PILOT STUDY--PHOSPHORYLATION OF P53 PROTEIN IN HUMAN BREAST CANCER CELLS

试点研究--人乳腺癌细胞中 P53 蛋白的磷酸化

• 批准号: 6353700
• 负责人: Bodiford Lee Stackhouse
• 金额: $ 17.24万
• 依托单位: WINSTON-SALEM STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2002-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6353700
• 关键词: breast neoplasms; cell growth regulation; cell transformation; gel electrophoresis; human tissue; immunocytochemistry; immunoprecipitation; mammary epithelium; p53 gene /protein; phosphorylation; radionuclides; tissue /cell culture; transcription factor; tumor suppressor proteins; western blottings

The long term objective of this project is to investigate the role of protein phosphorylation in breast cancer. This study will investigate the effect of controlled dephosphorylation of protein p53 on human breast tumor cell lines T47D, MCF-7, and normal mammary epithelial cells. In addition, we will measure both qualitatively and quantitatively, p53 phosphorylation status in 50 primary human breast cancer specimens. The effect of inhibiting dephosphorylation of p53 on the cell cycle will be defined in multiple cultures of tumor cells and of normal cells previously exposed to graded concentrations of okadaic acid (OA) and calyculin A (CL-A). The growth response of these cells to OA and CL-A will be determined. These results will be compared to hyperphosphorylation and subcellular localization of protein p53 in these cells. We will determine the amount of immuno- precipitated p53 in all cells (cytoplasmic plus nuclear p53) to give an indication of whether inhibiting dephosphorylation results in repression of the p53 gene. Protein extracts from human breast tumor cell lines, normal mammary epithelial cells, and of primary tumor tissues will be analyzed by 2-D electrophoresis and immunoblotting. The pattern of p53 on these 2-D gels will be compared to the pattern in cell lines that contain normal or hyperphosphorylated p53. By comparing these patterns, we will derive a measure of the level of phosphorylation. In addition, the specific spots will be catalogued for each tumor and used as a qualitative measure of phosphorylation. The results of this study will define the effects on the availability of wild-type p53 caused by the inhibition of the dephosphorylation process. Should the p53 profile of normal breast epithelial cells overlap or significantly mimic that of the tumor cells, a possible mechanism through which normal breast cells become malignant will have been defined.

本项目的长期目标是研究 乳腺癌中蛋白质磷酸化。 本研究将 研究蛋白质受控去磷酸化的效果 p53在人乳腺癌细胞系T47 D、MCF-7和正常人中的表达 乳腺上皮细胞。 此外，我们还将测量 定性和定量地，p53磷酸化状态在50 原发性人类乳腺癌标本。 的抑制作用 细胞周期中p53的去磷酸化将在 肿瘤细胞和正常细胞的多次培养 暴露于不同浓度的冈田酸（OA）， calyculin A（CL-A）。 这些细胞对OA的生长反应， 将确定CL-A。 这些结果将与 p53蛋白过度磷酸化与亚细胞定位 在这些细胞中。 我们将确定免疫- 在所有细胞中沉淀p53（细胞质加细胞核p53）， 给出抑制去磷酸化是否导致 抑制p53基因。 正常人乳腺肿瘤细胞系的蛋白质提取物 乳腺上皮细胞和原发性肿瘤组织将被 通过2-D电泳和免疫印迹分析。 图案 p53在这些二维凝胶上的模式将与细胞中的模式进行比较。 含有正常或过度磷酸化的p53的细胞系。 通过 比较这些模式，我们将得出一个衡量的水平， 磷酸化 此外，具体景点将在 对每个肿瘤进行分类，并用作 磷酸化 这项研究的结果将确定 对野生型p53可用性的影响， 抑制去磷酸化过程。 如果p53 正常乳腺上皮细胞的轮廓重叠或显著 模仿肿瘤细胞的机制，这是一种可能的机制， 正常的乳腺细胞变成恶性将被定义。