Post-transcriptional destabilization of estrogen receptor mRNA in MCF-7 cells by 12-O-tetradecanoylphorbol-13-acetate.

M Saceda,C Knabbe,R.B Dickson,M.E Lippman,D Bronzert,R.K Lindsey,M.M Gottardis,M.B Martin

doi:10.1016/s0021-9258(18)55199-1

Abstract

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the regulation of the estrogen receptor (ER) was investigated in this study. After treatment with 100 nM TPA the concentration of receptor protein was measured using an enzyme immunoassay. By 24 h the receptor protein declined by about 80% from a level of approximately 236 fmol of ER/mg of protein in control cells to 50 fmol of ER/mg of protein in cells treated with TPA. Similar results were obtained with an estrogen receptor ligand binding assay. After removal of TPA, the level of ER returned to control values. 4-alpha-Phorbol, a compound related to TPA, had no effect on ER. The effects of TPA on ER expression appear to be mediated by activation of protein kinase C as H-7, an inhibitor of protein kinase C, blocks these effects. In addition to the effect on ER protein, TPA treatment also resulted in a decrease in the steady-state level of ER mRNA as determined by a RNase protection assay. The metabolic inhibitor cycloheximide was unable to prevent the TPA-induced decrease in ER mRNA. Transcription run-off experiments demonstrated that TPA had no effect on ER gene transcription. A half-life study demonstrated that TPA decreased ER mRNA half-life by a factor of 6 from approximately 4 h in control cells to 40 min in TPA-treated cells. These data suggest that the decline in ER expression is mediated by post-transcriptional destabilization of ER mRNA.

Highlights

T o obtain total protein kinasCe activity thecells were homogenized by sonication in a buffer (50 mM Tris/HCl, pH 7.5, containing 5 mM EDTA, 10 mM EGTA, 0.3% (w/v) P-mercaptodata presented in Fig. 1 show that 100 nM TPA treatment resulted in a decline in total receptor protein by about 80%
Receptor protein declined from a level of approximately 235 fmol of estrogen receptor (ER)/mg of protein in control cells to 50 fmol of ER/ mg of protein incells treated with TPA
The supernatant was saved, and the TPA, the normal level of ER protein was achievedby 48 h in pellet was treated for 20 min a t 4 "C with the same buffer with the addition of 0.1% of Nonidet P-40 and centrifuged a t 100,000 X g for 1 h a t 4 "C

Summary

Introduction

Ahalf-life tration and binding capacity, the steady-stalteevels of recepstudy demonstrated that TPA decreased ER mRNA half-life by a factor of 6 from approximately 4 h in control cells to 40 min in TPA-treatedcells These data suggest that the decline in ER expression is mediated by post-transcriptional destabilizationof ER mRNA. The Phorbol esters are tumor promoters that display a wide decrease in ER mRNA was not accompaniedbyasimilar range of activities in mammalian tissues ancedlls They have decrease in ER gene transcription, as determined by run-off been shown to produce different and often opposibteiological experiments, suggesting thatthepredominantmechanism responses in different types of cells. The H-7, a known inhibitorof protein kinaseC [22].precise mechanism of action of phorbol esters remains un- this report confirms the observation that TPA induces epiknown it is believed that phorbol esters may mediate many dermal growth factor receptor (EGFr) and c-myc transcripof their effects by regulating the activity and expression of tion. S.).The costs of publication of this article were defrayed in part by the payment of page charges

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