Evidence for a Novel Pituitary Factor That Potentiates the Mitogenic Effect of Estrogen in Human Breast Cancer Cells1

Estrogen, prolactin, and other tissue-derived factors are impli cated in the etiology and pathophysiology of human breast cancer (HBC). In a previous study, we demonstrated that a factor(s) secreted by rat pituitary tumor cells (GH3) synergizes with estrogen to induce growth of HBC cells (T-47D) transplanted into athymic nude mice. The present studies were carried out to characterize further this pituitary growth factor. Pituitary tumor cell lines (GH3, GHi, 235-1, and AtT-20) and normal rat pituitaries were transplanted s.c. into estrogen-treated (estradici valerate injection, 500 ng/i 4 days) athymic nude mice which also received T-47D cells. The influence of the presence of these normal and tumorous pituitary cells on growth (size and weight) of T-47D tumors was monitored for 49 to 56 days. The results indicate that factor(s) from normal rat pituitary glands as well as from the GHi and GH3 but not 235-1 and AtT-20 pituitary tumor cells were able to potentiate the growth of T-47D tumors in estrogenized mice. To ascertain whether or not prolactin and/or growth hormone are responsible for the growth-promoting activity, pu rified human and ovine growth hormone and ovine prolactin were administered to estrogenized athymic nude mice either by daily s.c. injection (100 ^g/day) or by constant infusion using Alzat osmotic minipumps (1.25 and 5.0 ¿¿g/h) for 49 to 56 days. None of these treatments stimulated the growth of the T-47D tumors, suggesting that prolactin, growth hormone, and their intermedi ates may not be directly involved. We further determined whether the factor from pituitary tumor cells was present in serum-free conditioned medium and could stimulate the growth of HBC cells in vitro. Conditioned medium from GH3 and GHt but not from 235-1 and AtT-20 pituitary cells significantly stimulated growth of T-47D cells in the presence of estradici (10~10M)after 12 days of culture in a serum-free medium (Dulbecco's modified Eagle's medium containing bovine serum albumin, 0.5 mg/ml). Optimal serum-free growth of T-47D cells (2-fold above control) was observed in the presence of estradici (10"1°M) and conditioned medium (30% v/v) from 48-h cultures of GH3 cells. The bovine serum albumin concentration of the serum-free medium (Dul becco's modified Eagle's medium) was also important: optimal T-47D cell proliferation was observed with BSA between 0.5 and 2.0 mg/ml. Conditioned medium preparations from serum-pretreated flasks (without cells) from GH3 cell monolayers for zero time and from actinomycin D plus cycloheximide-inhibited GH3 cells were inactive. In addition, the GH3 pituitary factor poten tiated the estrogen effect only in those HBC cells that contained estrogen receptors (T-47D, T-47D clone 11, MCF-7, ZR-75-1); ' Supported by Medical Research Council of Canada. 2 Postdoctoral Fellow, supported by the Manitoba Health Research Council and recipient of Royal Society (London) Anglo-Canadian Traveling Fellowship. To whom requests for reprints should be addressed. 3 Scientist, Medical Research Council of Canada. Received 12/6/84; revised 4/4/85; accepted 4/8/85. estrogen receptor-negative tumor cells (BT-474, MDA-MB-231, BT-20) and the nontumour, breast milk-derived cell line HBL-100 were not responsive to estrogen, whether or not the pituitary factor was present. These results demonstrate that the simul taneous presence of estrogen and pituitary growth factors are required for maximal growth of T-47D human breast cancer cells both in vivo and in vitro and support our hypothesis that a novel pituitary growth factor for estrogen-responsive HBC may exist.