Costlow M E, Gallagher P E, Koseki Y
Mol Cell Endocrinol. 1979 Apr;14(1):81-97. doi: 10.1016/0303-7207(79)90060-1.
7,12-Dimethylbenz[alpha]anthracene-induced rat mammary tumors were dissociated with collagenase and hyaluronidase and placed into primary culture. In most cultures, specific binding of 125I-labeled ovine prolactin was (i) lower than that for the original tumors unless bovine prolactin (1 microgram/ml) had been added to the dissociation medium, and (ii) varied with the type of growth medium used. The level of prolactin binding in cultured cells was relatively constant for the first 7-10 days. Prolactin binding in cultured cell homogenates was maximal at pH 7.0, proportional to cell protein, specific for prolactin, and reached a steady state by 12 h at 22 degrees C. The half-maximum inhibition of 125I-labeled prolactin binding by unlabeled prolactin was 100 ng/ml for cells grown in 5-1000 ng of prolactin/ml. After prolactin was removed from the growth medium, the level of available binding sites progressively increased, reached a maximum at 48 h and then declined. At 48 h, the dissociation constant for prolactin binding (Kd approximately 1 x 10(-10) M) was comparable to that in tumors. In some cultured tumors, a 48-h treatment with 0.5 or 1.0 ng of prolactin/ml caused an apparent increase in the level of prolactin binding. Prolactin increased DNA synthesis and its removal caused a reduction in [3H]estradiol and [3H]-R5020 binding to cultured cell cytosols.
用胶原酶和透明质酸酶将7,12-二甲基苯并[a]蒽诱导的大鼠乳腺肿瘤解离，并进行原代培养。在大多数培养物中，125I标记的绵羊催乳素的特异性结合（i）低于原始肿瘤，除非在解离培养基中添加了牛催乳素（1微克/毫升），并且（ii）随所用生长培养基的类型而变化。培养细胞中催乳素结合水平在最初7至10天相对恒定。培养细胞匀浆中催乳素结合在pH 7.0时最大，与细胞蛋白成比例，对催乳素具有特异性，并在22℃下12小时达到稳定状态。对于在5至1000纳克/毫升催乳素中生长的细胞，未标记的催乳素对125I标记的催乳素结合的半数抑制浓度为100纳克/毫升。从生长培养基中去除催乳素后，可用结合位点水平逐渐增加，在48小时达到最大值，然后下降。在48小时时，催乳素结合的解离常数（Kd约为1×10-10 M）与肿瘤中的相当。在一些培养的肿瘤中，用0.5或1.0纳克/毫升催乳素处理48小时导致催乳素结合水平明显增加。催乳素增加DNA合成，去除催乳素导致[3H]雌二醇和[3H]-R5020与培养细胞胞质溶胶的结合减少。
