8226/LR5 cells release earlier from melphalan-induced cell-cycle inhibition compared with drug-sensitive 8226/S cells. (A) Flow cytometric analysis of cell-cycle phases. DNA histograms of untreated and melphalan-treated 8226/S (top) and 8226/LR5 cells (bottom) show the dose-response effects of melphalan on cell-cycle progression. Representative data are shown. (B) Time course analysis of cell-cycle progression in 8226/S and 8226/LR5 cells with and without melphalan treatment. The dot plots depict BrDU incorporation (S phase cells) detected with an FITC anti-BrDU antibody on the y-axis, and propidium iodide (PI) to detect DNA content on the x-axis. The distribution of DNA content is represented by the inset histograms. Shown is 1 of 3 representative experiments. 8226/LR5 cells are able to progress through the cell cycle following melphalan exposure; whereas 8226/S cells arrest at G2/M (72 hours). (C) Quantitative analysis of the delay of progression through the cell cycle. To study cell-cycle progression delay in early and late S phases, DNA content was divided into 6 sections and quantified as G1, S1, S2, S3, S4, and G2 phase using FlowJo 4.4.4 software Watson (Pragmatic) model. Within 12 hours following melphalan treatment, 8226/S and 8226/LR5 cells significantly accumulated at early S phase compared with non-drug-treated cells. (D) At 24 hours after melphalan treatment, 8226/S cells significantly accumulated at S2 compared with 8226/LR5, while 8226/LR5 cells significantly accumulated at late S phase (S3) compared with 8226/S cells. The mean values and standard deviations from 3 independent experiments are shown. Student t test was used for statistical analysis. *P < .05.
