INSULIN INDUCTION OF PROLACTIN MRNA IN GH3 CELLS

生长激素细胞是大鼠垂体来源的细胞系(GH1、GC、GH3和GH4)。 它们会产生生长激素和/或催乳素 荷尔蒙。初步数据显示，胰岛素和三碘甲腺原氨酸 (T3)在无血清条件下诱导催乳素合成和催乳素 在GH3细胞中，信使核糖核酸水平提高了3到14倍。这些荷尔蒙共同诱导了 戏剧性的20到100倍的协同反应。首先，这项研究将 尝试定义此响应。动力学将由In确定 滤膜体外转录和体内[~3H]尿苷标记研究 杂交法测定特异性总催乳素水平 序列。对胰岛素或其他物质的反应的特异性 胰岛素样生长因子(IGF)将通过剂量反应来确定 研究胰岛素、胰岛素类似物、胰岛素样生长因子-I和胰岛素样生长因子-II。研究到 确定胰岛素和T3对催乳素RNA加工和 还提出了半衰期的概念。最后，对反应的去归纳 激素去除后将进行调查。第二，我们将尝试 明确胰岛素可能通过以下途径调节催乳素mRNA的积累 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。顺应式活动 将通过构建嵌合质粒来研究调节序列 5‘-催乳素基因组DNA与细菌黄嘌呤-鸟氨酸的关系 磷酸核糖转移酶基因及其激素调控的研究 XGPRT在转基因细胞中的表达及缺失突变体 这一质粒。将尝试确定任何角色的变装 调节分子对这一系统的抑制研究和两个 标记核蛋白的双向凝胶电泳法。最后，我们 将尝试确定荷尔蒙对染色质结构的影响 催乳素基因对脱氧核糖核酸酶I的敏感性研究 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.