Paradoxical production of target protein using antisense RNA expression vectors.
We used antisense RNA in a protocol designed to reduce estrogen receptor (ER) content in human breast cancer cells and observed paradoxical increases in ER levels. ER protein activity was measured using a highly sensitive reporter gene assay that relies on the ability of functional ER to bind a consensus estrogen response element (ERE) and drive the production of chloramphenicol acetyl-transferase (CAT). Upon transient transfection of ER-positive cell lines with three different vectors containing the full-length ER cDNA cloned in an antisense orientation, we observed unexpected increases in ER-driven CAT activity. To further investigate this phenomenon, expression from the antisense ER vectors was studied in an ER-negative breast tumor cell line, MDA-MB-453. ER activity was observed in these ER-negative cells upon transient transfection with each of three antisense ER vectors, but not from control vectors. Expression of ER from antisense constructs was 30-100-times less efficient than ER expression from isogenic sense constructs. The paradoxical ER activity was consistent with expected ER behavior in that it exhibited characteristic binding to the natural ligand, 17 beta-estradiol (E2), and it was inhibited by the antiestrogens, 4-hydroxy-tamoxifen (OHT) and ICI 164384 (ICI). Control vectors containing a truncated antisense ER cDNA produced no ER activity. Although the mechanism for this ER expression has not been determined, it appears likely that it is due to transcription off the opposite strand of the antisense construct.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmacy and Pharmaceutical Chemistry, University of California, San Francisco 94143-0446.
R
R
Williard
C C
CC
Benz
J D
JD
Baxter
P
P
Kushner
C A
CA
Hunt
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D016372:RNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D016372:RNA, Antisense; D011960:Receptors, Estrogen