Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor activity

The life span (Hayflick limit) of primary mouse embryo fibroblasts (ME F) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point ME F were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immor tal. With this protocol ME F lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary ME F senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized ME F inhibited cell proliferation thus displaying a tumor suppressor-like activity.