Steroid stimulation of plasminogen activator production in a human breast cancer cell line (MCF-7).

The production of plasminogen activator by the human breast cancer cell line MCF-7 was stimulated by physiological concentrations of estradiol under conditions where the growth of the cells was neither dependent on nor stimulated by estradiol. Stimulation was measurable within 8 hr after the addition of estradiol and was evident in both the level of plasminogen activator released into the culture medium and the level within the cells. The level of production varied with cell density, but production was stimulated by estradiol at all densities tested. The antiestrogen tamoxifen inhibited estrogen stimulation, and this inhibition could be overcome by increased concentrations of estradiol. Production was also stimulated by progesterone and could be stimulated by lower levels of progesterone in cells pretreated with estradiol or tamoxifen, both of which have been reported to increase the level of progesterone receptor in these cells. It has been reported that estrogen is essential and that progesterone is stimulatory for the formation of tumors by MCF-7 cells in athymic mice. The ability of these same two hormones to stimulate the production of plasminogen activator by these cells, under conditions where they have no effect on cell growth, raises the possibility that estrogen may not play a mitogenic role in the growth of these tumors. Rather, it may support tumor growth by inducing the cells to produce products, such as plasminogen activator, and possibly take on other characteristics essential to the malignant state.

Prognostic value of concanavalin A reactivity of primary human breast cancer cells.

A prospective, double-blind study was carried out to determine whether activity with concanavalin A (Con A) of human breast cancer cells was related to early disease recurrence. Mammary epithelial cells were isolated from 138 primary human breast cancers. The cells were placed in culture, and their reactivity with Con A was determined with a hemadsorption assay in which human erythrocytes treated with various concentrations of Con A were incubated with the test (mammary epithelial) cells in situ. The Con A half-maximum value was determined as the concentration of Con A at which approximately 50% of the test cells adsorbed erythrocytes. Con A reactivity of the tumors was classified as high or low (half-maximum value less than or equal to 30 or greater than 30 microgram/ml, respectively). Patients were followed for 2 to 60 months after primary surgery (median, 22 months). Those patients having tumors that were highly reactive with Con A were at significantly greater risk of developing early recurrence of their cancers than were those patients with low-reactivity tumors. No correlation was found between Con A reactivity and the age of the patients, their menopausal status, the number of axillary lymph nodes infiltrated with tumor, the number of axillary lymph nodes infiltrated with tumor, the estrogen receptor content of the tumor, or the clinical stage of the disease. These data show that Con A reactivity is an independent discriminator for identifying those breast cancer patients who are at high risk of developing early recurrent disease.

Growth of normal and malignant human mammary epithelial cells in culture.

Normal and malignant human mammary epithelial cells were placed in culture. Normal cells were recovered from late-lactation milk and breast fluids, and malignant cells were isolated from primary breast tumors by collagenase digestion. The concentration of cells obtained from breast fluid samples was inversely proportional to the volume of fluid secreted. Most of these cells adhered rapidly to the substrate, did not replicate, displayed Fc receptor-dependent phagocytic activity, and were thus identified as macrophages. The remaining cells grew out into large islands comprised of one or two distinct morphologic types of mammary epithelial cells. Optimum growth of these cells was obtained in medium buffered to pH 6.8, and the epidermal growth factor markedly prolonged the exponential growth phase of the cells. Two morphologically distinct populations of epithelial cells were also observed in cultures established from individual breast tumors. Growth of the malignant cells was relatively unaffected by the pH of the culture medium, and the cells were unresponsive to exogenously added hormones. Overgrowth of malignant epithelial cells in primary cultures by stromal fibroblasts was retarded by replacement of standard growth medium with fresh medium containing a serum substitute; growth of the malignant epithelial cells was unaffected. A feeder layer of mitomycin C-treated human fibroblasts increased the plating efficiency of both normal and malignant cells in primary culture and also facilitated passage of these cells to secondary and tertiary cultures.

Concanavalin A-mediated hemadsorption by normal and malignant human mammary epithelial cells.

The concanavalin A (Con A) reactivity of malignant and normal human mammary epithelial cells in culture was determined with a hemadsorption assay. Human erythrocytes were treated with various concentrations of Con A, and these indicator Red Blood Cells were incubated with the test cells in situ in culture dishes. The Con A concentration at which approximately 50% of the test cells adsorbed erythrocytes ([Con a] 1/2 max) was determined. Five malignant epithelial cell lines and the primary cultures derived from 3 pleural effusions and 20 solid tumors were tested. Primary cultures of normal epithelial cells were established from human milk samples obtained from 3 separate donors. The average [Con A] 1/2 max value for the 5 cell lines and the pleural effusion cultures was 6 and 5 microgram/ml, respectively. The average [Con A] 1/2 max value for the 20 solid breast tumors was 20 microgram/ml. In contrast to the malignant cells, normal mammary epithelial cells did not adsorb erythrocytes coated with as much as 100 microgram Con A per ml. These results show that Con A reactivity distinguishes normal from malignant human mammary epithelial cells.

Control of cell growth. I. Estrogen-dependent growth in vivo of a rat pituitary tumor cell line.

