increases during tumor development; it may reflect the ability of normal cells to limit, or of tumor cells to enhance, regulation of gene expression by specific steroid hormone receptors as has been suggested previously (22Vivanco M.d.M. Johnson R. Galante P.E. Hanahan D. Yamamoto K.R. EMBO J. 1995; 14: 2217-2228Crossref PubMed Scopus (31) Google Scholar). Such a phenomenon may be a result of increased expression of steroid hormone receptor co-factors during tumor development, and indeed co-activators such as E6-AP and AIB1 are overexpressed in certain tumor types (50Sivaraman L. Nawaz Z. Medina D. Conneely O.M. O'Malley B.W. Breast Cancer Res. Treat. 2000; 62: 185-195Crossref PubMed Scopus (19) Google Scholar,51Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1422) Google Scholar). It is interesting to consider that the discovery of regulation of Bcl-xL expression by glucocorticoids during tumorigenesis may have clinical relevance. Glucocorticoids such as dexamethasone are used as anti-emetics in the treatment of several cancers. One study has identified a strong negative correlation between Bcl-xLexpression and sensitivity to a wide variety of cytotoxic agents in 60 cancer cell lines (18Amundson S.A. Myers T.G. Scudiero D. Kitada S. Reed J.C. Fornace Jr., A.J. Cancer Res. 2000; 60: 6101-6110PubMed Google Scholar). Our results suggest that Bcl-xLexpression (and thus cell survival) may be promoted by dexamethasone. Therefore, glucocorticoid treatment of tumors may reduce the cytotoxic effects of chemotherapy on tumor cells and may be contraindicated in those tumors with highly inducible Bcl-xL expression. A similar situation may exist in malignant gliomas, where glucocorticoids have been reported to interfere with chemotherapy response (52Weller M. Schmidt C. Roth W. Dichgans J. Neurology. 1997; 48: 1704-1709Crossref PubMed Scopus (91) Google Scholar). Importantly, indications that expression of Bcl-xL may be more glucocorticoid-dependent in tumor cells than in normal cells suggest that glucocorticoid therapy could have a certain degree of tumor specificity. The enhanced expression of Bcl-xL by GR during fibrosarcoma development reflects the unexpected transition in the transcriptional activity of GR and reveals an alternative mode of regulation for steroid hormone receptors. In summary, increasing GR transcriptional activity is likely to exert a protective effect by enabling the reduction of apoptosis at the tumor stage of fibrosarcoma development and, therefore, prolonging tumor cell survival. We are very grateful to Gabriel Nuñez (University of Michigan Medical School, Ann Arbor) for providing the mouse Bcl-x(3.2) reporter, to Leanne Wiedemann (Stowers Institute for Medical Research, Kansas City, MO) for the human Bcl-xLcDNA, and to Roussel-Uclaf for RU 40555. We also thank Ramon Roca (Institute of Cancer Research, London) for help with various aspects of this work and Francesca Buffa (Institute of Cancer Research, London) for help with the statistical analysis. We very much appreciate Alan Ashworth and Pascal Meier for their critical reading of the manuscript.
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