Assay development for high throughput screening of p21(Waf1/Cip1) protein expression in intact cells using fluorometric microvolume assay technology.

Abstract

The p21(Waf1/Cip1) protein represents a broad-acting cyclin-dependent kinase inhibitor that plays a key role in cell cycle regulation. Furthermore, p21(Waf1/Cip1) protein has been described as a direct participant in regulating genes involved in growth arrest, senescence and aging. In response to genotoxic insults (e.g., following chemotherapeutic treatment), p21(Waf1/Cip1) protein accumulates mainly through p53-mediated transcriptional activation and is also regulated at the post-transcriptional level. In tumor cells, p53 is frequently mutated leading to reduced p21(Waf1/Cip1) protein induction that may contribute to resistance to treatment by DNA-damaging agents. In order to identify compounds capable of restoring p21(Waf1/Cip1) protein level, we have developed a 96-multi-well plate-based high throughput screening assay in intact cells using the Applied Biosystems Fluorometric Microvolume Assay Technology (FMAT) macro-confocal system. Briefly, following incubation with test compounds, human MCF7 breast carcinoma cells were fixed and p21(Waf1/Cip1) protein was detected using anti-p21(Waf1/Cip1) monoclonal antibody and anti-mouse IgG conjugated to the red fluorescent dye Alexafluor 633. FMAT provides a set of raw images at a high magnification, in which fluorescence concentrated in a cell is detected as a specific signal. The mean fluorescence of a population of cells is calculated independently of the number of cells as with a classical FACS analysis. This is of particular interest for screening anticancer drugs that may affect cell number and therefore may impact on the readout. This assay was validated using reference compounds such as camptothecin and actinomycin D, known inducers of p21(Waf1/Cip1) protein.