FIG. 9.

Failure to degrade cyclin D1 compromises the intra-S-phase checkpoint response to DNA damage and sensitizes cells to CPT. (A) The replication factor Cdt1 is stabilized following DNA damage in cells expressing cyclin D1T286A. HeLa cells were transfected with wild-type or cyclin D1T286A, CDK4, and Cdt1. Cells were treated with 10 μg of bleomycin/ml as indicated, and Cdt1 levels were assessed by immunoblotting. (B) Cyclin D1 stabilization promotes maintenance of MCM proteins on chromatin. Esophageal carcinoma cell lines expressing endogenous wild-type or D1P287A were irradiated and harvested at the time points indicated. Cell lysates were fractionated into chromatin-bound and soluble fractions; MCM3 binding to chromatin was assessed by immunoblotting on chromatin-bound fractions. Ponceau-S stain served as a control for equal loading of chromatin-bound fractions, and PCNA is a representative chromatin-bound protein. (C) Expression of D1T286A results in RDS. RDS assays were performed in synchronous parental NIH 3T3 cells or 3T3-D1 or 3T3-D1T286A cells. Equal concentrations of DNA were counted for [³H] and [¹⁴C]thymidine incorporation. DNA synthesis, calculated as the ratio of [³H] to [¹⁴C], was normalized to the nonirradiated control for each cell line. (D) Disruption of SCF^{Fbx4-αBcrystallin} promotes RDS. RDS assays were performed as in panel C utilizing cells stably expressing luciferase, αB crystallin, or Fbx4-specific shRNA. (E) Fbx4 attenuation in cyclin D1-null cells does not result in RDS. RDS assays as in panel C were performed in wild-type or cyclin D1-null MEFs stably expressing empty vector or Fbx4-specific shRNAs. (F) Fbx4 levels are attenuated in cyclin D1-null MEFs stably expressing Fbx4-specific shRNA. (G) Failure to degrade cyclin D1 sensitizes cells to the CPT. 3T3-D1 or D1T286A cells were treated with CPT as indicated for 24 or 48 h. Cells were fixed and stained with propidium iodide and subjected to FACS analysis; a sub-G₁ population is indicative of apoptotic cells.