ONCOGENE-TRANSFORMED GRANULOSA-CELLS AS A MODEL SYSTEM FOR THE STUDY OF STEROIDOGENIC PROCESSES
ONCOGENE-TRANSFORMED GRANULOSA-CELLS AS A MODEL SYSTEM FOR THE STUDY OF STEROIDOGENIC PROCESSES
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DOI：
10.1016/0960-0760(92)90315-a
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发表时间：
1992-12-01
影响因子：
4.1
通讯作者：
STRAUSS, JF
中科院分区：
文献类型：
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作者：
AMSTERDAM, A;HANUKOGLU, I;STRAUSS, JF
通过用 SV40 DNA 和 Ha-ras 癌基因转染来自排卵前卵泡的原代颗粒细胞来建立高类固醇生成颗粒细胞系。通过用 8-Br-cAMP、毛喉素和霍乱毒素进行长时间（> 12 小时）刺激，这些细胞中的黄体酮产量可提高至与正常类固醇生成细胞相当的水平，从而提高细胞内 cAMP。各个细胞系的类固醇生成能力与 ras 癌基因产物 (p21) 的表达和细胞的形态相关。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。由于胆固醇输入线粒体对于类固醇生成至关重要，因此对这些细胞中外周苯二氮卓受体 (PBR) 和甾醇载体蛋白 2 的表达进行了表征。编码这两种蛋白质的基因表达的诱导似乎至少部分是由cAMP介导的。特定激动剂对 PBR 的刺激增强了这些细胞中孕酮的产生。尽管细胞内 cAMP 水平增强，佛波酯 12-O-十四酰基-佛波醇 13-乙酸酯 (TPA) 仍显着抑制 cAMP 诱导的类固醇生成，表明 TPA 可以改变 cAMP 的作用。 cAMP 刺激抑制转化细胞的生长，同时诱导类固醇生成。转化的细胞缺乏天然兴奋剂促性腺激素的受体。用促黄体激素 (LH) 受体表达质粒转染细胞后，重建 LH 和 hCG 反应。在这些新建立的细胞系中，促性腺激素能够以剂量依赖性方式刺激cAMP和孕酮的形成，并具有天然受体的ED50特征。正如在正常细胞中观察到的那样，高剂量导致对促性腺激素的脱敏。这些新建立的癌基因转化的颗粒细胞系可以作为研究诱导类固醇生成以及癌基因表达对该过程的影响的有用模型。
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.