SAHA minimally affected cyclin D1 mRNA levels and stability of cyclin D1 protein. (A) Northern blot analysis of cyclin D1 in Jeko1 cells treated with SAHA (5 μM, 8 hours). Levels of 18S ribosomal RNA (18S) are shown as an internal control. Intensity of bands was quantified, and levels of cyclin D1 relative to 18S rRNA are shown. Cyclin D1 mRNA/S18 in nontreated cells is regarded as 1.00. (B) Jeko1 cells were pretreated with SAHA (0, 5, or 50 μM; 4 hours); then, the protein synthesis inhibitor cycloheximide was added to each well (CHX, 100 μM). Cells were collected at indicated time points, and cyclin D1 and β-actin proteins were detected by Western blot analysis. Levels of cyclin D1 protein decreased, whereas β-actin did not change. In the presence of SAHA (5 or 50 μM), stability of cyclin D1 slightly increased (0 μM SAHA, 34% protein levels at 30 minutes; 5 μM SAHA, 47% protein at 30 minutes; 50 μM SAHA, 69% protein at 30 minutes.) Protein levels at 30 minutes are expressed as a percentage of levels at time 0, and the data are shown graphically. (C) Result of pulse-chase assay. Both SAHA and [³⁵S]methionine were added to culture media containing Jeko 1 cells at the beginning of the incubation. Jeko1 cells plus or minus SAHA (5 μM) were labeled with [³⁵S]methionine for 4 hours (Pulse), washed 3 times with culture medium, and released in normal culture medium plus or minus SAHA (5 μM) for indicated times (Chase) followed by immunoprecipitation of the cyclin D1 protein with anti-cyclin D1 antibody and fractionated in SDS-PA gel. Signal of cyclin D1 proteins was captured on a X-ray film, and intensity of the bands were quantified and plotted graphically. Intensity at time 0 is regarded as 1.0. (D) Jeko1 cells were treated with SAHA (5 μM) and/or the proteosome inhibitor, PS341 (also known as bortezomib [Velcade]) (10 μM) for 6 hours. Cellular lysate was Western-blotted and probed with antibodies to cyclin D1 and β-actin. No tx, cellular lysate of cells treated with diluent.