Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence.

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by "knocking-in" a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16INK4a locus. We used this allele (p16tdTom ) for the enumeration, isolation, and characterization of individual p16INK4a -expressing cells (tdTom+). The half-life of the knocked-in transcript was shorter than that of the endogenous p16INK4a mRNA, and therefore reporter expression better correlated with p16INK4a promoter activation than p16INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryo fibroblasts from p16tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated ß-galactosidase (SA-ß-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.

Chemotherapy and Stem Cell Transplantation Increase p16INK4a Expression, a Biomarker of T-cell Aging.

The expression of markers of cellular senescence increases exponentially in multiple tissues with aging. Age-related physiological changes may contribute to adverse outcomes in cancer survivors. To investigate the impact of high dose chemotherapy and stem cell transplantation on senescence markers in vivo, we collected blood and clinical data from a cohort of 63 patients undergoing hematopoietic cell transplantation. The expression of p16INK4a, a well-established senescence marker, was determined in T-cells before and 6months after transplant. RNA sequencing was performed on paired samples from 8 patients pre- and post-cancer therapy. In patients undergoing allogeneic transplant, higher pre-transplant p16INK4a expression was associated with a greater number of prior cycles of chemotherapy received (p=0.003), prior autologous transplantation (p=0.01) and prior exposure to alkylating agents (p=0.01). Transplantation was associated with a marked increase in p16INK4a expression 6months following transplantation. Patients receiving autologous transplant experienced a larger increase in p16INK4a expression (3.1-fold increase, p=0.002) than allogeneic transplant recipients (1.9-fold increase, p=0.0004). RNA sequencing of T-cells pre- and post- autologous transplant or cytotoxic chemotherapy demonstrated increased expression of transcripts associated with cellular senescence and physiological aging. Cytotoxic chemotherapy, especially alkylating agents, and stem cell transplantation strongly accelerate expression of a biomarker of molecular aging in T-cells.

Effect of cytotoxic chemotherapy on markers of molecular age in patients with breast cancer.

BACKGROUND: Senescent cells, which express p16 (INK4a) , accumulate with aging and contribute to age-related pathology. To understand whether cytotoxic agents promote molecular aging, we measured expression of p16 (INK4a) and other senescence markers in breast cancer patients treated with adjuvant chemotherapy. METHODS: Blood and clinical information were prospectively obtained from 33 women with stage I to III breast cancer at four time points: before anthracycline-based chemotherapy, immediately after anthracycline-based chemotherapy, 3 months after anthracycline-based chemotherapy, and 12 months after anthracycline-based chemotherapy. Expression of senescence markers p16 (INK4a) and ARF mRNA was determined using TaqMan quantitative reverse-transcription polymerase chain reaction in CD3(+) T lymphocytes, telomere length was determined by Southern analysis, and senescence-associated cytokines were determined by enzyme-linked immunosorbent assay. Findings were independently assessed in a cross-sectional cohort of 176 breast cancer survivors enrolled a median of 3.4 years after treatment; 39% previously received chemotherapy. All statistical tests were two-sided. RESULTS: In prospectively analyzed patients, expression of p16 (INK4a) and ARF increased immediately after chemotherapy and remained elevated 12 months after treatment. Median increase in log2 p16 (INK4a) was 0.81 (interquartile range = 0.28-1.62; Wilcoxon signed-rank P < .001), or a 75% absolute increase in expression, equivalent to the increase observed over 14.7 years of chronological aging. ARF expression was comparably increased (P < .001). Increased expression of p16 (INK4a) and ARF was associated with dose-dense therapy and hematological toxicity. Expression of two senescence-associated cytokines (VEGFA and MCP1) was durably increased by adjuvant chemotherapy. Telomere length was not affected by chemotherapy. In a cross-sectional cohort, prior chemotherapy exposure was independently associated with a log2-increase in p16 (INK4a) expression of 0.57 (repeated measures model, P < .001), comparable with 10.4 years of chronological aging. CONCLUSIONS: Adjuvant chemotherapy for breast cancer is gerontogenic, inducing cellular senescence in vivo, thereby accelerating molecular aging of hematopoietic tissues.

p16INK4a reporter mice reveal age-promoting effects of environmental toxicants.

