FIG.9.
Effects of UV stimulation on cyclin D1 and p52. (A) UV treatment down regulates cyclin D1 protein levels. U-2 OS cells were treated with 40-J/m2 UV radiation for the indicated times. Whole-cell lysates were prepared and immunoblotted for cyclin D1 and a β-actin control as indicated. (B) UV treatment inhibits the cyclin D1 promoter in a manner dependent upon the proximal κB element. One and a half micrograms of each of the cyclin D1 (−66) and cyclin D1 (−66 mut) luciferase reporter plasmids were transfected into U-2 OS cells. Cells were treated with 40-J/m2 UV radiation for 6 h as indicated. Results are expressed as change in activation or repression (n-fold) relative to levels seen in the relevant untreated cell controls. luc, luciferase. (C) UV treatment induces Ser-15 phosphorylated endogenous p53 and down regulates Bcl-3 levels. U-2 OS cells were treated with 40-J/m2 UV radiation for the indicated times. Nuclear protein extracts were prepared and immunoblotted for p53, phospho-Ser-15-modified p53, and Bcl-3 as indicated. (D) UV treatment results in loss of p52/Bcl-3 complexes. U-2 OS cells were either left untreated or stimulated with 40-J/m2 UV radiation for 6 h, and nuclear protein extracts were prepared. p52 or Bcl-3 was then immunoprecipitated from 100 μg of protein extract. The immunoprecipitated complex was then resolved by SDS-PAGE and immunoblotted for for the reciprocal protein, as indicated. A sample of input material is shown (10 μg). (E) Inhibition of cyclin D1 promoter activity following UV treatment can bereversed by expression of Bcl-3. One and a half micrograms of cyclin D1-luciferase reporter plasmid was cotransfected with 1.5 μg of Bcl-3 or control RSV expression plasmid into U-2 OS cells. Cells were treated with 40-J/m2 UV radiation for 6 h as indicated. Results are expressed as change in activation or repression (n-fold) relative to levels seen in the relevant untreated cell controls. (F) Down regulation of cyclin D1 protein levels following UV treatment can be reversed by expression of Bcl-3. U-2 OS cells were transfected with 1.5 μg of Bcl-3 or control RSV expression plasmid. After 36 h, cells were treated with 40-J/m2 UV radiation for 6 h as indicated. Whole-cell lysates were then prepared and analyzed by immunoblotting with the indicated antibodies. (G) UV treatment increases the association of p52 with HDAC1. U-2 OS cells were either left untreated or stimulated with 40-J/m2 UV radiation for 6 h, and nuclear protein extracts were prepared. p52 was then immunoprecipitated with the polyclonal antibody, together with an immunoglobulin G control, from 100 μg of protein extract. The immunoprecipitated complex was then resolved by SDS-PAGE and immunoblotted for HDAC1. A sample of input material is shown (10 μg).