Effects of dexamethasone, heat shock, and serum responses on the inhibition of Hsc70 synthesis by antisense RNA in NIH 3T3 cells.
A dexamethasone (Dex)-inducible antisense RNA expression vector was constructed that contains the 5'-untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand-specific probes was used to demonstrate the Dex-inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex-treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex-treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex-treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex-treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum-stimulated cultures expressing antisense RNA.
Li
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Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3044, USA.
Hightower
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LE
D016475:3T3 Cells; D000818:Animals; D001790:Blood Physiological Phenomena; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D006358:Hot Temperature; D051379:Mice; D012313:RNA / Q000378:metabolism; D016372:RNA, Antisense / Q000502:physiology*; D012769:Shock / Q000378:metabolism*; D014158:Transcription, Genetic
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D016372:RNA, Antisense; D012313:RNA; D003907:Dexamethasone