Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induce d apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MATK pathway. Radiation induced p21 protein expression i n A431 cells within 6 h via secondary activation of the MAPK pathway. Withi n 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inh ibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apop tosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decr eased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatmen t or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the a bility of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/r elease of cells through G(2)/M phase after radiation exposure and that an i mpairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to-increase the p roportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the con trol level of apoptosis as well as the abilities of both radiation and MEK1 /2 inhibitors to cause apoptosis. In addition, loss: of basal p21 expressio n significantly reduced the capacity of MAPK inhibition to potentiate radia tion-induced apoptosis. Collectively, our: data argue that MAPK signaling a nd p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibi tion of MAPK increases the proportion of irradiated;cells in G(2)/M, and ba sal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regula ting cell cycle progression that affect cell survival after radiation expos ure.