Figure 8.

Caffeine treatment of cells, or washing to remove the MEK1/2 inhibitor, 6 h after irradiation abrogates the ability of MAPK inhibition to potentiate enhanced G₂/M cell numbers and reduces the level of apoptosis at 24 h. A431-TR25-EGFR-AS cells were pretreated for 30 min before irradiation with a MEK1/2 inhibitor (PD98059, 10μM) or DMSO control. (A) Cells were irradiated (2 Gy), and 6 h laterthey were subjected to medium replacement containing PD98059 (control wash), medium replacement with no PD98059 addition in fresh medium (wash out), or medium replacement containing PD98059 supplemented with 1 mM caffeine. The cell cycle profiles under each condition were determined 18 h after washing (24 h after irradiation) by flow cytometry. Data are the means of four separate experiments (±SEM). (B) Cells from each treatment and condition in A were examined for apoptosis 18 h after washing (24 h after irradiation) by TUNEL assay. Cells were trypsinized from their dishes, and portions (∼10,000 cells) were spun onto glass slides, followed by TUNEL staining for double-stranded DNA breaks. Randomly selected fields of fixed cells (n = 5 per slide) were counted initially with the use of propidium iodide counter stain, followed by examination and counting of TUNEL positive-staining cells of the same field under FITC/florescent light. Data are the means of multiple individual points from three separate experiments (±SEM).