A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM in vitro. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.

Kinase Activity-dependent Nuclear Export Opposes Stress-induced Nuclear Accumulation and Retention of Hog1 Mitogen-activated Protein Kinase in the Budding Yeast Saccharomyces cerevisiae

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1–green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1–GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1–GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Mechanism of Ca2+-dependent nuclear accumulation of calmodulin

The intracellular Ca2+ receptor calmodulin (CaM) coordinates responses to extracellular stimuli by modulating the activities of its various binding proteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between cytoplasm and nucleus. Here we show that Ca2+-dependent nuclear accumulation of CaM can be reconstituted in permeabilized cells. Accumulation was blocked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca2+-dependent influx of CaM into nuclei was not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells. Fluorescence recovery after photobleaching studies of CaM in intact cells showed that influx is a first-order process with a rate constant similar to that of a freely diffusible control molecule (20-kDa dextran). Studies of CaM efflux from preloaded nuclei in permeablized cells revealed the existence of three classes of nuclear binding sites that are distinguished by their Ca2+-dependence and affinity. At high [Ca2+], efflux was enhanced by addition of a high affinity CaM-binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the nucleus act as a sink for Ca2+-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca2+.

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFκB

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin’s inhibition of NFκB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFκB, or via the “inflammatory” cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFκB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFκB.