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

这个项目的长期目标是调查 蛋白磷酸化在乳腺癌中的作用。这项研究将 研究蛋白质受控去磷酸化的效果 人乳腺癌细胞系T47D、MCF-7和正常乳腺癌细胞株P53的表达 乳腺上皮细胞。此外，我们还将衡量这两项指标 定性和定量检测50例患者中P53的磷酸化状态 原代人类乳腺癌标本。抑制的效果 P53在细胞周期中的去磷酸化将在 以前的肿瘤细胞和正常细胞的多重培养 暴露于分级浓度的冈田酸(OA)和 毛盏花素A(CL-A)。这些细胞对油酸和维生素A的生长反应 CL-A将被确定。这些结果将与 P53蛋白的过度磷酸化及其亚细胞定位 在这些牢房里。我们将测定免疫量- 所有细胞中的沉淀P53(细胞质和核P53) 给出抑制去磷酸化结果的迹象 对p53基因的抑制作用。 人乳腺肿瘤细胞系蛋白质提取物，正常 乳腺上皮细胞和原发肿瘤组织的 用双向电泳法和免疫印迹法进行分析。这种模式 这些二维凝胶上的P53的含量将与细胞内的模式进行比较 含有正常或过度磷酸化P53的品系。通过 比较这些模式，我们将得出一个衡量 磷酸化。此外，具体的地点将是 为每个肿瘤编目，并用作定性衡量标准 磷酸化。这项研究的结果将定义 对野生型P53利用的影响 抑制去磷酸化过程。P53是否应该 正常乳腺上皮细胞轮廓重叠或显著 模仿肿瘤细胞，一种可能的机制，通过 正常的乳腺细胞变得恶性将被定义为。

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.