Vol. 10, Issue 12, 4231-4246, December 1999

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

Jong-Sung Park,^* Steven Carter,^* Dean B. Reardon,^* Rupert Schmidt-Ullrich,^* Paul Dent,^* and Paul B. Fisher

 ^*Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298; and  Department of Urology and Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032

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-induced 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 MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G₁ that were p21 and MAPK dependent, whereas the elevation of cells present in G₂/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G₂/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G₂/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G₂/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G₂/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G₁/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G₂/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.