Although numerous immortalized cell lines have been established from various domestic animals, such as bovine, equine, ovine, avian, canine and porcine [10, 15, 26–29], here, we first report establishment of the spontaneous immortalized cells derived from miniature pigs.
One of the major regulatory pathways for replicative senescence is known to come from activation of p53, one of the best characterized cell cycle checkpoints and tumor suppressors [9, 30]. p53 functions as transcriptional activator or repressor and plays a crucial role in cell proliferation and transformation by tightly regulating expression of the various cell cycle negative regulators and apoptosis-inducing factors such as p21WAF1 and BAX, respectively [31–33]. It has been documented that p53 activity is markedly elevated in the senescent cells, whereas loss of p53 is sufficient to escape senescent barriers and ultimately become immortalized in a variety of cells [34–36].
A shortening of telomere length by inactivation of telomerase should also be associated with senescent-mediated growth arrest in a variety of species [9, 36]. In normal somatic cells, the telomere shortening occurs in each cell division as a result of the end-replication problem of DNA polymerase . Critically shortened telomeres trigger loss of chromosomal integrity and concomitantly activate DNA damage response that eventually results in irreversible cell cycle arrest (senescence) through activation of p53 function .
In the present study, the primary MPF cells showed slower growth rate and shorter in vitro lifespan compared to the primary PF cells. The shorter lifespan in the primary MPF cells might be caused by activation of p53 as judged by relative expression levels of p53 and p21WAF1 (one of the p53-downsteam target genes). Although expression of p53 was elevated in immortalized MPF cells compared to their counterpart cells, expression of p21WAF1 was shown to be dramatically decreased in these cells. However, there is a question of how p21WAF1 is downregulated in the immortalized MPF cells possessing a significantly higher steady-state level of p53 protein. This converse expression pattern of p53 and p21WAF1 in the immortalized MPF cells has been commonly observed in other immortalized cells [39, 40]. It is also well-characterized that when p53 protein is inactivated by point mutation, its stability is known to increase owing to evasion of MDM2-dependent ubiquitin-mediated proteolysis . Therefore, we speculate that p53 function in the immortalized MPF cells might be inactivated by point mutation.
In accordance with previous data , our results have indicated that the activation of p53 signaling should be a major determinant for the replicative senescence in the cells derived from pigs, because inactivation of the p53 signaling by SV40LT was shown sufficient for both primary MPF and PF cells to escape from the senescent barrier. However, we can not rule out that a variety of other genetic alterations, such as inactivation of Rb by SV40LT or less well characterized associations of p300/CBP and Bub1 with SV40LT, might be also involved in cellular immortalization . Furthermore, since the spontaneously immortalized PF cells are recognized to possess functional p53 activity, it is also possible that cellular immortalization of pig cells might be occurred by external microenvironmental changes [44, 45] or other endogenous genetic alterations such as loss of Ink4a/Arf tumor suppressor, without loss of p53 function .
In the case of telomerase activity, endogenous telomerase activity in the all primary and immortalized pig cells used in the present study was found to possess minimum basal levels that are similar to that in the human BJ fibroblast cells. Moreover, we failed to reconstitute telomerase activity in the PF and MPF cells by overexpression of human telomerase catalytic subunit (hTERT), although expression of exogenous hTERT in those cells was verified by RT-PCR (data not shown). A couple of research groups have demonstrated that most somatic cells derived from pigs, unlike human somatic cells, are known to possess endogenous telomerase activity [47, 48]. However, recent reports have also demonstrated that endogenous telomerase activity was shown to be restricted in the particular tissues/organs of pigs, and introduction of hTERT failed to reconstitute telomerase activity in the cells derived from pig [30, 49, 50]. These results suggest that telomerase activity might be required for cellular immortalization in the pig cells, depending on cell types, tissue origins or culture conditions.
Since it has been documented that p53 protein is stabilized and activated through its phosphorylation under the various cellular insults such as DNA damage and oncogenic stresses , the relatively increased p53 level in the primary MPF cells of early passage (P3) compared to primary counterpart PF cells (P3) reflects that the primary MPF cells might be prone to possess relatively higher cellular stresses, compared to the primary PF cells. Among various cellular stresses that enable to activate p53, chromosome-spindle attachment might be improperly regulated in the primary MPF cells, by which a relatively higher steady-state level of the p53 protein would be maintained in these cells. This speculation is further supported in that cells containing abnormal chromosome numbers (3N) are dramatically increased in the immortalized MPF cells possessing inactivated p53 function, and deregulation of chromosome-spindle attachment could activate not only p53-dependent checkpoint pathway but also stimulate replicative senescence [52, 53]. Therefore, we assume that the loss of the p53 activity in the immortalized MPF cells should increase chromosome abnormality, ultimately leading to cellular transformation.