Cyclin D1 Overexpression Increases Chemosensitivity Via Prolonged S-Phase and TRAIL Signal in Myeloma Cell.

The mechanism of oncogenesis of plasma cells remains unclear. Since tumor cells are post-germinal center B cells, reciprocal chromosomal translocations between the IgH gene located on chromosome 14q32 and other chromosomal partners such as cyclin D1 located on chromosome 11q23, which are supposed to be candidate oncogene, arise in myeloma cells during the procedure of isotype switching. However, a recent study demonstrated that multiple myeloma (MM) with cyclin D1 overexpression belongs to the low-risk group. Previously, we reported that cyclin D1 overexpression downregulated cyclin D2 expression in myeloma cells and increased the sensitivity to treatment with anti-myeloma agents such as bortezomib (PS-341), dexamethasone, melphalan, and immunomodulatory thalidomide analogs in a comparison between RPMI8226 transfected cyclin D1 and mock. Here we have analyzed characteristics of RPMI8226 transfected cyclin D1. This cyclin D1 transfectant did not induce cell growth advantage, and cell cycle analysis by bromodeoxyuridine (BrdU) stimulation showed significant increase cell number in S-phase without an increase of that in G₂/M-phase and decrease of cell number in G₀/G₁-phase. Therefore, Cyclin D1 overeexpression prolonged the S-phase. Western blot analysis demonstrated an increase in the hyperphosphorylated form of retinoblastoma protein (ppRb), and this ppRb was also found in KMS12BM, KMS21BM, in which myeloma cell line cyclin D1 overexpresion was detected due to t(11;14). Considering that ppRb releases free E2F, the increase in E2F could be expected to upregulate apoptosis-related genes. However, expressions of p53, Bcl-2, Bax, Bad, Bim, Mcl-1, p16, and CDK4 were not changed in cyclin D1 transfectant besides the decrease in p27 expression compared with those of the mock and parent cells. Furthermore, cyclin D1 overexpression that alone did not induce apoptosis because there were no such cells detected in sub-G₀/G₁ by BrdU stimulation without treatment by anti-myeloma agents. On the other hand, treatment with anti-myeloma agents induced both the intrinsic and extrinsic pathways earlier in the cyclin D1 transfectant. And this cyclin D1 transfectant cells easily lost viability in confluent samples. Interestingly, expression of the TRAIL receptors (DR4 & DR5) were significantly higher in cyclin D1 transfectant cells and treatment with recombinant TRAIL induced apoptosis earlier compared with those of mock and parent cells. There was no difference of TRAIL expression in these cells by western blot. These findings suggest that high sensitivity to anti-myeloma agents in myeloma cell with cyclin D1 overexpressin might be due to the prolonged S-phase duration and high expression of TRAIL receptor. We speculate that high ppRb induces gene instability via high E2F and leads to progressive disease in MM. This might be a reason why cyclin D1 overexpression caused by t(11;14) or hyperdiploidy is an early event in the progression of MM.