Polo-like kinase 1 (Plk1) depletion by RNA interference (RNAi) induced G2/M arrest and apoptosis in cancer cells. Cancer cells with a p53-dificient background were more sensitive to Plk1 depletion-induced cell death than those with wt p53. By lentivirus-based RNAi, a series of Plk1 hypomorphs were generated in HeLa cells. Study with these hypomorphs demonstrated that different levels of Plk1 were required for different stages of mitosis, with the level required for mitotic entry lower than that necessary for mitotic progression. In striking contrast to cancer cells, normal nontransformed MCF1 0A and hTERT-RPE1 cells survived Plk1 depletion levels that would result in cancer cell death, supporting the potential of Plk1 as a target for cancer therapy. When p53 was co-depleted with Plk1, MCF10A cells underwent G2/M arrest and apoptosis. Study on cloned individual Plk1-depleted MCF1 0A cells revealed a minimal requirement for Plk1 for normal cell proliferation, although the requirement was much lower than that of cancer cells. Cloned Plk1-depleted hTERT-RPE1 cells displayed reduced proliferation rates due to a delay in cell-cycle progression at early S phase, suggesting possible functions of Plk1 in DNA synthesis. Plk1 depletion induced DNA damage and activated the DNA-damage checkpoint. Caspase 2 was partially responsible for the apoptotic signaling after Plk1 depletion in p53-null cancer cell, H1299.