Development of a real-time in vivo transcription assay: application reveals pregnane X receptor-mediated induction of CYP3A4 by cancer chemotherapeutic agents.
We report the development of a rapid real-time assay that measures the transcription of luciferase reporter genes in transduced mouse hepatic cells in vivo. Luciferase activity is noninvasively measured by whole-body optical imaging within hours of the hydrodynamic injection of as little as 1 microg of naked DNA. Transcription of genes introduced as linearized DNA can be serially assayed for weeks in each animal. Transcription was quantified by extracorporal monitoring of bioluminescence as well as or better than by traditional in vitro bioluminescence assay. Our assay allows the measurement of transcription as it occurs, under the most informative biological conditions (i.e., in a living, intact organ). Furthermore, it substantially reduces the cost, time, and number of animals required for analysis of gene expression. The utility of the method is demonstrated in the discovery that topotecan and etoposide are ligands of pregnane X receptor that induce CYP3A4 transcription.
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. erin.schuetz@stjude.org
Erin
E
Schuetz
Lubin
L
Lan
Kazuto
K
Yasuda
Richard
R
Kim
Thomas A
TA
Kocarek
John
J
Schuetz
Stephen
S
Strom
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D013629:Tamoxifen / Q000494:pharmacology; D019772:Topotecan / Q000494:pharmacology; D014158:Transcription, Genetic
D000970:Antineoplastic Agents; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D013629:Tamoxifen; D005047:Etoposide; D019772:Topotecan; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human