Sci. Aging Knowl. Environ., 3 November 2004
Vol. 2004, Issue 44, p. pe40
[DOI: 10.1126/sageke.2004.44.pe40]


The Search for Biomarkers of Aging: Next Stop INK4a/ARF Locus

Goberdhan P. Dimri

The author is in the Division of Cancer Biology, Department of Medicine, Evanston, Northwestern Healthcare Research Institute, Feinberg School of Medicine and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL 60201, USA. E-mail: gdimri{at}

Key Words: p16INK4a • ARF • replicative senescence • tumor suppressor • biomarker • caloric restriction • SA-{beta}-Gal


Aging is a complex process whereby all living organisms exhibit a time-dependent progressive decline in various bodily functions and an increased risk of mortality. Although aging is not a disease per se, it increases the risk of developing age-related pathologies. How do we estimate the progression of aging and approximate when the onset of age-related pathologies might occur? The obvious answer is to determine the chronological age of an individual--in other words, the amount of time that has passed since birth. In a biological setting, aging occurs at every level of organization, from cells to tissues and organs. The aging of these individual components may not necessarily correlate with the chronological age of the whole organism. However, it can profoundly affect the physiology of an organism and in fact determine the onset of age-related pathologies. Because of the enormous cost associated with the treatment of age-related pathologies, as well as their potential value in aging-related research, it is desirable to have biomarkers that can define the aging of various cells, tissues, and organs of an individual (see Miller Perspective). Although there are several potential markers to measure aging (1), a recent article from Sharpless's laboratory focuses on two well-known regulators of replicative senescence, p16INK4a and ARF (2).

After a finite number of cell divisions, most normal cells enter a stage of permanent growth arrest known as replicative senescence (3) (see "More Than a Sum of Our Cells"). This phenomenon was first described in the early 1960s by Hayflick and Moorehead (4). It is now generally believed that replicative senescence contributes to aging and the development of age-related pathologies (5). If replicative senescence does indeed contribute to aging in vivo, one can hypothesize that the molecular effectors of replicative senescence could serve as meaningful biomarkers of aging. This hypothesis was probed by Krishnamurthy et al. from Sharpless's laboratory (2).

Tumor Suppressors and Replicative Senescence

Apart from having a probable causal role in aging, replicative senescence is a strong tumor suppressor mechanism (3, 5, 6) (see Sharpless Perspective and "Backup Plan: Senescence fights tumors when apoptosis fails"). Tumor suppressors play an important role in the genesis and maintenance of the senescent phenotype (6, 7). For example, tumor suppressors p53 and/or the retinoblastoma protein (pRb) are required for the induction of senescence in various cell types (3, 6, 7). The downstream targets of these two tumor suppressors are the cyclin-dependent kinase inhibitors (CDKIs) p21 and p16INK4a, respectively (3, 6, 7). CDKIs function to inhibit the progression of the cell division cycle. In some settings, p21, p16INK4a, and other CDKIs can induce a phenotype similar to replicative senescence (8). In addition to CDKIs, the ARF tumor suppressor, which regulates p53, is also an important mediator of senescence (3).

Hyperactivity of p53 has been recently reported to cause accelerated aging and generate age-related pathologies in three separate studies of transgenic mice (9-12) (see Campisi Perspective, Martin Perspective, and "Tumor-Free, But Not in the Clear"). Similarly, increased expression of p16 caused by deficiency of the SNF2-like gene PASG was shown to cause premature aging in mice (13). Very recently it was shown that p16INK4a and ARF, which are transcribed from the common Ink4a/ARF locus (14), limit hematopoietic and neural stem cell proliferation in vivo (15, 16). p16INK4a and ARF expression have also been shown to increase with age in certain tissues in mice (17, 18). Keeping these findings in mind, Sharpless's laboratory rightly focused on p16INK4a and ARF (2).

