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Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: analysis of breast cancer cell mixtures and genetic instability of the T47D line.

A flow cytometry assay that can simultaneously measure progesterone receptors (PR) and DNA indices in breast cancers would be a valuable clinical tool. We have developed a prototype assay that has proven useful in studies of the cell biology of breast cancer cell lines. The assay uses PR-specific monoclonal primary antibodies and fluorescein-conjugated secondary antibodies to measure PR, and propidium iodide to measure DNA. We find that the specific PR fluorescence generated by labeling PR-rich T47D human breast cancer cells is located predominantly in nuclei. The flow cytometry assay can quantitatively measure large fluctuations in intracellular PR levels: an apparent increase in PR following acute progestin treatment that cannot be documented by ligand binding assays; and the receptor down-regulation that follows chronic progestin treatment. The assay can identify fewer than 10% PR-positive cells in a population of PR-negative cells having the same DNA content, and it can sort PR-positive and PR-negative cells from cell mixtures having different DNA indices. Gating allows quantitative analysis of these mixed cell populations by ploidy, cell-cycle phase, and PR content. Finally, the assay has allowed us to monitor the gradual emergence of a stable hypertetraploid cell population, designated T47Dv, from the wild-type hyperdiploid T47Dco stocks. The new cells have unchanged estrogen receptors but even higher PR levels than the parental cells. They have five to ten copies of chromosome 11, site of the PR gene and other genes of interest in breast cancer.