Biochimica et Biophysica Acta, 1008 (1989) 315-321 315 Elsevier BBAEXP 91972 Thyroid hormone effect on a-fetoprotein and albumin coordinate expression by a human hepatoma cell line Raffaella Conti, Costante Ceccarini and Mario F. Tecce Cell Biology Laboratory. Sclavo Research Center. Siena (Italy) (Received 2 February 1989) Key words: Gene expression; a-Fetoprotein: Albumin; Thyroid hormone; Triiodothyronine; (Human hepatoma cell) The action of triiedathymnine on flae production of ¢-fetoprotein and albumin in serum-free cultures of Hep G2 hub,an hepatoma cells was examined. Our da~ showed that a marked inhibition ~up to 8-fold) of a-fetoprotein secretion and an increase in albumin (up to &fold) are produced by 10-8 M ~|odoflty~nine. These effects were slow in their onset and for completion required 20-25 days of treatment with the hormone. However, an exposure of the cel|s to t~- iedathyronine for only the first 4 h was sufficient to affect, in a similar way, the secretion of a-fetoprotein and albumin when measm~d |5 days after treatment. The secretion of the two proteins pal'a[|els their intraeeH~ur ieve|s. The daerease in a-fetoproteln production can be explained by a reduction of the RNA coding for the protein. The same is ¢~enfiaHy true also for albumin hlcreased secretion and related mRNA expression. Introduction Triiodothyronine is the active form of thyroid hormone in viva [1,2]. This hormone exerts its biological activity via nuclear receptors which have been shown to share common features with nuclear steroid receptors, which seem likely to represent the normal cellular coun- terpart of the viral erb-A oncogene protein [3-5]. We have previously described the purification of high quantities of pure a-fetoprotein from the serum-free culture medium of the human hepatoma cell fine, Hep G2 [6]. Since these cells are induced to secrete a higher concentration of a-fetoprotein by the subtraction of serum, we hypothesized that serum components, such as thyroid hormones, might account for this effect, i.e., the suppression of a-fetoprotein synthesis. In mammals, this protein is regulated with regards to development: during the fetal life it is ~he major serum protein secreted by fiver and yolk sac. In contrast, albumin rises from very low levels to the adult levels soon after birth and replaces a-fetoprotein in serum [7]. Various proofs indi- cate that the expression of the two proteins occurs in an alternative fashion and that the related genes are coor- Abbreviations: PBS, phosphate-buffered saline; Hepes, 4-(2-hydroxy- ethyl)-I-piperazineethanesulfonic acid. Correspondance: M.F. Tecce~Sclavo Research Center, Via Fiorentina 1, 53100, Siena, Italy. dinately regulated [8-10]. Since a number of hepatic proteins show a regulation ~y thyroid hormone [11-14], we attempted to determ'~ine whether triiodothyronine could influence albumin and a-fetoprotein production in human hepatoma cells. The use of various concentrations of the hormone in our cell culture system showed that triiodothyronine effectively inhibits a-fetoprotein production and, at the same time, stimulates the secretion of albumin. The RNAs from treated cells have been analyzed by dot-blot hybridization and Northern blot, showing a decrease in a-fetoprotein rnRNA levels, while albumin mRNA was increased. Materials and Methods Cell culture and adaptation in serum-free medium. Stock cultures of Hep G2 cells (obtained from Dr. B.B. Knowles, The Wistar Institute of Anatomy, Phila- delphia, PA [15]) were transferred every 6 or 7 days at 5- 10 4 cells/cm 2 in minimal essential medium (Gibco) supplemented with 10~ (v/v) fetal calf serum (Seromed). Adaptation to the absence of serum was achieved by transferring confluent cells to serum-free RPMI 1640 (Gibco) supplemented with 3.10 -8 M Na2SeO3, 50 ~g/l~ penicilfin-G and 50 pg/ml strepto- mycin [6]. From each confluent flask, three experimen- tal flasks were prepared. In these conditions, cells in the flasks continue to grow and detach, so that the total amount of cells remains relatively constant. 0167-4781/89/$03.50 © 1989 E]sevier Science Publishers B.V. (Biomedical Division)