First, the authors determined the changes in expression of the genes encoding a number of different senescence-related proteins, including p16INK4a and ARF, as well as the CDKIs p15INK4b, p18INK4c, p19INK4d, p21, and p27, that occur during aging in rat and murine tissues (2) by real-time reverse transcriptase polymerase chain reaction (RT-PCR), a technique in which the concentration of product is monitored during each PCR cycle to give a measure of the initial template concentration. Out of all these potential biomarkers, only the genes encoding p16INK4a and ARF showed a marked increase (>30-fold) in expression in 26 of 27 aged versus young organs analyzed. The expression of genes encoding other INK4 family members showed either no up-regulation or organ-specific up-regulation to a certain degree in aged tissues. For example, expression of the p15INK4b gene was only significantly up-regulated in the aged heart. The p21 gene also exhibited some degree of age-related increase in expression; nevertheless, this increase was much less compared to that displayed by the INK4a/ARF locus. The degree of up-regulation of p16INK4a and ARF expression in aging tissues was much less than the degree of up-regulation observed in cultured senescent mouse embryonic fibroblasts, suggesting that this further increase in p16INK4a and ARF expression was induced by in vitro culture conditions. The real time RT-PCR data were verified by immunohistochemistry (IHC) in selected organs, where it was found that p16INK4a and ARF protein abundance increases in aged versus young tissues.

It is possible that p16INK4a and ARF are expressed in certain specific cell types or compartments (cellular or stromal) within a tissue and that the tissue composition changes with increasing age. Because the authors initially used a ground mixture of various tissues to detect p16INK4a and ARF, the increased p16INK4a and ARF abundance might simply reflect changes in tissue composition rather than increased expression with age. Thus, it was imperative that the authors show age-specific increases of these markers in stromal and cellular compartments as well as various cell types. Overall, IHC and mRNA quantification data suggest that the abundance of these markers does increase at the protein as well as the mRNA level in individual compartments and cell types of various aged organs.

What is the functional significance of increased p16INK4a and ARF abundance in aged tissues and their cells? Are these cells replicatively senescent or simply quiescent? (Quiescent cells, although not undergoing repeated cell division cycles at a given time, can be stimulated to do so, unlike senescent cells.) The senescence-associated beta galactosidase (SA-{beta}-Gal) marker can differentiate between these two growth-arrest phases (19). This marker can also be used to identify senescent cells in a few selected tissues in vivo in mouse models as well as aging organs in primate and nonprimate models (11-13, 19-25). Krishnamurthy et al. demonstrated a significant increase in SA-{beta}-Gal activity in the kidneys of aged versus young mice and rats. This finding suggests that increased expression of ARF and p16INK4a is caused by the accumulation of senescent cells, at least in the kidneys.

Effects of Caloric Restriction

Perhaps the most important finding described by Sharpless's laboratory is that a decrease in the expression of ARF and p16INK4a, as well as a decrease in the level of SA-{beta}-Gal activity, occurs in calorie-restricted animals. Caloric restriction (CR) is a robust intervention shown to increase both mean and maximum life span and delay the onset of numerous age-related pathologies in a variety of animal species (26-29) (see Masoro Subfield History). In a CR regimen, animals are maintained on a diet that contains 30 to 50% fewer calories than an unrestricted [ad libitum (AL)] diet (26-29). Although molecular effectors of CR are not very well understood, biomarkers of aging are likely to be involved in the CR-mediated increase in life span. If p16INK4a and ARF were to serve as true biomarkers of aging, the fair assumption would be that in animals undergoing CR (CR animals), there will be a less pronounced increase in p16INK4a and ARF expression as compared to age-matched AL-fed animals (AL animals). When the authors compared p16INK4a and ARF expression in various aging tissues of CR and AL animals, a limited number of tissues indeed showed 2- to 16-fold attenuation of the age-induced expression of p16INK4a (the heart, kidney, ovary, and testis). However, no decrease in the expression of p16INK4a and ARF was observed in certain other organs such as the lung, lymph nodes, spleen, and liver in the CR versus AL animals. Decreased expression of p16INK4a and ARF also correlated with the decreased incidence of nephritis (inflammation of the kidney), Leydig cell hyperplasia (hyperproliferation of interstitial cells of the testis, which increases the risk of developing Leydig cell tumors), and the overall tumor burden that occur in CR versus AL animals. In agreement with this finding, the authors also report a high spontaneous incidence of Leydig cell tumors in AL rats, which correlated with the increased expression of p16INK4a in rat testis.

