Effects of tamoxifen and 4-hydroxytamoxifen on synchronized cultures of the human breast cancer cell line MCF-7.

The effect of antiestrogens on cultures of synchronized MCF-7 human breast cancer cells was studied. Cultures were synchronized by an eighteen hour block with 1.5 mM hydroxyurea. Tamoxifen or 4-hydroxytamoxifen was added at various times following removal of the hydroxyurea block and the cell number and rate of incorporation of 3H-thymidine by the cultures were followed for 48 hours through two waves of DNA synthesis. Antiestrogens added when hydroxyurea was removed inhibited only the second wave of DNA synthesis. Inhibition of the second wave only occurred if the antiestrogen was added before or at about the time of mitosis. To determine whether antiestrogen had to be present before mitosis in order to block the next round of DNA synthesis, cells in mitosis were selected by shaking them loose. The subsequent division of these cells was followed to determine the effect of treating them with antiestrogens before, after, or both before and after mitosis. The addition of antiestrogens at the time of mitosis proved sufficient to block the next round of cell division. Thus antiestrogens are cell cycle specific agents and appear to act at a point early in G1 to block the next round of DNA synthesis and cell division.

Relation of in vitro properties to tumorigenicity for a series of sublines of the human breast cancer cell line MCF-7.

The relation of in vitro properties to tumorigenicity was studied using eight sublines of the human breast cancer cell line MCF-7. Four of the eight were tumorigenic in estrogen-treated nude mice. The sublines differed for each of the in vitro properties measured, and no property correlated perfectly with tumorigenicity. Cytochalasin B-induced multinucleation was a property of all four tumorigenic sublines but of only one of the four nontumorigenic ones. Anchorage-independent growth and concanavalin A-mediated hemadsorption levels were higher in all sublines than reported levels for nontransformed fibroblasts and normal human or mouse mammary epithelial cells. The production of both plasminogen activator and a plasminogen-independent fibrinolytic activity showed no relationship to tumorigenicity but was higher in those sublines producing more invasive tumors. It appears that no one of these in vitro properties is sufficient to make a subline tumorigenic. Rather, the first three properties studied here and, perhaps, also production of plasminogen activator may each be necessary, but not sufficient, to make a subline tumorigenic. In addition, properties such as production of plasminogen activator and other proteases, while perhaps not essential to tumorigenicity, may confer characteristics, such as invasiveness, on the tumors produced by a given subline.

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.

Effects of serum and insulin on the sensitivity of the human breast cancer cell line MCF-7 to estrogen and antiestrogens.

The effect of the antiestrogens tamoxifen and nafoxidine on the growth of the human breast cancer cell line MCF-7 is modified by both serum and insulin. Tamoxifen inhibition of the growth of MCF-7 cells in culture is reduced as the concentration of serum in the medium is increased from 0.1% to 5 to 10%. Estradiol does not stimulate cell growth over the same range of serum levels. Insulin changes the sensitivity of MCF-7 cells to both estrogen and antiestrogens. Cells growing in media containing insulin are less sensitive to inhibition by either tamoxifen or nafoxidine than are cells growing in its absence. In addition, higher concentrations of estradiol are required to stimulate the production of plasminogen activator when cells are grown in media containing insulin. This effect of insulin can be accounted for by the finding that insulin lowers the level of estrogen receptor in MCF-7 cells without altering the binding constant for the hormone. Cells grown with insulin have an average of 21,000 +/- 4,700 (S.D.) estrogen binding sites/cell compared to 62,000 +/- 9,700 sites/cell in cells grown in the absence of insulin. This difference in receptor level is sufficient to account for the difference in the concentration of estradiol needed for equivalent induction of plasminogen activator in cultures with or without insulin. These results indicate that the level of estrogen receptor in breast cancer cells can be changed and that the sensitivity of such cells, both to estrogen and to antiestrogens, is altered by changes in the level of estrogen receptor. They also have implications concerning the mechanism by which antiestrogens act to inhibit the growth of mammary tumor cells.

Epidermal chalone--past to present concept.

How the constantly renewing epidermis maintains its homeostatic state remains a mystery. A "negative feedback" mechanism of control with chalone, a tissue-specific, species-nonspecific inhibitor of mitosis, has been suggested by Bullough and Laurence. Progress in isolating, purifying, and characterizing epidermal chalone has been slow and difficult because of the lack of a fast, assurate, and meaningful assay system. A G2 assay (depending upon mitotic counts) was used to obtain the data which served as the basis for postulating the existence of chalone. Since a control point with the G1 phase of the cell cycle is considered to be the one with physiologic implications, in vivo and in vitro assays with pure primary epidermal cell cultures have been used to investigate a G1 block. The incorporation of tritiated thymidine into DNA is often used as a measure of the rate of prokiferation. However, inhibition of thymidine transport and phosphorylation have been encountered in several systems and negate this method as an accurate measure of cell proliferation.