A subclone of rat pituitary tumor cells, designated GH3/C14, was isolated from the parent population of GH3 pituitary cells and was estrogen-dependent for growth in vivo. GH3/C14 cells inoculated into host animals formed tumors in intact females, estrogen-treated ovariectomized females, and estrogen-treated males, but not in untreated intact or castrated males, or untreated ovariectomized females. Exogenous treatment with estrogens supported tumor formation in male animals. Radioimmunoassay of the average serum estradiol concentrations that support tumor growth in intact females, estradiol-treated intact females, and estradiol-treated intact males gave values of 41 +/- 4, 1,130 +/- 150, and 730 +/- 140 pg/ml, respectively. Tumor formation by GH3/C14 cells inoculated into male animals was not supported by either exogenous progesterone or hydrocortisone acetate. Further, these two steroid hormones had no significant effect on the estrogen-promoted growth in males or females. Exogenous testosterone treatment promoted tumor formation in males and ovariectomized females, but dihydrotestosterone was completely ineffective. Radioimmunoassay of the serum from tumor-bearing animals and the medium from the cells in culture showed that the cells produced growth hormone in vivo and in vitro but did not produce measurable amounts of luteinizing hormone or follicle-stimulating hormone. The groth of GH3/C14 cells in culture was examined in medium without added estrogen, and with estradiol added to the level of either the normal intact female rat or the estradiol-treated animals. No direct growth stimulation by estrogens could be detected in culture; the data suggested an indirect growth control mechanism.

Control of cell growth. II. Requirement of thyroid hormones for the in vivo estrogen-dependent growth of rat pituitary tumor cells.

Further examination of rat pituitary cell line GH3/C14 showed that at least the physiologic concentration of L-thyroxine was required for estrogen-dependent growth in vivo. Two L-thyroxine synthesis inhibitors, 6-n-propyl-2-thiouracil (propylthiouracil) and 1-methylimidazole-2-thiol (methimazole), were administered concurrently with estrogen to GH3/C14-inoculated hosts. Propylthiouracil administration to estrogen-treated males, intact females, and estrogen-treated ovariectomized females inhibited tumor formation by 93, greater than 95, and 68%, respectively, as compared to tumor formation in controls not treated with propylthiouracil. Methimazole treatment of estrogen-primed males and intact females inhibited tumor formation by 78 and 95%, respectively. Concentrations of total L-thyroxine and free L-thyroixine in sera from normal and inhibitor-treated hosts were depressed 70-80% by propylthiouracil and 60-70% by methimazole. Administration of either drug caused greater inhibition of tumor growth than of total body weight gain. In addition, the administration of a combination of L-thyroxine and L-triiodothyronine to male rats promoted tumor formation even in the absence of exogenous estrogen.

Control of cell growth. III. Direct mitogenic effect of thyroid hormones on an estrogen-dependent rat pituitary tumor cell line.

A possible direct estrogen requirement for growth of GH3/C14 rat pituitary tumor cells was evaluated in culture medium supplemented with estrogen-depleted serum prepared by a 56 degree C charcoal extraction procedure, and with serum obtained from ovariectomized sheep and ovariectomized adrenalectomized sheep. Growth of the GH3/C14 cells in culture medium in which the final estrogen concentration was 2 pg/ml or less was equal to growth in medium with normal serum and equal to growth in the presence of estrogen-depleted serum to which estradiol was added back at concentrations of 10-1,000 pg/ml. Under no conditions could a direct estrogen requirement for growth be demonstrated. The function of thyroid hormones in cell growth was examined in culture medium supplemented with serum from thyroidectomized sheep. In such medium the growth of the GH3/C14 cells was stimulated 3.5-fold by addition of 1.0 X 10(-8) M L-thyroxine (T4) or 1.0 X 10(-9) m L-triiodothyronine (T3). Investigation of the possible synergistic effects of estrogens and T4 revealed that combinations of estrogen and T4 or T3 did not stimulate growth over that seen with T4 or T3 alone. These data indicated that estrogens are not direct growth requirements for these cells but instead operate in vivo through secondary or indirect mechanisms; in contrast, thyroid hormones are directly mitogenic in vitro.

Control of cell growth. IV. Growth properties of a new cell line established from an estrogen-dependent kidney tumor of the Syrian hamster.

A permanent cell line has been established in culture from an estrogen-dependent kidney tumor of the Syrian hamster. The cells were selected from mixed populations by three methods: 1) animal passage enrichment, 2) growth on petri dishes, and 3) serum-type dependence. The inoculation of these cells, designated the H-301 line, into host animals showed tumor formation in estrogen-treated males or estrogen-treated females, but no tumor formation in normal or castrated hamsters of either sex. The estrogen-dependent tumor formation in male hamsters by H-301 cells was inhibited by progesterone, hydrocortisone, and testosterone. When H-301 cell growth was examined in culture, the addition of estrogens or other steroid hormones to the medium over the range of 0.1 to 10 ng/ml showed no growth-stimulating or inhibiting effects. Medium prepared with charcoal-extracted serum (to reduce by 90% the endogenous levels of steroid hormones) supported growth as well as medium prepared with normal serum. Furthermore, the addition of either estrogens, testosterone, or antiestrogens to medium prepared with charcoal-extracted serum showed no significant effects upon growth. We have shown also that culture medium prepared with serum from young calves did not support the growth of the cells, and the addition of estrogens to this medium did not change this growth pattern. However, the addition of fetal calf serum to medium already containing calf serum promoted maximal growth. Evidence is presented that the growth-promoting agent in fetal calf serum was inactivated by a 90 C heat treatment. Furthermore, the ether-soluble fractions from fetal calf serum and horse serum did not significantly improve growth when added to calf serum containing medium. We conclude that estrogens or other steroid hormones alone are not sufficient to promote the growth of the H-301 cells, but that heat-labile serum factors are required.