Two-fold elevation of expression of FoxM1 transcription factor in mouse embryonic fibroblasts enhances cell cycle checkpoint activity by stimulating p21 and Chk1 transcription

Objectives: Forkhead Box M1 (FoxM1) transcription factor regulates expression of cell cycle effective genes and is stabilized by checkpoint kinase 2 (Chk2) to stimulate expression of DNA repair enzymes in response to DNA damage. This study intended to test whether FoxM1 is involved in cell cycle checkpoint pathways. Materials and methods: Analysis of senescence and cell proliferation in FoxM1 transgenic (TG) mouse embryonic fibroblasts (MEFs) with 2-fold elevation of FoxM1, and overexpression or knockdown of FoxM1 in an inducible FoxM1 expression cell line, or FoxM1 siRNA. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and cotransfection to determine FoxM1 transcription targets, as well as RNase protection assays and western blot analysis, were performed. Results: Two-fold elevation of FoxM1 in FoxM1-TG-MEFs resulted in low levels of cell proliferation and increase in permanent cell cycle arrest at early passages (from passage 6 to 9). These phenotypes correlated with increased phosphorylation of p53 on Ser15, elevated expression of cell cycle inhibitor p21 and Chk1 at passage 3. FoxM1 was stabilized in response to DNA damage in MEFs and FoxM1 overexpression induced p21. Knockdown of FoxM1 resulted in decrease in Chk1. ChIP, EMSA and cotransfection assays confirmed that FoxM1 stimulated promoters of p21 and Chk1. Conclusions: Chk1 and p21 are direct transcription targets of FoxM1 and FoxM1 participates in transcriptional responses to stress in normal cells.