Cell Cycle Analysis of Estrogen Receptor Positive Breast Cancer Cells Using Flow Cytometry

In cases of estrogen receptor positive (ER+) breast cancer, the antiestrogen Tamoxifen, a selective estrogen receptor modulator, in its active form (4-hydroxytamoxifen) has been used as a long-term treatment to inhibit tumor growth. However, a large portion of patients develop resistance to tamoxifen overtime by unknown mechanisms. Another breast cancer, Triple Negative Breast Cancer (TNBC), is characterized by the lack of expression of three receptors: human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor alpha (ERa). Since these receptors are commonly targeted in breast cancer, there are limited treatments for TNBC. Our laboratory hypothesized that the accumulation of insulin-like growth factor binding protein 3 (IGFBP-3) contributes to the development of Tamoxifen resistance, and this hypothesis has been supported by previous PCR and immunoblot analysis. In this project, we perform cell cycle analysis on two ER+ cell lines developed to overexpress IGFBP-3. We do this by performing cell cycle synchronization at the G1 or G2 phase using the chemical synchronization drugs Lovastatin and Mevalonic acid. Flow cytometry was used to confirm synchronization. Successful cell cycle arrest and release were observed in the samples treated with Lovastatin and Mevalonic acid, respectively. In future experiments, this protocol will be used to perform direct comparison between triple negative and MCF7 breast cancer cells.