Mechanisms of the sustained expression of cyclin D1 and bcl-2 in M1-G1 during the treatment with IL-6.

(A) Changes in expression levels of cyclin D1 and bcl-2 messenger RNA during the culture in the presence of actinomycin D. M1-V1 and M1-G1 cells were pretreated with 10 μg/mL of actinomycin D for 2 hours and then subjected to the cultures with or without recombinant human (rh) interleukin 6 (IL-6) in the presence of actinomycin D. Expression of cyclin D1 and bcl-2 was examined by Northern blot analysis at the time indicated. (B) Protein turnover of cyclin D1 and bcl-2 in M1-V1 and M1-G1 during the culture with or without rhIL-6. To examine the half-lives of cyclin D1 and bcl-2 proteins, the cells (1 × 10⁷ cells for each sample) were radiolabeled with ³⁵S]methionine for 30 minutes (for cyclin D1) or 6 hours (for bcl-2). Then, the cells were washed, resuspended in DMEM containing 2 mmol/L of unlabeled methionine and cultured with or without rhIL-6 for the time indicated. Cyclin D1 and bcl-2 were immunoprecipitated from total cellular lysates and subjected to SDS-PAGE. The gels were dried and subjected to the autoradiography. The amounts of the radioactivity of cyclin D1 and bcl-2 were measured by a densitometric analysis.