Evaluating Small Molecule Cell Cycle and Chromatin Condensation Modulators Using a Novel Green Fluorescent DNA-Binding Probe: Potential Application to Compound Screening

When eukaryotic cells undergo replication, they pass through a tightly regulated series of events known as the cell cycle, marked by distinctive characteristics as the DNA is replicated. Cell cycle checkpoints at specifi c points in the process prevent cells from progressing to the subsequent phase of the cell cycle in the event of DNA damage or other adverse conditions that might impact overall survival. Considerable progress has been made in the analysis of the stages of cell cycle progression, with a number of cell permeable small molecules identifi ed to modulate the process. Flow cytometry is a commonly implemented platform for cell cycle analysis, but the most widely employed dye for the instrument, propidium iodide, requires permeabilization or fi xation of cells. A number of newer cell-permeable dyes have subsequently been introduced but most require very restrictive staining conditions and laborious standardization of techniques. We have introduced a novel fl uorescent probe that facilitates cell cycle analysis in live cells. The green fl uorescent probe can be used in a mix and read format over a wide concentration range (5-20 µM) employing a wide range of cell densities (1x 10 5 -1x10 6 cells/ml). Moreover the dye provides substantial fl exibility with respect to the incubation medium, time and temperature used in the analysis. Live cell cycle analysis was benchmarked using a panel of 12 small molecule cell cycle modulators known to perturb cells at the G0/G1, S or G2/M phases in a concentration- dependent manner. Apoptotic cell death was monitored as well through protocol modifi cations allowing sub G0 analysis and chromatin condensation determination. The described fl uorescent probe should be applicable to the analysis of the phases of the cell cycle, especially as applied to the identifi cation of small molecule modulators for use in treatment strategies targeting cell cycle checkpoints.