Pilot Study - Phosphorylation of P53 Protein in Human Breast Cancer Cells

Stackhouse, Bodiford

Abstract

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.