p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

Introduction

Recent evidence suggests that tissue accumulation of senescent p16^INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16^INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16^INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).

Methods

We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16^INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16^INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.

Results

p16^INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16^INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16^INK4a. Accordingly, p16^INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.

Conclusions

We disclosed herein a new role of the senescence marker p16^INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.     

p16INK4a deficiency promotes DNA hyper‐replication and genetic instability in melanocytes

Activated oncogenes restrict cell proliferation and transformation by triggering a DNA damage‐dependent senescence checkpoint in response to DNA hyper‐replication. Here, we show that loss of the p16^INK4a cyclin‐dependent kinase inhibitor and melanoma tumour suppressor facilitates a DNA damage response after a hyper‐replicative phase in human melanocytes. Unlike cells expressing activated oncogenes, however, melanocytes depleted for p16^INK4a display enhanced proliferation and an extended replicative lifespan in the presence of replication‐associated DNA damage. Analysis of human benign naevi confirmed that DNA damage and loss of p16^INK4a expression co‐segregate closely. Thus, we propose that loss of p16^INK4a facilitates tumourigenesis by promoting the proliferation of genetically unstable cells.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

Whole‐body senescent cell clearance alleviates age‐related brain inflammation and cognitive impairment in mice

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro‐inflammatory senescence‐associated secretory phenotype (SASP), which is a major contributor to aging and age‐related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single‐nuclei and single‐cell RNA‐seq in the hippocampus from young and aged mice. We observed an age‐dependent increase in p16^Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK‐ATTAC mice, in which p16^Ink4a‐positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16^Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof‐of‐concept for senolytic interventions'' being a potential therapeutic avenue for alleviating age‐associated cognitive impairment.     

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16^Ink4a and p21^Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1^?/? and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16^Ink4a and p21^Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1^?/? mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16^Ink4a and p21^Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21^Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1^?/Δ mice with a p16^Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1^?/? and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.     

Human chondrocyte senescence and osteoarthritis

Although osteoarthritis (OA) is not an inevitable consequence of aging, a strong association exists between age and increasing incidence of OA. We hypothesized that this association is due to in vivo articular cartilage chondrocyte senescence which causes an age-related decline in the ability of the cells to maintain articular cartilage, that is, increasing age increases the risk of OA because chondrocytes lose their ability to replace their extracellular matrix. To test this hypothesis, we measured senescence markers in human articular cartilage chondrocytes from 27 donors ranging in age from one to 87 years. The markers included expression of the senescence-associated enzyme beta-galactosidase, mitotic activity measured by 3H-thymidine incorporation, and telomere length. beta-galactosidase expression increased with age (r=0.84, p=0.0001) while mitotic activity and mean telomere length declined (r=-0.774, p=0.001 and r=-0.71, p=0.0004, respectively). Decreasing telomere length was strongly correlated with increasing expression of beta-galactosidase and decreasing mitotic activity. These findings help explain the previously reported age related declines in chondrocyte synthetic activity and responsiveness to anabolic growth factors and indicate that in vivo articular cartilage chondrocyte senescence is responsible, at least in part, for the age related increased incidence of OA. The data also imply that people vary in their risk of developing OA because of differences in onset of chondrocyte senescence; and, the success of chondrocyte transplantation procedures performed to restore damaged articular surfaces in older patients could be limited by the inability of older chondrocytes to form new cartilage. New efforts to prevent the development or progression of OA might include strategies that delay the onset of chondrocyte senescence or replace senescent cells.     

CBX7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a

Polycomb repressive complexes (PRC1 and PRC2) are epigenetic regulators that act in coordination to influence multiple cellular processes including pluripotency, differentiation, cancer and senescence. The role of PRCs in senescence can be mostly explained by their ability to repress the INK4/ARF locus. CBX7 is one of five mammalian orthologues of Drosophila Polycomb that forms part of PRC1. Despite the relevance of CBX7 for regulating senescence and pluripotency, we have a limited understanding of how the expression of CBX7 is regulated. Here we report that the miR‐9 family of microRNAs (miRNAS) downregulates the expression of CBX7. In turn, CBX7 represses miR‐9‐1 and miR‐9‐2 as part of a regulatory negative feedback loop. The miR‐9/CBX7 feedback loop is a regulatory module contributing to induction of the cyclin‐dependent kinase inhibitor (CDKI) p16^INK4a during senescence. The ability of the miR‐9 family to regulate senescence could have implications for understanding the role of miR‐9 in cancer and aging.     

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long‐lived and cancer‐resistant subterranean wild rodent,Spalax

The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.     

Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration

Current evidence implicates intervertebral disc degeneration as a major cause of low back pain, although its pathogenesis is poorly understood. Numerous characteristic features of disc degeneration mimic those seen during ageing but appear to occur at an accelerated rate. We hypothesised that this is due to accelerated cellular senescence, which causes fundamental changes in the ability of disc cells to maintain the intervertebral disc (IVD) matrix, thus leading to IVD degeneration. Cells isolated from non-degenerate and degenerate human tissue were assessed for mean telomere length, senescence-associated β-galactosidase (SA-β-gal), and replicative potential. Expression of P16 ^INK4A (increased in cellular senescence) was also investigated in IVD tissue by means of immunohistochemistry. RNA from tissue and cultured cells was used for real-time polymerase chain reaction analysis for matrix metalloproteinase-13, ADAMTS 5 (a disintegrin and metalloprotease with thrombospondin motifs 5), and P16 ^INK4A . Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. In non-degenerate discs, there was an age-related increase in cellular expression of P16 ^INK4A . Cells from degenerate discs (even from young patients) exhibited increased expression of P16 ^INK4A , increased SA-β-gal staining, and a decrease in replicative potential. Importantly, there was a positive correlation between P16 ^INK4A and matrix-degrading enzyme gene expression. Our findings indicate that disc cell senescence occurs in vivo and is accelerated in IVD degeneration. Furthermore, the senescent phenotype is associated with increased catabolism, implicating cellular senescence in the pathogenesis of IVD degeneration.     

