CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Abstract

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner. Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.

Keywords: CDK1 (cyclin-dependent kinase) inhibitor; RO-3306; cell cycle arrest; epithelial cells; fibroblasts; radiation; radiosensitivity; radiotherapy.

Figure 1.

CDK1 inhibition sensitizes tumor cells but not normal cells to radiation. (a) Clonogenic survival assay of (a) tumor cells (HeLa, T24, and SQ20B) and (b) normal cells (HFL1, MRC-5 and RPE) treated with RO-3306 prior to radiation. Cells were seeded as single cells and treated with 5 µM RO-3306 from 3 h after seeding. Cells were irradiated 20 h after compound addition followed by a medium change. Colonies were stained at 7 – 13 days after seeding. Data are representative of three independent experiments, and are presented as mean ± SD from triplicate wells, p-values were derived through two-way ANOVA, testing whether the curves are statistically different. PE = plating efficiency; SER₁₀ = sensitization enhancement ratio at a surviving fraction of 10%.

Figure 2.

RO-3306 treatment causes inhibition of downstream signaling and cell cycle arrest in both tumor and normal cells. (a) Western blot stained with the MPM-2 antibody, recognizing phosphorylated CDK1 substrates. Cultures were enriched for mitotic cells by overnight nocodazole treatment followed by 2 h treatment with 5 µM RO-3306 in the continuous presence of nocodazole. Both floating and adherent cells were lysed and subjected to immunoblotting. Image is representative of three independent experiments. For clarity, a longer exposure is shown for the normal cell lysates; a comparison of different exposure times is shown in Supplementary Figure 2. (b) Cell cycle profile of tumor and normal cells treated with RO-3306. Cells were seeded in 6-well plates and treated with 5 µM RO-3306 for 20 h. Cells were lifted, fixed in 70% ethanol and stained with propidium iodide for analysis by flow cytometry. Representative histograms are shown (dark grey: G1, white: S; light grey: G2/M). Graph shows percentages in each cell cycle phase determined through curve fitting with ModFit and show the mean +/- SD from two independent experiments. Significant increases in G2/M proportion after RO-3306 treatment are indicated (* p < 0.05).

Figure 3.

Stalling of cell cycle progression in normal cells causes insensitivity to CDK1 inhibition. (a) Investigation of the cell cycle progression of untreated HFL1, MRC-5 and RPE cells plated for colony formation assays. Cultures were grown for three days to 80% confluence, lifted and reseeded in the absence of drug. Cells were then fixed for cell cycle analysis at the time points indicated. Representative histograms are shown (dark grey: G1, white: S; light grey: G2/M). Graphs indicate percentages in each cell cycle phase over time determined through curve fitting with ModFit and show the mean +/- sd from three independent experiments. Significant differences in G2/M proportion from t = 0 are indicated, * p < 0.05. (b) Clonogenic survival assay of HFL1, MRC-5 and RPE cells treated with RO-3306 at different time points. Cultures were grown for three days to 80% confluence, lifted and seeded as single cells. RO-3306 was added at a concentration of 5 µM at 3 or 24 h after seeding, followed by a medium change after 20 h of compound incubation. Colonies were stained after 12 days. Data are representative of three independent experiments, and is presented as mean +/- SD from triplicate wells, *p < 0.05.

Figure 4.

Normal and tumor cells are equally sensitive to continuous CDK1 inhibition. (a) Clonogenic survival assay of tumor and normal cells treated for 20 h or continuously with increasing concentration of RO-3306. Cultures were grown for three days to 80% confluence, lifted and seeded as single cells. RO-3306 was added 3 h after seeding and either removed after 20 h or left for the duration of the experiment. Colonies were stained after 12 days. Representative of three independent experiments, data is presented as mean +/- SD from triplicate wells. (b) Comparison of IC50 values for tumor and normal cells. IC50 values were determined for each cell line using the data in panel A. Mean IC50 was calculated for both tumor and normal cells grouped together. Statistical differences were confirmed by t-tests, ** p < 0.01.

Figure 5.

CDK1 inhibition sensitizes normal cells to radiation in a cell cycle dependent manner. (a) Clonogenic survival assay of cells treated with RO-3306 at different time points followed by radiation. Cells were grown in T75 flasks for three days, lifted and plated as single cells in 6-well plates. RO-3306 (5 µM) was added either at 3 h after seeding (for stalled cells) or at 24 h after seeding (when many cells are entering G2), and cells were irradiated 20 h later followed by a medium change. Colonies were stained after 12 days. Representative of three independent experiments, data is presented as mean +/- SD from triplicate wells. p-values were derived through two-way ANOVA, testing whether the curves are statistically different. PE = plating efficiency; SER₁₀ = sensitization enhancement ratio at a surviving fraction of 10%.