title: Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.
abstract: Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.
authors.affiliation: The Molecular Imaging Program at Stanford (MIPS), The Department of Radiology and Bio-X Program, Stanford University, Stanford, CA 94305-5344, USA.
authors.forename: Manickam
authors.initials: M
authors.lastname: Krishnan
authors.forename: Jinha M
authors.initials: JM
authors.lastname: Park
authors.forename: Feng
authors.initials: F
authors.lastname: Cao
authors.forename: Dongxu
authors.initials: D
authors.lastname: Wang
authors.forename: Ramasay
authors.initials: R
authors.lastname: Paulmurugan
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Tseng
authors.forename: Mark L
authors.initials: ML
authors.lastname: Gonzalgo
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000235:genetics*; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D006877:Hydroxamic Acids / Q000494:pharmacology; D049409:Luciferases, Firefly / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D032386:Myoblasts, Cardiac / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D006877:Hydroxamic Acids; C012589:trichostatin A; D014212:Tretinoin; D049409:Luciferases, Firefly; D015254:DNA Modification Methylases; D001374:Azacitidine