Cell cycle synchronization induced by tamoxifen and 17 beta-estradiol on MCF-7 cells using flow cytometry and a monoclonal antibody against bromodeoxyuridine

Abstract

Cell cycle synchronization of MCF-7 hormone-sensitive human breast cancer cells has been evaluated after sequential treatment with tamoxifen and 17 beta-estradiol. The analysis was performed by flow cytometry. Two methods were used, one for single-parameter DNA content analysis, and one for bivariate analysis of DNA content and amount of incorporated bromodeoxyuridine (BrdUrd) into DNA using a specific monoclonal antibody. According to the BrdUrd method, tamoxifen was found (over a 30h period) to decrease (with respect to cells grown in control medium) the fraction of cells in S phase from 45% to 20%, to increase cells in G0 + G1 from 47% to 68%, and to induce a slight build-up of cells in G2 + M. Subsequent addition of estradiol resulted in partial synchronous recruitment of the cells from G0 + G1 to progress through the S phase; after 6-8 h delay time, the percentage of cells in G0 + G1 decreased by 50% and cells in S increased by 175%. The bivariate BrdUrd technique offered more reliable and detailed information than the single-parameter DNA analysis for differentiating and measuring the time course of estrogen-recruited cells as they progressed through early and late S phase, and has the potential for a very detailed cell kinetic analysis of both in vitro and in vivo hormone-sensitive cells.