A clonogenic assay of long-term breast cancer cell cultures in vitro has been developed to provide a highly reproducible method with which to quantitate tumor cell killing by hormones and/or cytotoxic chemotherapeutic agents. Monolayer cultures of estrogen receptor-positive MCF-7 human breast cancer cells and of estrogen receptor-negative Evsa T cells are harvested by treatment with 0.01% trypsin:0.02% EDTA in Hanks' balanced salt solution. Cell suspensions are treated with drug or hormone in serum-free medium for 1 hr at 37°; treated cells are washed, plated, and cultured for approximately 14 days; and colonies consisting of ≥30 cells are counted. Compared to estrogen receptor-positive cells, estrogen receptor-negative cells were 2-fold more sensitive to melphalan but were conversely 1.9-fold more resistant to Adriamycin; these differences were statistically significant (p < 0.001). Thus, response to cytotoxic chemotherapeutic agents appeared to be independent of estrogen receptor status.

For cells treated with diethylstilbestrol, the dose of drug or hormone reducing the surviving cell fraction to 1/e (D0) for estrogen receptor-positive cells was 2.27 nmol/ml, and that for estrogen receptor-negative cells was 2.80 nmol/ml; this difference was not statistically significant. However, with tamoxifen therapy, the D0 for estrogen receptor-positive cells was 0.601 nmol/ml, and that for estrogen receptor-negative cells was 3.64 nmol/ml; this 6-fold greater degree of resistance to tamoxifen of estrogen receptor-negative cells was highly significant (p < 0.001). Treatment of cells for 24 hr with 17β-estradiol stimulated proliferation not only of estrogen receptor-positive cells but also of estrogen receptor-negative cells. However, estradiol at concentrations up to 200 µm had no apparent cytocidal activity, as measured by the clonogenic assay. Furthermore, treatment of MCF-7 cells simultaneously with estradiol and either diethylstilbestrol or tamoxifen failed to reverse the cytocidal activity of those two agents. These findings suggest that, in the clonogenic assay described herein, diethylstilbestrol and tamoxifen may kill human breast cancer cells by an independent mechanism of action and that the cytocidal activity of diethylstilbestrol and the proliferative effect of 17β-estradiol appear to be independent of estrogen receptor status.

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This work was supported by a grant from the National Cancer Institute of Canada.

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