CCD alters time-dependent anticancer activity of PD-0332991, a potent CDK4/6-specific inhibitor, in U2OS cells.

(A) Western blot analysis of the dose dependency of PD-0332991 action on RB phosphorylation in U2OS cells. After a 48-hour incubation with increasing doses of PD-0332991, cell extracts were harvested for immunoblot analysis of protein abundances of phosphorylated RBs at multiple sites (pRB-S807/811, pRB-S795, pRB-S780, pRB-S612), total RB, cyclin D1, CDK4, and CDK6 using specific antibodies. Anti-GAPDH (αGAPDH) was used for loading control. (B) Schematic of the experimental schedule to determine time dependence of the antiproliferative effect of PD-0332991 on U2OS cells. After 24 hours of dex (100 nM, yellow bolt) synchronization, the cells were treated with vehicle or PD-033291 (0.01–5 μM, red bolt) at 6-hour intervals over the course of 24 hours and subjected to an MTT cell proliferation assay at the indicated time points 48 hours later. (C) Graph of experimental procedures in (B) to show time-dependent antiproliferative effects of PD-0332991 at various doses. **p < 0.001, ***p < 0.0001; two-way ANOVA and Tukey multiple comparisons test. p^MetaCycle < 0.05 denotes results of MetaCycle analysis, identifying a 24-hour rhythm in drug sensitivity of cells treated with 0.1 μM PD-0332991 (p = 0.0146). Data were normalized to represent the average ± SD; n = 3 in all time points. The result is representative of three independent experiments. (D) Twenty-four hours after the final dex stimulation depicted in Fig 1A, Control (CTL) and Jet lag (JL) U2OS cells stably expressing siRNA against GFP (si-GFP^stable) or BMAL1 (si-BMAL1^stable) were subject to a time course of vehicle or PD-0332991 (0.1 μM, 1 μM) treatment, and subsequent MTT analysis was performed as described in (B). Data were normalized to represent the average ± SD; n = 3 in all time points. p^MetaCycle < 0.001 denotes results of MetaCycle analysis, identifying a 24-hour rhythm in drug sensitivity of cells treated with 1 μM PD-0332991 (p = 0.00019). The result is representative of two independent experiments. Underlying data for this figure can be found in S3 Data. BMAL1, brain and muscle arnt-like protein-1; CCD, chronic circadian desynchrony; CDK, cyclin dependent kinase; dex, dexamethasone; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; MTT, thiazolyl blue tetrazolium bromide; RB, retinoblastoma; siRNA, small interference RNA U2OS, human U2 osteosarcoma.