A team of researchers led by Dr. Judith Campisi of the Buck Institute has discovered valuable information about how cells express the senescence marker p21 .
Two genetic loci, one protein
Cellular senescence is partially determined by two cyclin-dependent kinase inhibitor proteins: p16 and p21. The former is encoded by a genetic locus known as Cdkn2a, and p16 has been successfully used as a biomarker of senescence, as it is strongly dependent on age and responds to caloric restriction. On the other hand, p21, which is encoded by Cdkn1a, has shown less success as a biomarker; it seems to be entirely tissue dependent, and it is unaffected by caloric restriction.
These researchers have noted two different genetic loci that produce the same p21 protein. Cdkn1a variant 1 has been the most commonly studied, as it normally produces significantly more p21 than Cdkn1a variant 2.
However, the expression of variant 1 does not seem to change with age, as determined in the liver tissue of 2-month-old and 24-month-old mice. Instead, dermal fibroblast testing showed that this locus is immediately sensitive to stressors, such as radiation and doxorubicin, which is known to induce cellular senescence. Unlike variant 2, it also varies greatly with circadian rhythm; mice produce eight times as much p21 from this locus in liver tissue at 6 AM as they do at 3 PM, and its expression in fat and kidney tissue is similar.
On the other hand, variant 2 of Cdkn1a is significantly less sensitive to these stressors in the short term. Instead, this variant is much more aligned with aging, as it is more than twice as active in 24-month-old mice than 2-month-old mice, as determined by samples of multiple tissues. Its expression is strongly suppressed by the senolytic ABT-263, while the first variant is largely unaffected by this drug.
Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo, variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice.
A noisy signal
This research is highly illuminating as to the nature of p21 expression, but it doesn’t make the p21 protein itself any more useful as a biomarker. Variant 2 produces significantly less p21 than variant 1 overall; even aged mice produce less than half as much p21 from variant 2 as from variant 1. Because the average value of p21 significantly varies from sample to sample, it would be easy to lose such a signal in the general noise.
Instead, as the researchers point out, more investigation is needed to determine the effects of the ten currently known p21 loci that exist in human beings, which have been discovered by previous research  and may be part of determining cell fate in cancer. The researchers note that murine variant 2 and human variant 4 share certain transcriptional regulators, so it is possible that senescence-based research in that direction may bear fruit.
Despite the fact that its results prove an unpleasant fact for the longevity community, namely that p21 by itself is not of much value as a biomarker, this research has provided the scientific community with an enormous boon: we now know significantly more about why p21 is the way it is, and this approach might also be useful in determining the value of other biomarkers.
The potential value of this locus-based technique isn’t restricted to p21 or even to senescent cells as a whole; if multiple genetic loci are responsible for other proteins whose aging-related effects have been overlooked, such loci might also be worth studying. While searching for useful information gene by gene is expensive, time-consuming, and difficult, such exploration may help us to better determine, and ultimately combat, the root causes of aging themselves.
Literature López-Domínguez, J. A., Rodríguez-López, S., Ahumada-Castro, U., Desprez, P. Y., Konovalenko, M., Laberge, R. M., … & Campisi, J. (2021). Cdkn1a transcript variant 2 is a marker of aging and cellular senescence. Aging, 13.
 Radhakrishnan SK, Gierut J, Gartel AL. Multiple alternate p21 transcripts are regulated by p53 in human cells. Oncogene. 2006; 25:1812–5. https://doi.org/10.1038/sj.onc.1209195 [PubMed]