Construction of a system that simultaneously evaluates CYP1A1 and CYP1A2 induction in a stable human-derived cell line using a dual reporter plasmid.
Human CYP1A1 and CYP1A2 genes are in a head-to-head orientation on chromosome 15 and are separated by a 23-kb intergenic space. To our knowledge, this is the first report on a stable cell line that contains the 23-kb full-length regulatory region and is able to simultaneously assess the transcriptional activation of CYP1A1 and CYP1A2 genes. The stable cell line that constitutively expresses the reporter activities was constructed by inserting the dual reporter plasmid containing the 23-kb region between the CYP1A1 and CYP1A2 genes into the chromosome. Transcriptional activation of the CYP1A1 and CYP1A2 genes was measured simultaneously using luciferase (Luc) and secreted alkaline phosphatase (SEAP) activities, respectively. To demonstrate the utility of the stable cell line, CYP1A1/1A2 induction by the majority of compounds previously identified as CYP1A1/1A2 inducers was measured. The results clearly show that all compounds caused induction of reporter activities. In addition to assessing transcriptional activation of the CYP1A1 and CYP1A2 genes by measuring reporter activities, we determined the intrinsic CYP1A1 and CYP1A2 mRNA levels by treating them with the same compounds. The results suggest that this stable cell line may be used to rapidly and accurately predict CYP1A1/1A2 induction.
Sato
Wataru
W
Department of Environmental and Health Science, Tohoku Pharmaceutical University, Sendai, Japan.
Suzuki
Hiroyuki
H
Sasaki
Takamitsu
T
Kumagai
Takeshi
T
Sakaguchi
Shuhei
S
Mizugaki
Michinao
M
Miyairi
Shinichi
S
Yamazoe
Yasushi
Y
Nagata
Kiyoshi
K
D002460:Cell Line; D002884:Chromosomes, Human, Pair 15; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D011995:Recombination, Genetic; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2