Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth.

The effect of 17 beta-estradiol (E2) on growth of GH4C1 rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1:1 (vol/vol) mixture of F12-DME supplemented with 50 micrograms/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 micrograms/ml insulin, 10 micrograms/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3'-triiodothyronine (T3), 50 microM ethanolamine, and 500 micrograms/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E2 responsive only when growth was retarded by reducing the T3 concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E2 to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E2 to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors.