| Effects of c-Myc expression were investigated on the DNA damage-induced checkpoints at the G1/S and at the G2/M boundaries in human mammary epithelial cells (HMECs). We initially used flow cytometric analysis to identify a c-Myc-induced alteration of the G1/S checkpoint in two nontransformed HMEC lines—184A1N4 and MCF10A. These results were confirmed using a BrdU incorporation assay, measuring de novo DNA synthesis. A transient excess of c-Myc activity, via the regulatable c-MycER, was, similarly, able to attenuate G1/S arrest, following ionizing radiation (IR). The c-Myc-induced G1/S alteration was also observed in finite lifespan, normal HMECs. In a molecular analysis, we observed deregulated hyperphosphorylation of Rb and then the reappearance of cyclin A, as the c-Myc-overexpressing cells entered into S phase, following IR. One additional finding from G1/S study is a dramatic increase in the cellular contents of multiple micronuclei, following IR, selectively in cells with excess c-Myc activity. Since micronucleation is a post-mitotic event, the results suggest a possibility of altered G2/M checkpoint by c-Myc. Therefore, we hypothesized that c-Myc alters the G2/M checkpoint, as well. We investigated the effects of c-Myc overexpression on the G2/M arrest following IR by employing a mitotic trapping assay, and obtained pilot data supporting our hypothesis. The MCF10A cells, overexpressing either c-Myc or dominant negative p53DD, demonstrated a significant percentage (roughly 10% in each) of cells in mitosis (based on rounded cell morphology), following prolonged post-IR incubation with nocodazole. In contrast, cells infected with an empty vector or a c-MycS-containing vector did not show significant numbers of rounded-up cells. Finally, mitoses of irradiated MCF10A-MycER cells were directly examined by time-lapse videomicroscopy. After prolonged mitoses, following IR, mitotic cells eventually were observed to carry out unbalanced cytokineses. Taken together, these results suggest that c-Myc alters safeguard mechanisms for genomic stability, both at the G1/S and at the G2/M boundaries. Alterations of the DNA damage-induced checkpoints, such as the one studied here, have been identified as crucial prerequisites for certain types of genomic instability, such as gene amplification. Therefore, based on these findings, we propose that deregulated c-Myc abrogates a safeguard mechanism(s) for genomic stability. |
