Abstract

Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM-2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774-788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM-2, phospho-S10-histone-H3, and the phospho-serine peptide, TRPPTLSPIPHIPRC (phospho-S780-Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho-S780-Rb and the relationship between reactivity with this antibody and MPM-2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho-S780-Rb epitope. Cell cycle related phospho-S780-Rb immunofluorescence correlated strongly with that of MPM-2. Laser scanning cytometry showed that phospho-S780-Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho-S780-Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post-cytokinetic ("newborn") cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.

(c) 2007 International Society for Analytical Cytology