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CAMA-1 cells, isolated from malignant pleural effusion, are grown in long-term cultures as monolayers. The rate of growth is dependent upon fetal bovine serum and estradiol. These cells also exhibit a dose response to added 17 beta-estradiol with respect to the incorporation of radiolabeled thymidine into cells. The uptake is increased at low levels of estradiol and decreased at pharmacological levels of estradiol. The uptake of uridine and leucine is also stimulated by estradiol in a dose-related manner. Induction of precursor uptake is not observable until cells have been exposed to estradiol for approximately 10 hr or longer. Cells plated for different periods in steroid-stripped serum remain sensitive to estrogenic stimulation and show similar lag time in response. Estrogenic effect is not noticeable in the absence of serum. Addition of prolactin can partially restore estrogenic stimulation of thymidine uptake in serum-free medium. Like other estrogen target tissues, these cells contain cytoplasmic and nuclear estrogen receptors. These results demonstrate that the CAMA-1 cell line is estrogen dependent and that these cells may provide a promising model for the in vitro investigation of the mode of estrogen action in human breast cancer.

Hormonal influences on dimethylbenz(a)anthracene-induced tumor growth were investigated in detail by endocrine ablation and replacement of hormones. The majority of tumors regressed following ablation and most of them were reactivated by subsequent administrations of estrogen (0.1 to 5 mug) or prolactin (2 mg). Increasing numbers of tumors, however, were not stimulated by prolactin when administration was delayed, and a basal level of estradiol (0.01 mug) in addition to prolactin was required for reactivation of tumors. Nafoxidine hydrochloride, a competitor of estrogen at the receptor sites, arrested growth of a large portion of dimethylbenz(a)anthracene-induced tumors in intact animals but failed to retard growth of prolactin-stimulated tumors. On withdrawal of prolactin-nafoxidine, rapid regression of tumor occurred and readministration of prolactin failed to activate most of the tumors for as long as 28 days. Our results give good supporting evidence that estrogen plays a primary role in tumor growth. The interactions of prolactin and estrogen at tumor sites are necessary for regulatory events related to tumor growth.

Two sublines, CAMA-1R and CAMA-1N, were derived from the original CAMA-1 cells after continuous passages in our laboratory. 1R cells were supplemented in culture with estradiol and retained the properties of the original cell line, whereas 1N cells, not supplemented with estradiol, had a partial loss of estrogenic responsiveness. The doubling times for 1R and 1N cells were 2 and 2.4, respectively. These two sublines showed different effects on thymidine uptake after estradiol stimulation. Cells of 1R had a 2-fold greater thymidine incorporation than did the control without estradiol. In contrast, no difference of thymidine uptake was demonstrated in cells of 1N in the presence or absence of estradiol. Both cell lines contained cytoplasmic progesterone receptors with a Kd of 0.56 nM and a sedimentation coefficient of 6 to 7S on a 5 to 20% sucrose gradient. The progesterone receptor level in 1N was about 8-fold higher than in 1R cells. Both sublines exhibited a dose-dependent increase in progesterone receptor levels in response to estradiol with the maximal stimulation (8-fold increase) reached by using 5 nmol of estradiol. The rate of increase for estradiol-induced progesterone receptor appears to be the same for both sublines; a liner increase was demonstrated between Days 3 and 7 following plating. Our results show that estrogen regulation of thymidine uptake and progesterone receptor induction may be mediated through two separate mechanisms. Thus, following a chronically deficient supply of estradiol to 1N cells, there is apparent loss of responsiveness to estradiol-induced thymidine uptake, but the ability of these cells to respond to estradiol induction of progesterone receptor was retained.