Restoration of senescent human diploid fibroblasts by modulation of extracellular matrix
Hae Ri Choi, Kyung a Cho, Hyun Tae Kang, Jung Bin Lee, Matt Kaeberlein, Yousin Suh, In Kwon Chung and Sang Chul Park
Aging Cell 2011, 10, ppp148-157 (doi:10.1111/j.1474-9726.2010.00654.x)
Thoughts/Implications/Summary
Cells not expressing telomerase eventually begin to show evidence of a DNA damage response (DDR) resulting in terminal arrest of the cell cycle limiting further replication. This replication limit is known as the 'Hayflick' limit. Senescence is the upshot of reaching the Hayflick limit and senescence is associated with phenotypic traits including an increase in senescence associated β galactosidase staining, increase in intracellular reactive oxygen species (ROS), decrease in BrdU staining, decreased response to apoptotic signals, and decreased response to epidermal growth factor (EGF). Additional markers include sets of secretory proteins, structural changes in nuclear lamina, and gamma-H2AX staining on uncapped telomeres. It is thought that the senescent state is one that is, for the most part, irreversible. However, previously a study by Conboy, et al. provided evidence that it may be possible to restore old (and, possibly, senescent) cells to a more youthful state[1]. The mechanism mediating a reversion from a senescent state to a more youthful state could be contingent on the microenvironment which is composed of the extracellular matrix (ECM). Building on previous observations the authors sought to test the idea that old (i.e., senescent) cells that have reached their replication limit can be restored to a more youthful state.
In this paper the authors show data supporting the hypothesis that young ECM can induce cells that are in a replicative senescent state to overcome constraints on further proliferation and return to a more youthful phenotypic state.
The authors' main experiment compares and contrasts the effects of culturing old (i.e., senescent) cells on young ECM and old ECM and young cells on young ECM and old ECM. They document a reversion of phenotype of old cells on young ECM by assessing changes in the following known markers associated with cellular senescence.
Restored cells show an increase in telomere length and a dramatic decrease in DDR. Within the context of a dramatic reduction in DDR, a comet assay was used to observe the high levels of DDR in senescent cells and subsequent abrogation of this finding in restored cells. gamma-H2AX foci were reduced in restored cells to similar levels seen in young cells. In the paper the authors point out that a lack of gamma-H2AX foci can be interpreted as telomeres being restored or a masking of the DDR is taking place in the presence of short telomeres. To test these two possibilities the authors utilized the telomeric repeat amplification protocol (TRAP) assay and RT-PCR. Results from these assays suggest that telomeres were being elongated, but they were unable to detect telomerase activity during the restoration process. This indicates that there may be a telomerase-independent restoration of telomeres. This finding is very intriguing, because it would be interesting to determine if this putative telomerase-independent mechanism of telomere lengthening is different from what is observed in some malignancies. The basis for this assertion is that restored cells did not exhibit colony formation in soft agar indicating that restored cells were not being transformed to a (pre-)malignant state. They did not assess whether or not aneuploidy was occurring by way of chromosome fusion, a phenomenon seen in telomerase-independent lengthening of telomeres in malignant cells, but they did show that restored cells did revert to an apparently normal senescent state. These findings suggest that the integrity of the genome is maintained to the extent that restored cells do not show any evidence of promotion/progression towards a malignant state.
Given the absence of telomerase dependent lengthening of telomeres and previous knowledge they decided to assess the role of Ku70 in restoration of senescent cells. They wanted to answer the question: Is Ku70 is necessary for restoration? In senescence cells, Ku70 levels decrease but this decrease in expression was reversed in restored cells. To address Ku70's role, the authors examined whether siRNA knockdown in young cells affected the ability of young ECM to restore senescent cells to a proliferative state. They found that young cells with siRNA knockdown of Ku70 produced ECM that was not able to induce restoration of senescent cells. This is a very interesting finding, because Ku70 cells have defects in telomere end capping and DNA damage signaling which result in genomic instability and an acceleration of cellular senescence. Thus, the young cells deficient in Ku70 are (perhaps) either producing an ECM lacking in a required signaling molecule or are producing a dominant-negative signaling molecule inhibiting the restoration process. There may be another mechanism at play, too.
To further assess the mechanism(s) at play in the restoration of senescent cells they looked at the role of SIRT1 in mediating the restoration process. It is known that SIRT1 is required for efficient DNA double strand break repair playing a role in genomic stability and is known to deacetylate and activate Ku70. Additionally, like Ku70, SIRT1 levels decrease during senescence. They also state that Ku70 acetylation status is inversely correlated with SIRT1 levels. To test the idea that SIRT1 plays a role in the restoration process they used an inhibitor of SIRT1, EX-527, to diminish the levels of SIRT1 in young cells. This resulted in young ECM that was unable to restore senescent cells. Together with the Ku70 results, this finding implicates Ku70 and SIRT1 in the restoration process and as being necessary for young cells to produce ECM capable of restoring senescent cells to a proliferative state.
SIRT1 catalytic activity is essential for the restoration process. SIRT1 inhibition suggests that the deacetylase activity of SIRT1 of Ku70 is required for the production of restorative ECM by young cells. Additional SIRT1 deacetylation targets might also play a role. Further elucidation of SIRT1's role is required.
Conclusion: It may be possible to affect the cellular microenvironment allowing for possible rejuvenation of tissues.
Additional Questions/Ideas
Do they see changes in telomere organization and lamina organization in the rejuvenated cells? This would suggest that there is a reversion in telomere organization and changes in lamina organization both of which are implicated in senescence at least in mesenchymal cells[3].
How does mixing cells affect the ability of young cell ECM to promote the of restoration of old cells?
Does the restoration of mitochondrial membrane potential indicate that inefficient mitochondria are being removed by autophagy and being replaced?
Does the defective young ECM (i.e., Ku70- and SIRT1-deficient) affect the growth rate of some epithelial cancer cell lines? Other cell lines?
Obviously serial passaging of restored cells on young ECM is required to assess the ability of restored cells to proliferate indefinitely.
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