Abstract DNA strand breakage and perturbation of cell-cycle progression contribute to gene amplification events that can drive cancer. In cells lacking p53, DNA damage does not trigger an effective cell-cycle arrest and in this setting promotes gene amplification. This is also increased in cells harboring oncogenic Ras, in which cell-cycle arrest is perturbed and ROS levels that cause DNA single strand breaks are elevated. This study focused on the effects of v-K-ras and p53 on Methotrexate (MTX)-mediated DHFR amplification. Rat lung epithelial cells expressing v-K-ras or murine lung cancer LKR cells harboring active K-ras continued cell-cycle progression when treated with MTX. However, upon loss of p53, amplification of DHFR and formation of MTX-resistant colonies occurred. Expression levels of cyclin A, Geminin, and Cdt1 were increased in v-K-ras transfectants. Geminin was sufficient to prevent the occurrence of multiple replications via interaction with Cdt1 after MTX treatment, and DHFR amplification proceeded in v-K-ras transfectants that possess a functional p53 in the absence of geminin. Taken together, our findings indicate that p53 not only regulates cell-cycle progression, but also functions through geminin to prevent DHFR amplification and protect genomic integrity. Cancer Res; 72(23); 6153–62. ©2012 AACR.