Kallistatin reduces vascular senescence and aging by regulating microRNA‐34a‐SIRT1 pathway

Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)‐induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF‐α‐induced cellular senescence in EPCs, as indicated by reduced senescence‐associated β‐galactosidase activity and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked TNF‐α‐induced superoxide levels, NADPH oxidase activity, and microRNA‐21 (miR‐21) and p16^INK4a synthesis. Kallistatin prevented TNF‐α‐mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR‐34a synthesis, whereas miR‐34a overexpression abolished kallistatin‐induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR‐34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ‐induced aortic senescence, oxidative stress, and miR‐34a and miR‐21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild‐type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR‐34 or sir‐2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR‐34, but stimulated sir‐2.1 and sod‐3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR‐34a‐SIRT1 pathway.     

Dysregulation of the Bmi‐1/p16Ink4a pathway provokes an aging‐associated decline of submandibular gland function

Bmi‐1 prevents stem cell aging, at least partly, by blocking expression of the cyclin‐dependent kinase inhibitor p16^Ink4a. Therefore, dysregulation of the Bmi‐1/p16^Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi‐1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi‐1/p16^Ink4a pathway occurs during aging in vivo. Using real‐time in vivo imaging of p16^Ink4a expression in Bmi‐1‐KO mice, we uncovered a novel function of the Bmi‐1/p16^Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16^Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging‐related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.     

Transthyretin deposition promotes progression of osteoarthritis

Deposition of amyloid is a common aging‐associated phenomenon in several aging‐related diseases. Osteoarthritis (OA) is the most prevalent joint disease, and aging is its major risk factor. Transthyretin (TTR) is an amyloidogenic protein that is deposited in aging and OA‐affected human cartilage and promotes inflammatory and catabolic responses in cultured chondrocytes. Here, we investigated the role of TTR in vivo using transgenic mice overexpressing wild‐type human TTR (hTTR‐TG). Although TTR protein was detected in cartilage in hTTR‐TG mice, the TTR transgene was highly overexpressed in liver, but not in chondrocytes. OA was surgically induced by destabilizing the medial meniscus (DMM) in hTTR‐TG mice, wild‐type mice of the same strain (WT), and mice lacking endogenous Ttr genes. In the DMM model, both cartilage and synovitis histological scores were significantly increased in hTTR‐TG mice. Further, spontaneous degradation and OA‐like changes in cartilage and synovium developed in 18‐month‐old hTTR mice. Expression of cartilage catabolic (Adamts4, Mmp13) and inflammatory genes (Nos2, Il6) was significantly elevated in cartilage from 6‐month‐old hTTR‐TG mice compared with WT mice as was the level of phospho‐NF‐κB p65. Intra‐articular injection of aggregated TTR in WT mice increased synovitis and significantly increased expression of inflammatory genes in synovium. These findings are the first to show that TTR deposition increases disease severity in the murine DMM and aging model of OA.     

Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype

Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16(INK4a), a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16(INK4a) and another cyclin-dependent kinase inhibitor, p21(CIP1/WAF1), induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest. Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16(INK4a). Cells induced to senesce by ectopic p16(INK4a) expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16(INK4a) by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16(INK4a) in suppressing the SASP. These findings suggest that p16(INK4a)-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16(INK4a) activation or senescence per se, but rather is a damage response that is separable from the growth arrest.     

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent‐associated secretory phenotype (SASP) and expression of p16^INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16‐3MR mice that allow elimination of p16^INK4a‐positive cells with exposure to ganciclovir, we show that impaired T‐cell proliferation is partially reversed, mechanistically dependent on p16^INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16^INK4a expression. Our results provide molecular insight on how senescence‐inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.     

Aged‐senescent cells contribute to impaired heart regeneration

Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.     

Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation

Systemic inflammation is central to aging‐related conditions. However, the intrinsic factors that induce inflammation are not well understood. We previously identified a cell‐autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it activates innate cytosolic DNA sensors that trigger inflammation. These results led us to hypothesize that DNA released after cumulative damage contributes to persistent inflammation in aging cells through a similar mechanism. Consistent with this notion, we found that older cells harbored higher levels of extranuclear DNA compared to younger cells. Extranuclear DNA was exported by a leptomycin B‐sensitive process, degraded through the autophagosome–lysosomal pathway and triggered innate immune responses through the DNA‐sensing cGAS‐STING pathway. Patient cells from the aging diseases ataxia and progeria also displayed extranuclear DNA accumulation, increased pIRF3 and pTBK1, and STING‐dependent p16 expression. Removing extranuclear DNA in old cells using DNASE2A reduced innate immune responses and senescence‐associated (SA) β‐gal enzyme activity. Cells and tissues of Dnase2a^?/? mice with defective DNA degradation exhibited slower growth, higher activity of β‐gal, or increased expression of HP‐1β and p16 proteins, while Dnase2a^?/?;Sting^?/? cells and tissues were rescued from these phenotypes, supporting a role for extranuclear DNA in senescence. We hypothesize a direct role for excess DNA in aging‐related inflammation and in replicative senescence, and propose DNA degradation as a therapeutic approach to remove intrinsic DNA and revert inflammation associated with aging.     

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.