Low necdin expression as a marker of senescence
Replicative senescence marks the end of the proliferative state in cells when telomeres reach a critical length
. This mechanism is relevant in limiting aberrant proliferation and may contribute to cancer prevention
. By comparing IMR90 cells at high or low population doublings, we demonstrate a reduction of the Necdin protein levels over time, while p16 increases, which is the mark of an irreversible arrest
[29, 37, 38]. Lee and al.
 identified Necdin among the gene expression profiles in skeletal muscle of aging mice, where Necdin levels decrease with age. In contrast, Necdin was upregulated upon caloric restriction, a process that retards aging in mice
. Our result confirmed that the decreased Necdin levels seen in aged mouse skeletal muscle can be reproduced with normal human fibroblasts in culture. The decrease in Necdin expression was also consistently observed in telomere-independent premature senescence induced by irradiation. It was also observed, but to a much lower extent, in premature senescence resulting from the expression of the activated oncogenic Ras (RasV12). Although the observed reduction in Necdin protein was subtle, another group has independently reported that Necdin mRNA was part of the list of downregulated genes following Ras-induced senescence
, supporting our general finding that Necdin is downregulated during cellular senescence.
In contrast to senescence, cellular quiescence is a reversible cell cycle arrest since cells may re-enter the cell cycle when the restriction is removed
[29, 37]. Interestingly, in a transient G1 arrest, Necdin remained expressed, suggesting that the decrease in Necdin is limited to permanent growth arrest and may be a marker of senescent cells. Necdin levels in other primary cells need to be analyzed since the molecular signature at senescence largely depends on the cell type
Like Necdin, many genes involved in cell cycle progression show altered expression in senescent cells as both proliferation-promoting proteins and their negative regulators decrease when human fibroblasts reach senescence, as exemplified by Rb and E2F1
[34, 41]. Necdin interacts with E2F1, E2F4 and also affects expression of the Rb family of genes
. One interpretation of these findings is that Necdin may affect cellular senescence. To test this hypothesis, ectopic expression of Necdin was used. We found that overexpression of Necdin did not extend the replicative life span of primary human fibroblasts. Moreover, the sustained expression of Necdin over time in this experiment suggested that the complete elimination of Necdin expression is not essential for senescence to occur.
There are various examples in the literature where proteins, possessing properties similar to Necdin, can induce premature senescence when depleted in IMR90 cells. The knock down of BS69 and p400, also known to interact with viral proteins, induced a premature senescence characterized by p21 and p53 upregulation
[43, 44]. BS69 and p400, like Necdin, can form a complex with p53 at the p21 promoter to repress transcription
[19, 44], although it is not known if Necdin is part of the same complex. Moreover Sirt1, a partner of Necdin
, can increase the risk of cancer due to its capacity to downregulate p53 activity by deacetylating this tumor suppressor
[24, 45, 46]. Accordingly, Sirt1 inhibition also induces a senescence-like phenotype in IMR90 cells
. From these data, we expected that knock down of endogenous Necdin by shRNA might also induce premature senescence resulting from p53 activation and p21 de-repression. However, shNDN-directed loss of Necdin expression did not induce premature senescence in IMR90 cells. Perhaps, under non-stress conditions, a complementary protein may contribute to maintaining Necdin function in its absence. For example, other members of the MAGE family like NDNL2, also known as MAGE-G1,
, are expressed in a wide variety of tissues including fibroblasts
 and shares many functions with Necdin. Alternatively, loss of Necdin might not be sufficient to activate p53 in the absence of others p53 stabilization signals.
Necdin is a maternally imprinted gene and its promoter contains many CpG sites for methylation regulation
. Consistent with this, the inhibition of DNA methyltransferase (DNMT) by 5-aza-2′-deoxycytidine (5AZA-dC) has been shown to induce Necdin expression in some cancerous cell lines
. During senescence establishment, epigenetic changes occur inducing important chromatin structure modifications; some at specific sites while other reflect a more global change. Global DNA methylation status decreases with aging by comparison to young counterparts and immortalized cells
[52, 53]. A specialized redistribution in chromatin heterochromatin, called senescence-associated heterochromatic foci (SAHF), is also associated with cellular senescence
. The mechanism is only partially understood; the genomic loci affected by this structure often contain proliferation-promoting genes such as E2F1 target genes
. Moreover, SAHF are not observed in quiescent cells. It is possible that the decrease in Necdin levels during aging could be the result of hypermethylation or others senescence associated modifications at specific sites.
Necdin expression confers resistance to ionizing irradiation
Necdin expression can exert an effect on normal cells as an increase in Necdin level confers resistance to ionizing radiation. At the employed dose, DNA double-strand breaks were induced in all cells and the physiological consequence is the induction of a DNA damage response activating p53. Thus, these damages caused cell cycle arrest and need to be repaired before cells can resume proliferation. When damages cannot be properly repaired, apoptosis or senescence are two possible cellular outcomes. Consequently, we observed that cells unable to form colonies showed enlarged and flat morphology of senescent cells in all populations tested (data not shown). This is expected for normal fibroblasts that are relatively resistant to apoptosis upon irradiation
. Apoptosis was probably also induced to a low level but could not be monitored due to the low cell densities used in these experiments.
An appropriate response to genotoxic stress is based on the capacity to sense the damage, to activate the cell cycle checkpoint and to repair the damage
. Two principal conditions may explain an increase in resistance to irradiation in a normal cell. First, an enhanced ability to repair the DNA damage could promote survival. Second, a failure in activating cell cycle checkpoints will contribute to the maintenance of cells in a proliferative state despite the presence of a genotoxic stress. This could result from inefficient sensing upstream of the p53 pathways or by a reduction in p53 downstream signaling. This is what was reproduced with the positive control expressing a peptide inhibitor of p53 (GSE22) resulting in a marked increase in colony formation consistent with the notion that a decrease in p53 function increases resistance to irradiation. We observed and others have shown previously that Necdin can interfere with p53-responses
[16, 19, 23]. These data suggest that increased radioresistance associated with Necdin may be related to its ability to directly influence the p53-response. Necdin may also confer radioresistance by inhibiting radiation-induced apoptosis since Necdin can negatively modulate caspase activation upon genotoxic stress
[56, 57], but this is unlikely in fibroblast in response to irradiation
. Further evidence supporting Necdin’s ability to contribute to radioresistance is a microarray analysis of the gene expression profiles produced by radiosensitive and radioresistant esophageal carcinoma cell lines. In this study, Necdin expression was higher in radioresistant cells
 which is consistent with the observation of the present study.