JOURNAL OF BIOSCIENCE AND BIOENOINEEKINO Vol. 91, No. 6, 576-580. 2001 Aurintricarboxylic Acid Exerts Insulin-Like Growth Stimulating Effects on Chinese Hamster Ovary Cells under Serum-Free Conditions CHI-HSIEN LIU, 1 I-MING CHU, 1. AND SHIAW-MIN HWANG 2 Department of Chemical Engineering, National Tsing-Hua University 1 and Food Industry Research and Development Institute, 2 Hsinchu, Taiwan 300, ROC Received 8 February 2001/Accepted 23 March 2001 When aurintricarboxylic acid (ATA) was added at a concentration of 30 mg/l in DME/F12 medium to Chinese hamster ovary (CHO) NTHU-108 cells in static six-well plates, some of the cells exhibited adherent growth while others grew in suspension. Beyond the critical concentration of ATA, CHO cells grew in single- cell suspension. In the same serum-free medium, insulin at a level of 0.5 mg/1 was found to be the most im- portant protein ingredient promoting cell growth. We used the respective kinase inhibitors to investigate their influence on the cell proliferation induced by ATA and insulin. It is interesting that the inhibition by seven kinase inhibitors of ATA-induced proliferation is similar to that of insulin-induced proliferation. It is possible that ATA mimics insulin and influences the mitogen activated signal transduction to induce the proliferation of ClIO cells. Although the actual mechanism of the proliferation of CHO cells by ATA is unclear, ATA sup- ported the long-term proliferation of CHO cells under serum-free conditions and thus could be used as a good substitute for insulin in the formulation of protein-free media. [Key words: aurintricarboxylic acid, insulin, fusion protein, CHO cells, serum-free media] Chinese hamster ovary (CHO) cells are often used for the production of genetically engineered proteins for therapeutics. Most processes using CHO ceils are deve- loped without serum in the medium. Since the response to the absence of serum can vary from one recombinant clone to the next, medium optimization is a necessary and time-consuming procedure (1). However, it is useful to screen serum substitutes using statistical methods (2) and to find new additives for simplifying serum-free me- dia development. Since some animal-derived ingredients in serum-free media still have problems such as being expensive and unknown contamination, the best choice for manufacturing pharmaceutical proteins is protein-free me- dium, Aurintricarboxylic acid (ATA) (Fig 1), a negatively charged triphenylmethane dye, is an inhibitor of nuclease (3) and topoisomerase II (4). ATA has been reported to prevent apoptosis in sympathetic neurons, PC12 cells deprived of nerve growth factor (5), and human neuroblastoma SH-SY5Y cells deprived of serum (6). Recently, the mechanism of the anti-apoptosis activ- ity of ATA was elucidated by Deng et al. (7). ATA induces Ser-70 phosphorylation of Bcl 2, functionally stabilizing the Bcl2-Bax heterodimerization. However, in this respect ATA acts indirectly. ATA has a molecular weight of 442 Da and can polymerize into larger poly- mers of up to 6000 Da (8). Because ATA can not pene- trate the intact cell membrane, it is presumably acting via transmembrane proteins (9). Three negatively charg- ed carboxylic acids of ATA facilitate extensive ionic binding of the molecule to positively charged groups of cellular proteins (10). Several studies have demonstrat- ed that ATA induced the cellular signal transduction system by increasing the level of phosphorylation of tyro- sine and serine residues in related proteins (6, 11-13). ATA has also been demonstrated to imitate by phos- * Corresponding author, e-mail: imchu@che.nthu.edu.tw phone: +886-3-5713704 fax: +886-3-5715408 phorylation the actions of several cytokines or growth factors such as epidermal growth factor (EGF) (14), prolactin (9) and interleukin-3 (IL-3) (12). In addition, ATA was reported to be an iron-presenting compound due to its mild chelating ability and could be used as a transferrin substitute in serum-free systems (15). We previously developed a serum-free medium suita- ble for the proliferation of and production of fusion pro- teins in CHO cells (2). However, this medium still con- tains some animal-derived proteins such as insulin, trans- ferrin, and linoleic acid-bovine serum albumin complex. In this study, insulin was found to be the most impor- tant exogenous ingredient for the growth of CHO cells in serum-free medium, and ATA was found to mimic the function of insulin and support the long-term prolifera- tion of CHO cells under protein-free and serum-free con- ditions. MATERIALS AND METHODS Cell line and cell culture Genetically engineered CHO NTHU108 cells were obtained from Dr. Tse-Wen Chang of the Department of Life Science at the National Tsing-Hua University (I-Isinchu, Taiwan). These cells pro- duced a fusion protein composed of CD20 and an IgG- Fc T4 fragment as the N-terminal fusion partner. The O ~ COOH FIG. 1. Structureof aurintricarboxylic acid. 576