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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 Line¹

Divisions of Medical Oncology [M. L. G.] and Endocrinology [K. E. D., K. B. H.], Department of Medicine, and Department of Pathology [K. B. H.], University of Colorado Health Sciences Center, Denver, Colorado 80262

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 T47D_v, from the wild-type hyperdiploid T47D_co 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.

¹ Supported by grants from the Cancer League of Colorado, Denver, CO, and NIH Grant GM 07063 (M. L. G.); NIH Grant CA-26869, National Science Foundation Grant DCB-8709790, and the National Foundation for Cancer Research (K. B. H.). The Bonhomie Foundation, Waverly, MN, supported the summer stipend of K. E. D. The flow cytometry analyses were performed by Phillip Jewett and Ellen Braunschweiger in the Flow Cytometry Core Laboratory of the University of Colorado Cancer Center (NIH CA-46934). The karyotype analysis was performed by Iris Hart of the Cytogenetics Laboratory, Eleanor Roosevelt Institute for Cancer Research, in collaboration with Dr. David Patterson. Submitted along with an accompanying paper by Graham et al. entitled, "Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: characterization of an assay in breast cancer cell lines."

² To whom requests for reprints should be addressed, at Box B151, Divison of Endocrinology, University of Colorado Health Science Center, 4200 East Ninth Avenue, Denver, CO 80262.

Received 12/27/88. Revised 3/28/89. Accepted 4/ 6/89.