Regulation of p16INK4a and ARF Expression in Aging Tissues

What are the factors responsible for the up-regulation of p16INK4a and ARF expression in aging tissues? To answer this question, the authors examined three known regulators of p16INK4a and ARF: (i) Bmi-1, a polycomb protein that represses expression of the p16INK4a/ARF locus in cells grown in culture as well as in vivo (15, 30, 31); (ii) Id-1, a transcriptional repressor; and (iii) Ets-1, an activator of p16INK4a expression (32, 33). Among these three regulators, Bmi-1 expression did not correlate with p16INK4a and ARF up-regulation, whereas decreased Id-1 expression only weakly correlated with increased p16INK4a and ARF expression in aged versus young animals. However, a strong correlation was found between p16INK4a and Ets-1 expression in various aging tissues. Accordingly, CR was also found to down-regulate Ets-1 expression. As expected, no correlation was found between Ets-1 and ARF expression. A closer look at the Pearson correlation coefficient, which measure the degree of linear relationship between two variables, suggests that Ets-1 regulates expression of p16INK4a expression in 38% of the tissues examined, whereas an unknown regulator, independent of Ets-1, must regulate the expression of p16INK4a and ARF in 49% of the cases. What about the remaining cases? Although the authors did not find any correlation between Bmi-1 expression and p16INK4a/ARF locus expression in tissues that were studied, such a correlation cannot be ruled out for other tissues. Perhaps other Bmi-1-like polycomb proteins such as Mel18 (34) and MBLR (35) could also play a role in modulating the expression of the p16INK4a/ARF locus in vivo.

Implications of Findings

What is the significance of the findings from Sharpless's laboratory? First, p16INK4a and ARF are expressed in a wide range of tissues in stroma as well as cellular compartments. Both of these proteins are expressed at levels that are several times higher in aged versus young tissues; the expression levels are easily quantifiable by real-time RT-PCR or IHC. The authors clearly showed that CR, a well-known intervention that increases life span, leads to the down-regulation of ARF and p16INK4a expression, suggesting that perhaps one of the downstream targets of CR is the p16INK4a/ARF locus. These studies also suggest that other known and potential regulators of aging, such as insulin-like growth factor 1, p66Shc, SirT1, and FOXO transcription factors (26), might also regulate the expression of the INK4a/ARF locus, although the relation between these proteins and INK4a/ARF expression is yet to be explored. Likewise, the connection between interventions that might affect the aging process, such as hormone therapy, and p16INK4a/ARF expression is unexplored. Nonetheless, the authors' work clearly suggests that p16INK4a and ARF could serve as molecular biomarkers of aging. Finally, the authors reasonably conjecture that INK4a/ARF expression could provide a surrogate marker for (i) determining tissue regenerative potential, (ii) forecasting disease progression in premorbid syndromes, (iii) predicting the toxic effects of radio- or chemotherapy, and (iv) determining the efficacy of antiaging therapeutics. An implicit suggestion in this study is that p16INK4a and ARF are molecular effectors of aging and possibly the cause of some age-related pathologies, including cancer.

In the last decade or so, we have learned a lot about cancer and aging from transgenic models. Is it possible to create transgenic mice overexpressing the proteins encoded by the INK4a/ARF locus and validate the findings from Sharpless's laboratory? In other words, would the overproduction of p16INK4a and ARF lead to a reduction in life span? Without a prejudice against the results of Sharpless et al., Serrano's laboratory came close in creating such a transgenic model (36). Matheu et al. created transgenic mice that harbor an extra copy of the INK4a/ARF locus in their genome (36). When compared to nontransgenic mice, these animals expressed only modestly high concentrations of p16INK4a and ARF proteins. The authors showed that the transgenic animals had a decreased incidence of spontaneous as well as chemically-induced tumors. Although the authors did not study the age-dependent increase in the expression from the native and foreign INK4a/ARF locus and its effect on the development of age-related pathologies in detail, the transgenic animals appear to have a normal life span. These studies suggest that a modest increase in p16INK4a and ARF abundance might not affect the life span but, instead, might be beneficial for fighting cancer. Nevertheless, the effect of overexpression of INK4a/ARF to the degree that occurs in aged animals remains to be studied in transgenic models. The take home message from these two studies (2, 36) might be that the old saying "Too much of a good thing is bad for you" is true not only philosophically but practically as well.

November 3, 2004
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Citation: G. P. Dimri, The Search for Biomarkers of Aging: Next Stop INK4a/ARF Locus. Sci. Aging Knowl. Environ. 2004 (44), pe40 (2004).

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