Simultaneous Measurement of Progesterone Receptors and DNA Indices by Flow Cytometry: Characterization of an Assay in Breast Cancer Cell Lines1

Progesterone receptors (PR) are the strongest predictors of response to hormone therapy in metastatic breast cancer, while PR and the DNA indices of cell ploidy and percentage of S phase are useful prognostic indicators in early stage breast cancer. We have developed a flow cytometry method to measure PR and DNA indices simultaneously using two aneuploid breast cancer cell lines—PR-positive T47D cells and PRnegative MDA-231 cells. Cells were pretreated with the progestin 17,21dimethyl-19-nor-4,9-pregnadiene-3,20-dione, harvested, counted, fixed with paraformaldehyde, and permeabilized with Triton X-100. To mea sure total PR, cells were first exposed to a mixture of the mouse anti-PR monoclonal antibody AB-52, which binds both Protein A and Protein B of human PR, and to monoclonal antibody B-30 or B-64, which bind only Protein B. Then the cells were treated with fluorescein isothiocyanateconjugated goat anti-mouse second antibody to produce a green fluores cence signal corresponding to PR. To measure nonspecific binding, cells were treated with mouse IgGl as the first antibody in a parallel incuba tion. Specific ¡mmunoassayable PR is the difference between total and nonspecific binding. Following the antibodies, the cells were treated with RNase A and propidium iodide to give a red fluorescence signal corre sponding to DNA content. Red and green fluorescence per cell was then quantified by flow cytometry. This method gives a strong specific signal for PR in several T47D cell sublines but no specific binding in MDA231 cells. Progestin treatment led to apparent increases in PR. The proportion of cells in the G0-G„S, and G2-M phases of the cell cycle was determined from DNA histograms and showed that both cell lines were hyperdiploid. The simultaneous flow cytometry method allowed assignment of relative PR levels in subsets of cells segregated by their DNA content. In T47D cells, PR were present throughout the cell cycle, and levels doubled in <; >and mitosis.