Estradiol-induced down-regulation of estrogen receptor. Effect of various modulators of protein synthesis and expression.
Incubation of MCF-7 cells with estradiol (E2) down-regulates estrogen receptor (ER) resulting in a progressive reduction of the capacity of cells to concentrate selectively [3H]E2. Scatchard plot analysis failed to detect any transformation of residual receptors into peptides of lower binding affinity. [3H]Estrone gave an identical ER disappearance pattern with an ER half-life comprised between 2 and 3 h. A similar value was established by incubating the cells with [3H]tamoxifenaziridine ([3H]TAZ) for 1 h before the addition of excessive unlabeled E2 which induced ER-down regulation and impeded any further labeling of the residual receptors. Submission of the [3H]TAZ labeled cell extracts to SDS-PAGE revealed no progressive emergence of low molecular weight cleavage products of the receptor (< 67 kDa). Two inhibitors of protein kinases, H-7 at 40 microM and H-89 at 20 microM, failed to block the E2-induced ER down-regulation. On the contrary, the protein phosphatases 1 and 2A inhibitor, okadaic acid, was effective with concentrations higher than 0.1 microM indicating that a dephosphorylation mechanism was involved in this phenomenon. Cycloheximide (CHX) also significantly reduced the receptor decrease at concentrations higher than 1 microM. G-C specific intercalating agents [actinomycin D (AMD) and chromomycin A3 at 1 microM] also prevented ER disappearance; ethidium bromide (EB) and quinacrine were ineffective. AMD and CHX operated immediately after their addition to the medium indicating an inhibitory action on the synthesis of an RNA and/or a peptide with high turnover rate involved in ER decline. Moreover, AMD produced its suppressive effects under conditions impeding any labeling of newly synthetized receptors (i.e. [3H]TAZ with an excess of unlabeled E2) rejecting the possibility of an increasing ER production which may partially hamper its disappearance. Finally, E2-induced ER mRNA down-regulation was similarly abolished by AMD while EB and CHX were devoid of effect.
Borrás
M
M
Laboratoire J. C. Heuson de Cancérologie Mammaire, Institut J. Bordet, Brussels, Belgium.
Hardy
L
L
Lempereur
F
F
el Khissiin
A H
AH
Legros
N
N
Gol-Winkler
R
R
Leclercq
G
G
D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology*; D004970:Estrone / Q000378:metabolism / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011960:Receptors, Estrogen / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured
D007364:Intercalating Agents; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D013629:Tamoxifen; D014316:Tritium; D003609:Dactinomycin; D004970:Estrone; D004958:Estradiol; C031859:tamoxifen aziridine; D003513:Cycloheximide; D010744:Phosphoric Monoester Hydrolases