Date Published: July 11, 2011
Publisher: Impact Journals LLC
Author(s): Milena Rizzo, Monica Evangelista, Marcella Simili, Laura Mariani, Letizia Pitto, Giuseppe Rainaldi.
The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF 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 MEF 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 MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity.
The Hayflick limit of primary mouse embryo fibroblasts (MEF) is variable since the growth conditions can either reduce or extend it [1-3]. We have reported that primary MEF expanded every three days (6T3 protocol) underwent four population cell doubling and thereafter cell proliferation was fully abolished and SA-β-gal+ cells induced . However it is known that MEF senescence can be induced prematurely, before the end of the physiological lifespan, by cellular stresses such as the over expression  or down regulation  of single oncogenes and DNA damaging drugs [7-9]. Recently, it has been reported that miRNAs are involved in senescence of MEF as well as of human diploid fibroblasts [10,11].
The in vitro life span defines the number of population cell doubling which primary cells can undergo in culture. We have reported that MEF under the 6T3 expansion regimen were able to undergo four population cell doubling before achieving senescence. In this work we reported that by modifying the propagation regimen and growth conditions (6T15 protocol) MEF slowed down without reaching a complete cell proliferation block and restarted proliferation after about eight population cell doubling (passage 3). These results suggest that activation of p53 with the consequent induction of p21 in this case may favor proliferation arrest rather than senescence as previously described . Interestingly the in vitro life span of MEF was extended both in terms of population cell doubling and in terms of days in culture, in keeping with the hypothesis that p53 activation, by inducing quiescence rather than senescence may increase the cell life span . The immortalization process is delayed accordingly, suggesting that it does not strictly depend on the number of cell doubling or the time that the cells spend in culture (45 culture days in this case).