To understand the mechanism by which the deletions lead to clonal B lymphoproliferations, Klein et al. [57] investigated whether miR-15a/16-1 deletions could affect proliferation of mouse B cells. BrdU incorporation assays, by measuring active DNA-synthesis, demonstrated that miR-15a/16-1−/− B cells begin to synthesize DNA earlier than wild type B cells. Mitogen-stimulated B cells purified from miR-15a/16-1−/− or Mdr−/− and wild type mice were analyzed for levels of phosphorylated retinoblastoma (p-Rb) protein, an indicator of entry into the cell cycle. P-Rb could be revealed at earlier time points in both miR-15a/16-1−/− and Mdr−/− compared with wild type B cells. Consistently, the expression of the cell cycle inhibitory protein cyclin-dependent kinase inhibitor p27-Kip1 appeared to follow similar kinetics. To unravel the individual contributions of DLEU2 versus the miR-15a/16-1 cluster to the lymphoproliferative phenotype, the authors created an inducible in vitro system in which the DLEU2 transcript and the miR-15a/16-1 cluster were separately re-expressed in the human I83E95 cell line that was derived from a 13q14−/− CLL. The results showed that miR-15a/16-1 expressing cells, but not those expressing DLEU2, were impaired in proliferation.
