Inhibition of the mitogen activated protein kinase pathway potentiates radiation-induced cell killing via cell cycle arrest at the G(2)/M transition and independently of increased signaling by the JNK/c-Jun pathway

The ability of low dose ionizing radiation (2 Gy) to modulate the activitie s of the mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kin ase (JNK1) cascades in human monocytic leukemia (U937/pREP4) cells and in c ells over-expressing dominant negative c-Jun (TAM67) (U937/ TAM67) was inve stigated. Radiation exposure caused prolonged (similar to 1 h) MAPK activat ions in U937 cells. In contrast, low dose irradiation weakly modulated JNK1 activity in these cells. Inhibition of the MAPK pathway by use of the spec ific MEK1/2 inhibitor (10 mu M PD98059) in both U937/pREP4 and U937/TAM67 c ells prior to radiation exposure permitted strong prolonged radiation-induc ed activations of JNK1. Expression of TAM67 decreased the ability of radiat ion to cause apoptosis compared to control transfected cells. However, comb ined MEK1/2 inhibition and radiation exposure in both cell types caused a l arge decrease in suspension culture growth and a large increase in apoptosi s, when compared to either treatment alone. Reduced proliferation after com bined irradiation and PD98059 treatment in both cell types correlated with prolonged cell cycle arrest in G(2)/M phase. Prolonged growth arrest was ab olished when MEK1/2 inhibitor was removed 6 h following irradiation, which was associated with a reduction in apoptosis. The ability of MEK1/2 inhibit ion to cause prolonged G(2)/M growth arrest was reduced in U937 cells stabl y transfected with a p21(Cip-1/WAF1) antisense construct of radiation-induc ed apoptosis and a reduced ability of MEK1/2 inhibition to potentiate apopt osis. Collectively our data demonstrate that inhibition of MEK1/2 function increases the radiation sensitivity of U937 cells, independently of c-Jun f unction, and decreases the ability of these cells to recover from the radia tion-induced G(2)/M cell cycle checkpoint arrest. In addition, our data als o demonstrate that the ability of MEK1/2 inhibition to potentiate radiation -induced cell death in U937 cells in part requires an ability of cells to e xpress low levels of p21(Cip-1/WAF1).