McManaway M E, Eckberg W R, Anderson W A
Howard University, Department of Zoology, Washington, D.C. 20059.
Exp Clin Endocrinol. 1987 Dec;90(3):313-23. doi: 10.1055/s-0029-1210707.
Cytosolic casein kinase type II activity has been identified in MCF-7 and MDA-MB-231 human breast cancer cells heterotransplanted into athymic nude mice. Sephacryl S-300 chromatography of MCF-7 and MDA-MB-231 tumor cytosols revealed a major peak of casein kinase activity with an estimated molecular weight of 150,000. This peak was further characterized and optimal conditions for breast tumor casein kinase activity were established. Polylysine (10 micrograms) acted as a potent stimulator with casein as the phosphate acceptor protein. This enzyme used both ATP and GTP as phosphate donors and the Km for GTP was 10 microM. The rate of phosphorylation with increasing concentrations of [gamma-32p]GTP revealed typical Michaelis-Menten kinetics and Vmax was approached at a concentration of 30 microM GTP. MgCl2 stimulated enzyme activity at concentrations between 10-20 mM. Quercetin, a bioflavonoid, inhibited casein kinase type II activity in a dose dependent manner. MCF-7 (hormone-dependent) human breast cancer cells (2-3 X 10(6)) were inoculated into the mammary fat pads of nude mice, supplemented with a 0.5 mg estradiol pellet. To determine the influence of various regulatory agents on casein kinase activity in vivo, tumor-bearing mice were treated for five days with estradiol, progesterone, dexamethasone or tamoxifen. Casein kinase type II was partially purified by gel filtration on a Sephacryl S-300 column and assayed in the presence of polylysine and casein. Dexamethasone treatment significantly decreased casein kinase II activity in MCF-7 tumors, which are receptor-positive for estrogen, androgen and glucocorticoid receptors.
在移植到无胸腺裸鼠体内的人MCF-7和MDA-MB-231乳腺癌细胞中，已鉴定出胞质酪蛋白激酶II型活性。对MCF-7和MDA-MB-231肿瘤细胞溶质进行Sephacryl S-300层析，显示出酪蛋白激酶活性的一个主要峰，估计分子量为150,000。对该峰进行了进一步表征，并确定了乳腺肿瘤酪蛋白激酶活性的最佳条件。聚赖氨酸（10微克）作为以酪蛋白为磷酸受体蛋白的强效刺激剂。该酶使用ATP和GTP作为磷酸供体，GTP的Km为10微摩尔。随着[γ-32P]GTP浓度增加的磷酸化速率显示出典型的米氏动力学，在30微摩尔GTP浓度时接近Vmax。MgCl2在10 - 20毫摩尔浓度之间刺激酶活性。生物类黄酮槲皮素以剂量依赖性方式抑制酪蛋白激酶II型活性。将MCF-7（激素依赖性）人乳腺癌细胞（2 - 3×10⁶）接种到补充有0.5毫克雌二醇丸粒的裸鼠乳腺脂肪垫中。为了确定各种调节剂对体内酪蛋白激酶活性的影响，对荷瘤小鼠用雌二醇、孕酮、地塞米松或他莫昔芬治疗五天。通过在Sephacryl S-300柱上进行凝胶过滤对酪蛋白激酶II型进行部分纯化，并在聚赖氨酸和酪蛋白存在的情况下进行测定。地塞米松治疗显著降低了MCF-7肿瘤中酪蛋白激酶II的活性，MCF-7肿瘤对雌激素、雄激素和糖皮质激素受体呈阳性。
