Department of Hormone Research, The Weizmann Institute of Science, Rehovot 76100, Israel.
J Steroid Biochem Mol Biol. 1992 Dec;43(8):875-84. doi: 10.1016/0960-0760(92)90315-A.
Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED₅₀ characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.
通过将 SV40 DNA 和 Ha-ras 癌基因转染到来自预排卵卵泡的初级颗粒细胞中，建立了高类固醇生成性颗粒细胞系。通过用 8-Br-cAMP、佛司可林和霍乱毒素（可提高细胞内 cAMP）对这些细胞进行长时间（>12 小时）刺激，将这些细胞中的孕激素产生水平提高到与正常类固醇生成细胞相当的水平。个别系的类固醇生成能力与 ras 癌基因产物（p21）的表达和细胞形态相关。类固醇激素的形成与线粒体细胞色素 P450scc 系统蛋白的从头合成有关。由于胆固醇向线粒体的导入对于类固醇生成是必需的，因此在这些细胞中表征了外周苯二氮䓬受体（PBR）和固醇载体蛋白 2 的表达。这两种蛋白编码基因表达的诱导似乎至少部分由 cAMP 介导。特异性激动剂对 PBR 的刺激增强了这些细胞中的孕激素产生。尽管细胞内 cAMP 水平升高，但佛波醇酯 12-O-十四烷酰佛波醇 13-乙酸酯（TPA）强烈抑制 cAMP 诱导的类固醇生成，表明 TPA 可以修饰 cAMP 的作用。cAMP 刺激与类固醇生成的诱导同时抑制转化细胞的生长。转化细胞缺乏天然刺激物，即促性腺激素的受体。在用促黄体激素（LH）受体表达质粒转染细胞后，恢复了 LH 和 hCG 反应。在这些新建立的细胞系中，促性腺激素能够以与天然受体特征一致的 ED₅₀ 剂量依赖性方式刺激 cAMP 和孕激素的形成。高剂量导致对促性腺激素的脱敏，如在正常细胞中观察到的那样。这些新建立的癌基因转化的颗粒细胞系可用作研究诱导性类固醇生成和癌基因表达对该过程影响的有用模型。
