INSULIN INDUCTION OF PROLACTIN MRNA IN GH3 CELLS

GH细胞是大鼠垂体衍生的细胞系（GH 1、GC、GH 3和GH4） 它们产生生长激素和/或催乳素， 荷尔蒙 初步数据表明胰岛素和三碘甲状腺原氨酸 (T3)在无血清条件下诱导催乳素合成， 在GH 3细胞中mRNA水平为3- 14倍。 这些激素共同诱导 惊人的20到100倍的协同反应。 首先，这项研究将 尝试定义这个答案。 动力学将通过以下方式确定： 使用滤膜进行体外转录和体内[3 H]尿苷标记研究 杂交以确定总催乳素特异性 序列的 对胰岛素或其他药物反应的特异性 然后通过剂量反应测定胰岛素样生长因子（IGF） 胰岛素、胰岛素类似物、IGF-I和IGF-II的研究。 研究来 确定胰岛素和T3对催乳素RNA加工的影响， 还提出了半衰期。 最后，反应的反诱导 激素去除后将进行研究。 第二，我们将努力 通过以下方式确定胰岛素对催乳素mRNA积累的可能调节： 研究胰岛素和T3对顺式活性DNA调控序列的影响， 反式调节分子或染色质结构。 顺式活性 将通过构建嵌合质粒来研究调控序列 5'催乳素基因组DNA和细菌黄嘌呤-鸟嘌呤 磷酸核糖转移酶（XGPRT）基因和研究激素调节 XGPRT在用该质粒和用XGPRT的缺失突变体转染的细胞中的表达 这个质粒 将尝试确定任何作用的trans-acting 通过抑制剂研究和两个 标记的核蛋白的三维凝胶电泳。 最后我们 将试图确定激素对染色质结构的影响， 催乳素基因的DNA酶I敏感性研究和检查 基因的甲基化状态。

The GH cells are rat pituitary derived cell lines (GH1, GC, GH3, and GH4) which produce growth hormone and or prolactin in response to many hormones. Preliminary data demonstrate that insulin and triiodothyronine (T3) under serum free conditions induce prolactin synthesis and prolactin mRNA levels 3- to 14-fold in GH3 cells. Together these hormones induce a dramatic 20- to 100-fold synergistic response. First, this research will attempt to define this response. The kinetics will be determined by in vitro transcription and in vivo [3H]uridine labeling studies using filter hybridization to determine the levels of total prolactin specific sequences. The specificity of the response for insulin or other insulin-like growth factors (IGF) will then be determined by dose response studies with insulin, insulin analogs, IGF-I and IGF-II. Studies to determine the effects of insulin and T3 on prolactin RNA processing and half-life are also proposed. Finally, the deinduction of the response after hormone removal will be investigated. Second, we will attempt to define possible insulin regulation of prolactin mRNA accumulation by studying effects of insulin and T3 on cis-active DNA regulatory sequences, trans-active regulatory molecules, or chromatin structure. Cis-active regulatory sequences will be studied by construction of a chimeric plasmid of 5' prolactin genomic DNA and the bacterial xanthine-guanine phosphoribosyl transferase (XGPRT) gene and studying hormonal regulation of XGPRT in cells transfected with this plasmid and with deletion mutants of this plasmid. Attempts will be made to identify any role of trans-acting regulatory molecules on this system by inhibitor studies and by two dimensional gel electrophoresis of labeled nuclear proteins. Finally, we will attempt to determine hormonal effects on the chromatin structure of the prolactin gene by DNase I sensitivity studies and examination of the methylatin state of the gene.