February 06, 2019

Author: Paul Held, BioTek Instruments, Inc., Winooski, VT, USA


Cell cycle progression is a tightly regulated process that involves the expression of specific proteins and the duplication of nuclear DNA content prior to cell division. The control mechanisms that regulate this process are often disrupted in tumor cells and serve as viable targets for therapeutic compounds in the treatment of cancer. Cell cycle progression has historically been monitored using flow cytometry. Here we describe the use of a microplate reader, to rapidly image and analyze immunofluorescent and nuclear stained PC-3 tissue culture cells for cell cycle analysis. PC-3 cells were synchronized using double thymidine block. Once released, cells were fixed with 4% PFA at 1-hour intervals using a BioSpa 8 system. At the completion of the experiment, plates were stained with Hoechst 33342 and imaged at 10X. After imaging, fluorescence intensity thresholds used to sort cells into different cell cycle stages were manually defined using histogram analysis of cell count vs total fluorescence of stained nuclei in Gen5 software.

Subpopulations G1, S, and G2 were counted automatically and their percentages plotted as a function of time from thymidine release. Cells were found to be nearly synchronous with respect to their DNA content. Initially, about 70% of the cells have a 2N chromosome number. Within 6 hours, this percentage has dropped to 33%, while the percentage of cells in S-phase has increased from near 0 to approximately 33%. By 12 hours, most cells have duplicated their DNA content and are in G2 phase of the cell cycle. During cellular mitosis, nuclear DNA is divided equally between the two daughter cells returning the cells to G1 phase, which is observed by the rapid decline in the G2 percentage after 15 hours concurrent with an increase in the percentage of cells with a G1 DNA content.

Estimates of cell cycle passage time-length in synchronized cells were made using nuclear DNA content as well as specific protein markers. Temporal peaks in G1 and G2 subpopulation percentages based on nuclear content, and the G1 and Mitosis specific protein markers, geminin and p-Histone H3, respectively, demonstrated a periodicity of approximately 20 hours in synchronized PC-3 cells. In asynchronous cells, calculations based on mitotic index returned a cell cycle time of 17.5 hours.

This analysis was also used to screen cyto-active compounds for their ability to halt cell cycle progression. Using the known cell cycle blocking agents, mevinolin (G1) and nocodazole (G2) as controls, 27 compounds were tested for their efficacy to block cell cycle progression. Of the compounds tested, 7 were shown to cause a significant increase in the number of cells in G2 and mevinolin, when treated as an unknown was independently identified as blocking cells in G1.