Hi,
I have measured the senescence-associated B-Gal activity in three different cancer cell lines comparing a chemotherapy with a combination treatment. The combination treatment showed significant reductions in B-Gal activity. However, when I checked the SASP (IL6, IL8, IL-1a, IL-1B) there are no changes between the two treatments. I have also checked p16INK4a and p21 expression and they are also either unchanged, or the combination treatment has increased mRNA expression, suggesting that there should be a higher level of senescence. What could be the explanation fo the contradicting results and what could have caused the reduction in B-Gal/lysosome activity?
Thanks.
Dear Harald,
thank you for your help. I have been staining fixed cells and manually counting 500-1000 cells per well from microscope images, with each treatment in triplicate. And I have been counting senescent cells according to their blue colour as well as their morphology and have significant reductions in the combination treatment.
Thanks again,
Mia
SA-beta-gal activity increases under confluent conditions, hypo-nutrient tumor microenvironment, and due to high level of oxidative stress, all of which enhances lysosomal beta-galactosidase in the absence of the genuine senescence. It has been demonstrated that cellular senescence is not the only factor that can enhance SA-beta-gal activity, thereby rendering SA-beta-gal activity non-specific for cell-aging, also referred to as pseudo-senescence without change in expression levels of p16 and p21 (1, 2).
To put it simply, SA-beta-gal activity alone is not sufficient to allow them to conclude with the confidence that endothelial cells underwent senescence, due to the fact that the positive SA-b-gal staining can also occur in biological contexts other than the response to genotoxic stress. That is why SA-beta-gal assay is reliable only when those confounding factors are carefully considered or excluded. Remarkably, the significant correlation between SA-b-gal activity and the absence of intra-nuclear high mobility group box 1 (HMGB1) has been recently demonstrated in vitro (3). Thus, this combination might allow more reliable identification of the bona fide senescent cancer cells compared with SA-b-gal assay alone.
[References]
1: Yang NC, Hu ML. The limitations and validities of senescence associated-beta-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells. Exp Gerontol 2005;40:813-9.
2: Collado M, Serrano M. Senescence in tumours: evidence from mice and humans. Nat Rev Cancer 2010;10:51-7.
3: Biran A, Zada L, Abou Karam P, et al. Quantitative identification of senescent cells in aging and disease. Aging Cell 2017;16:661-71.
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