Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents.
The effects of cyclic AMP (cAMP)-elevating agents on the activity of cis-acting gene promoter sequences are frequently studied using the luciferase-reporter-gene system. The aim of the present study was to assess whether cAMP-elevating agents induce any changes in the level of luciferase activity independently of a transcriptional activation of promoter elements. Chloramphenicol acteyltransferase (CAT) and luciferase reporter genes under the control of the same promoter elements were transiently expressed in primary cultures of human vascular smooth-muscle cells. Transfected cells were treated with a cell-permeable and non-hydrolysable cAMP analogue, 2'-O-monobutyryl-8-bromo cAMP, or with the cAMP-elevating agents forskolin and prostaglandin E1 (PGE1). Forskolin and PGE1 induced a significant increase in the level of luciferase activity, but had no effect on CAT activity. Conclusions based solely on the use of the luciferase-reporter-gene system in studies involving promoter activation by cAMP-elevating agents could therefore be misleading.
Benzakour
O
O
Charter Molecular Genetics Laboratory, Thrombosis Research Institute, London, U.K.
Kanthou
C
C
Dennehy
U
U
al Haq
A
A
Berg
L P
LP
Kakkar
V V
VV
Cooper
D N
DN
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000293:Adolescent; D000328:Adult; D000527:Alprostadil / Q000494:pharmacology; D002478:Cells, Cultured; D002648:Child; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D000527:Alprostadil