Systematic Analysis of Cell Cycle Effects of Common Drugs Leads to the Discovery of a Suppressive Interaction between Gemfibrozil and Fluoxetine

Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation

Gemfibrozil delays initiation of DNA replication.

<p>A, The critical cell size (shown in fl) of diploid BY4743 cells treated with DMSO, rapamycin (0.1 µg/ml) or gemfibrozil (50 µg/ml), was measured from synchronous elutriated cultures, in YPD medium. The data points shown were from three independent experiments in each case. The <i>P</i> values shown were calculated from paired, two-tailed <i>t</i> tests, assuming unequal variance. The data used to calculate these parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503.s001" target="_blank">Figure S1</a>. B, The specific rate of cell size increase constant <i>k</i> (in h⁻¹) was measured from the same elutriation experiments shown in a, assuming exponential growth. The data used to calculate these parameters are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503.s001" target="_blank">Figure S1</a>. C, The cell size distributions of the indicated cell populations, proliferating asynchronously in YPD medium, were measured using a channelyzer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#s4" target="_blank">Materials and Methods</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503-Hoose1" target="_blank">[22]</a>). Cell numbers are plotted on the y-axis and cell size (in fl) on the x-axis. Daughter “birth” size was defined as the maximum size of the smallest 10% of cells on the left side of the cell size distribution of each sample <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036503#pone.0036503-Hoose1" target="_blank">[22]</a>.</p

Representative DNA content histograms.

<p>Independent experiments of the indicated samples are shown in each case. Fluorescence is plotted on the x-axis, while the number of cells analyzed is on the y-axis. Reference samples were treated with DMSO, shown at the top. Examples of “High G1” profiles include cells treated with ketoconazole or gemfibrozil, while cells treated with fluoxetine give rise to a “Low G1” DNA content profile. At the bottom, we show a few examples of complex DNA content histograms that were unquantifiable. These include profiles of cells treated with suramin and 5-fluorouracil (antineoplastic agents), and flubendazole (a microtubule blocker used as anti-nematodal).</p

Decision flow-chart diagram of our primary analysis.

<p>This diagram summarizes our DNA content measurements using the <i>pdr5Δ, snq2Δ</i> strain. See text for details.</p

DNA content analysis identifies drug effects on cell cycle progression.

<p>A, Cumulative histogram displaying the percentage of cells in the G1 phase of the cell cycle (%G1), for cells treated with a panel of FDA-approved drugs. The bin width of the histogram is 1%, with each bin containing all the drugs with values within the bin boundaries. The black line superimposed to this histogram is the normal distribution fit of the %G1 values of the reference sample. Bins with values >2 sd from the mean of the wild type distribution are in grey (“Low G1” group) and black (“High G1” group). B, From all the samples we analyzed by flow cytometry, the %G1 is on the x-axis, and the forward angle scattering (FSC) values on the y-axis. We colored the data points of the sub-groups as in A.</p