Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis Toshio Tsutsui, a Hiroko Koide, a Hiroko Fukahori, a Katsuhiro Isoda, a Shinji Higashiyama, a Isamu Maeda, a Fumi Tashiro, b Eiji Yamato, b Jun-Ichi Miyazaki, b Junji Yodoi, c Masaya Kawase, a and Kiyohito Yagi a, * a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan b Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan c Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan Received 12 June 2003 Abstract A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recom- bination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyteapoptosis.Italsosignificantlyextendedthelifespanofhepatocytesculturedconventionallyonpolystyreneplates.Liver- specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Thioredoxin; Adenovirus; Hepatocytes; Bioartificial liver; Apoptosis; Necrosis; Gene transfection A bioartificial liver support system (BAL) using liver cells has been studied for the treatment of patients with fulminant hepatic failure or as a transitional treatment prior to liver transplantation [1]. As the first-generation BAL, clinical trials in the USA have been carried out using porcine hepatocytes cultured on collagen-coated dextran microcarriers and packed in hollow-fiber-type bioreactors [2]. With the aim of developing a high-per- formance BAL, our previous work was concerned with improving the scaffolds for hepatocyte cultivation. We showed that chitosan and a polyamidoamine dendrimer are effective in the preparation of scaffolds for hepato- cytes [3,4]. We also showed that chemical modifications of these polymers are useful for improving the cell attachment and the maintenance of cell viability and functions [5,6]. The development of a second-generation BAL will requireimprovementofthecellitself.Inapreviousstudy, the gene encoding glutamine synthetase was introduced into hepatoma cells, which were then packed in a biore- actor.TheserecombinantHepG2cellsimprovedammo- niaremovalwhenappliedtopigswithhepaticfailure[7]. Tzanakakis et al. [8] transfected hepatocyte spheroids withthecytochromeP4502B1gene.Inthisstudy,weused the unique strategy of not only enhancing one liver function but of maintaining all liver functions by intro- ducingintohepatocytesagenethatconfersresistanceto variouskindsofstress.Wechosethethioredoxin(TRX) gene as a promising candidate for conferring this pro- tection on primary cultured hepatocytes. TRX is a low- molecular-weight redox protein (12kDa) found in both prokaryoticandeukaryoticcells[9].Cysteineresiduesat Biochemical and Biophysical Research Communications 307 (2003) 765–770 www.elsevier.com/locate/ybbrc BBRC * Corresponding author. Fax: +81-6-6879-8195. E-mail address: yagi@phs.osaka-u.ac.jp (K. Yagi). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01253-1