Regulation of exogenous gene expression by superoxide.
OBJECTIVE
Regulation of gene expression after gene introduction is a problematic aspect of gene therapy. Transcription regulates gene-specific transcriptional factors, which bind to regulatory regions in the promoter. The cytomegalovirus long terminal repeat (CMV-LTR) has a TPA response element (TRE) as a binding site for activator protein 1 (AP-1), which is induced by oxidative stress. The purpose of this study was to regulate exogenous gene expression in a vector with CMV-LTR using oxygen radicals.


METHODS
We used two plasmids (1) pQBI25 encoding CMV-LTR and red-shift green fluorescent protein (rsGFP) cDNA and (2) pRc/CMV-SOD encoding CMV-LTR and human Cu, Zn-superoxide dismutase (SOD) cDNA. FR cells were transfected with pQBI25 (FR-pQBI25 cells), and L2 cells were transfected with pRc/CMV-SOD (L2-pRc/CMV-SOD cells). Each type of cell was exposed to oxygen radicals using paraquat for 24 h. Levels of c-fos, c-jun and rsGFP mRNAs were determined using reverse transcription polymerase chain reaction (RT-PCR). Levels of rsGFP protein were measured by fluorometry. Total SOD activity was measured using the nitrite method.


RESULTS
Levels of c-fos, c-jun (AP-1 composition protein) and rsGFP mRNA were induced significantly by oxygen radical exposure in FR-pQBI25 cells. A positive correlation was observed between levels of c-fos mRNA and rsGFP mRNA and also between levels of c-jun mRNA and rsGFP mRNA. Levels of rsGFP protein were also induced significantly. Total SOD activity was induced significantly by oxygen radical exposure in L2-pRc/CMV-SOD cells.


CONCLUSIONS
This study suggests that gene expression driven by the CMV- LTR promoter may be regulated by oxygen radicals.
Kinoshita
Atsushi
A
Department of Drug Informatics, Faculty of Pharmaceutical Sciences, Josai University, 1-1, Keyakidai, Sakado, Saitama, 350-0248, Japan.
Kobayashi
Daisuke
D
Saitoh
Yukiya
Y
Tanabe
Naoto
N
Uchino
Katsuyoshi
K
Nishiguchi
Kohshi
K
Okumura
Katsuhiko
K
Komada
Fusao
F
D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D016762:Genes, fos; D016758:Genes, jun; D015316:Genetic Therapy; D010269:Paraquat / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013482:Superoxide Dismutase / Q000096:biosynthesis; D013481:Superoxides / Q000494:pharmacology*; D020079:Terminal Repeat Sequences
D012333:RNA, Messenger; D013481:Superoxides; D013482:Superoxide Dismutase; D010269:Paraquat