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Prenylation inhibitors stimulate both estrogen receptor alpha transcriptional activity through AF-1 and AF-2 and estrogen receptor beta transcriptional activity.

Cestac P, Sarrabayrouse G, Médale-Giamarchi C, Rochaix P, Balaguer P, Favre G, Faye JC, Doisneau-Sixou S - Breast Cancer Res. (2004)

Bottom Line: In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha.The roles of both AF-1 and AF-2 are significant in this effect.Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département Innovation Thérapeutique et Oncologie Moléculaire, Centre de Physiopathologie de Toulouse Purpan, INSERM U563 and Institut Claudius Regaud, Toulouse, France. cestac.p@chu-toulouse.fr

ABSTRACT

Introduction: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta.

Methods: The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity.

Results: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established.

Conclusions: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

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Effects of prenyltransferase inhibitors on estrogen response element (ERE)-dependent luciferase activity (a) in stably transfected MELN cells and (b) in transiently transfected MCF-7 cells. Both MELN cells and MCF-7 cells are deprived of estradiol (E2) for 4 days, and the MCF-7 cells are transiently transfected with the ERE-β-glob-Luciferase and the Renilla luciferase plasmids. Three hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle). Twenty-four hours later, cells were stimulated with E2 (5 nM) or ethanol and were treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of two independent experiments. Results obtained show that prenylation inhibitors statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone in MELN cells only (* P < 0.02).
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Figure 1: Effects of prenyltransferase inhibitors on estrogen response element (ERE)-dependent luciferase activity (a) in stably transfected MELN cells and (b) in transiently transfected MCF-7 cells. Both MELN cells and MCF-7 cells are deprived of estradiol (E2) for 4 days, and the MCF-7 cells are transiently transfected with the ERE-β-glob-Luciferase and the Renilla luciferase plasmids. Three hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle). Twenty-four hours later, cells were stimulated with E2 (5 nM) or ethanol and were treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of two independent experiments. Results obtained show that prenylation inhibitors statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone in MELN cells only (* P < 0.02).

Mentions: We previously showed that both FTI-277 and GGTI-298 markedly enhance ER-mediated transcription in MELN cells [17], a clone of MCF-7 cells stably transfected with the ERE-β globin-luciferase reporter gene (Fig. 1a). After 5 days of E2 deprivation, cells were treated or not with 5 nM E2, in the presence of or in the absence of prenyltransferase inhibitors, and the luciferase activity was quantified 16 hours later. For optimal FTI-277 treatments, a 24-hour preincubation was systematically performed before E2 addition, whereas GGTI-298 was added simultaneously with E2 [17]. We confirmed the efficiency and specificity of FTI-277 and GGTI-298 to inhibit protein prenylation in MELN cells in these experimental conditions. Indeed, for this purpose, we checked whether HDJ2, a farnesylated protein, and Rap1A, a geranylgeranylated protein, were indeed not prenylated in the presence of 10 μM FTI-277 and 5 μM GGTI-298, respectively (data not shown).


Prenylation inhibitors stimulate both estrogen receptor alpha transcriptional activity through AF-1 and AF-2 and estrogen receptor beta transcriptional activity.

Cestac P, Sarrabayrouse G, Médale-Giamarchi C, Rochaix P, Balaguer P, Favre G, Faye JC, Doisneau-Sixou S - Breast Cancer Res. (2004)

Effects of prenyltransferase inhibitors on estrogen response element (ERE)-dependent luciferase activity (a) in stably transfected MELN cells and (b) in transiently transfected MCF-7 cells. Both MELN cells and MCF-7 cells are deprived of estradiol (E2) for 4 days, and the MCF-7 cells are transiently transfected with the ERE-β-glob-Luciferase and the Renilla luciferase plasmids. Three hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle). Twenty-four hours later, cells were stimulated with E2 (5 nM) or ethanol and were treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of two independent experiments. Results obtained show that prenylation inhibitors statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone in MELN cells only (* P < 0.02).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1064103&req=5

Figure 1: Effects of prenyltransferase inhibitors on estrogen response element (ERE)-dependent luciferase activity (a) in stably transfected MELN cells and (b) in transiently transfected MCF-7 cells. Both MELN cells and MCF-7 cells are deprived of estradiol (E2) for 4 days, and the MCF-7 cells are transiently transfected with the ERE-β-glob-Luciferase and the Renilla luciferase plasmids. Three hours after transfection, cells were treated or not with FTI-277 (10 μM or dithiothreitol/dimethylsulfoxide vehicle). Twenty-four hours later, cells were stimulated with E2 (5 nM) or ethanol and were treated or not with FTI-277 or GGTI-298 (10 μM or 5 μM, respectively, or dithiothreitol/dimethylsulfoxide vehicle). Luciferase activity was quantified 16 hours after E2 addition, as described in Materials and methods. Results are expressed in arbitrary units after normalization. Error bars indicate the mean values ± standard deviation from triplicate experiments, and the results are representative of two independent experiments. Results obtained show that prenylation inhibitors statistically increase the luciferase activity induced by E2 compared with the activity induced by E2 alone in MELN cells only (* P < 0.02).
Mentions: We previously showed that both FTI-277 and GGTI-298 markedly enhance ER-mediated transcription in MELN cells [17], a clone of MCF-7 cells stably transfected with the ERE-β globin-luciferase reporter gene (Fig. 1a). After 5 days of E2 deprivation, cells were treated or not with 5 nM E2, in the presence of or in the absence of prenyltransferase inhibitors, and the luciferase activity was quantified 16 hours later. For optimal FTI-277 treatments, a 24-hour preincubation was systematically performed before E2 addition, whereas GGTI-298 was added simultaneously with E2 [17]. We confirmed the efficiency and specificity of FTI-277 and GGTI-298 to inhibit protein prenylation in MELN cells in these experimental conditions. Indeed, for this purpose, we checked whether HDJ2, a farnesylated protein, and Rap1A, a geranylgeranylated protein, were indeed not prenylated in the presence of 10 μM FTI-277 and 5 μM GGTI-298, respectively (data not shown).

Bottom Line: In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha.The roles of both AF-1 and AF-2 are significant in this effect.Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département Innovation Thérapeutique et Oncologie Moléculaire, Centre de Physiopathologie de Toulouse Purpan, INSERM U563 and Institut Claudius Regaud, Toulouse, France. cestac.p@chu-toulouse.fr

ABSTRACT

Introduction: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta.

Methods: The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity.

Results: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established.

Conclusions: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

Show MeSH
Related in: MedlinePlus