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Effect of Ca²⁺ and Salt on Forms of Estradiol Cytoplasmic Receptor in Human Neoplastic Breast Tissue¹

Department of Breast Surgery and Breast Cancer Research Unit, Roswell Park Memorial Institute, Buffalo, New York 14263

After incubation with [³H]estradiol, low-salt sucrose gradient centrifugation of 250 human breast tumor cytosols demonstrated receptor-bound steroid in both 4 S and 8 S forms. Cytosols prepared from 24 of these receptor-positive tumors were examined in the presence of KCI, Ca²⁺, and the serine protease inhibitor, phenylmethylsulfonyl fluoride. After incubation with KCI (0.4 M), interconversion of 8 S and 4 S forms was found in one-half of the 24 cytosols examined whose original low-salt patterns contained well-defined 4 S and 8 S peaks. When cytosols were made 4 mM in Ca²⁺ 15 to 30 min before the addition of KCI, an additional 4.5 S form was observed in low-salt gradients in 18 tumors, and, in 6 tumors, inactivation of receptor occurred. The effects were not produced when Ca²⁺ and salt were added simultaneously. After 2 to 24 hr of preincubation with Ca²⁺ of the same cytosols under the same conditions, an increased recovery of the 4 S receptor complex was observed in lowsalt gradient. These Ca²⁺ effects were inhibited if phenylmethylsulfonyl fluoride and Ca²⁺ were added simultaneously to cytosols prior to the addition of salt. The data suggest that human mammary tumor cytosols may contain proteolytic activities that can be activated by Ca²⁺ and that can effect irreversible changes in the salt-dissociated steroid receptor proteins which can be detected in sucrose density gradients.

¹ This work was supported in part by Grant CA 14812-02, National Cancer Institute, NIH, USPHS.

² To whom requests for reprints should be addressed, at Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N. Y. 14263.

Received 12/29/75. Accepted 10/29/76.