Effects of estrogen and vanadate on the proliferation of newly established transformed mouse Leydig cell line in vitro.

A permanent cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor (T 124958-R) was established in culture. Although this cell line showed estrogen-induced enhancement of cell proliferation in vitro, a moderately high concentration (10(-8) M) of estradiol was required to stimulate maximum growth. A whole cell binding assay revealed that the B-1-A-2 cells contained estrogen receptors with relatively low affinity for estradiol (Kd, 10(-9)-10(-8) M). This estrogen receptor was found to have a mol wt of 65,000. In addition, incubation of the cells with [32P]orthophosphate and subsequent purification revealed that the estrogen receptor is phosphorylated. This finding prompted us to study the effect of phosphatase inhibitors on cell proliferation. The inclusion of vanadate (25 microM) in the culture medium resulted in a significant increase in estrogen sensitivity, showing a maximal growth stimulatory effect of estradiol at concentrations of 10(-10)-10(-9) M. Simultaneously, the conversion of the low affinity to the high affinity receptor (Kd, 10(-10)-10(-9) M) was induced by treatment of cells with vanadate. These data suggest that estrogen directly stimulates the growth of B-1-A-2 cells in culture, and that the sensitivity to estrogen can be altered by modulating the estrogen receptor possibly via a phosphorylation-dephosphorylation mechanism.