While murine-based systems to identify cancer-promoting agents (carcinogens) are established, models to identify compounds that promote aging (gerontogens) have not been described. For this purpose, we exploited the transcription of p16INK4a, which rises dynamically with aging and correlates with age-associated disease. Activation of p16INK4a was visualized in vivo using a murine strain that harbors a knockin of the luciferase gene into the Cdkn2a locus (p16LUC mice). We exposed p16LUC mice to candidate gerontogens, including arsenic, high-fat diet, UV light, and cigarette smoke and serially imaged animals to monitor senescence induction. We show that exposure to a high-fat diet did not accelerate p16INK4a expression, whereas arsenic modestly augmented, and cigarette smoke and UV light potently augmented, activation of p16INK4a-mediated senescence. This work provides a toxicological platform to study mammalian aging and suggests agents that directly damage DNA promote molecular aging.

Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model.

Monitoring cancer and aging in vivo remains experimentally challenging. Here, we describe a luciferase knockin mouse (p16(LUC)), which faithfully reports expression of p16(INK4a), a tumor suppressor and aging biomarker. Lifelong assessment of luminescence in p16(+/LUC) mice revealed an exponential increase with aging, which was highly variable in a cohort of contemporaneously housed, syngeneic mice. Expression of p16(INK4a) with aging did not predict cancer development, suggesting that the accumulation of senescent cells is not a principal determinant of cancer-related death. In 14 of 14 tested tumor models, expression of p16(LUC) was focally activated by early neoplastic events, enabling visualization of tumors with sensitivity exceeding other imaging modalities. Activation of p16(INK4a) was noted in the emerging neoplasm and surrounding stromal cells. This work suggests that p16(INK4a) activation is a characteristic of all emerging cancers, making the p16(LUC) allele a sensitive, unbiased reporter of neoplastic transformation.

Expression of p16(INK4a) as a biomarker of T-cell aging in HIV-infected patients prior to and during antiretroviral therapy.

The p16(INK4a) tumor suppressor gene is a mediator of cellular senescence and has been suggested to be a biomarker of 'molecular' age in several tissues including T cells. To determine the association of both active and suppressed HIV infection with T-cell aging, T-cell p16(INK4a) expression was compared between 60 HIV+ suppressed subjects, 23 HIV+ untreated subjects, and 18 contemporaneously collected HIV-negative controls, as well as 148 HIV-negative historical samples. Expression did not correlate with chronologic age in untreated HIV+ patients, consistent with an effect of active HIV replication on p16(INK4a) expression. In patients on cART with suppressed viral loads, however, p16(INK4a) levels were similar to uninfected controls and correlated with chronologic age, with a trend toward an inverse correlation with CD4 count. These data show that p16(INK4a) is a reliable biomarker of T-cell aging in HIV+ patients with suppressed viral loads and suggest that poor CD4 cell recovery on cART may be associated with increased T-cell expression of p16(INK4a), a marker of cellular senescence.

Cdkn2a is an atherosclerosis modifier locus that regulates monocyte/macrophage proliferation.

OBJECTIVE: Common genetic variants in a 58-kb region of chromosome 9p21, near the CDKN2A/CDKN2B tumor suppressor locus, are strongly associated with coronary artery disease. However, the underlying mechanism of action remains unknown. METHODS AND RESULTS: We previously reported a congenic mouse model harboring an atherosclerosis susceptibility locus and the region of homology with the human 9p21 locus. Microarray and transcript-specific expression analyses showed markedly decreased Cdkn2a expression, including both p16(INK4a) and p19(ARF), but not Cdkn2b (p15(INK4b)), in macrophages derived from congenic mice compared with controls. Atherosclerosis studies in subcongenic strains revealed genetic complexity and narrowed 1 locus to a small interval including Cdkn2a/b. Bone marrow (BM) transplantation studies implicated myeloid lineage cells as the culprit cell type, rather than resident vascular cells. To directly test the role of BM-derived Cdkn2a transcripts in atherogenesis and inflammatory cell proliferation, we performed a transplantation study using Cdkn2a(-/-) cells in the Ldlr(-/-) mouse model. Cdkn2a-deficient BM recipients exhibited accelerated atherosclerosis, increased Ly6C proinflammatory monocytes, and increased monocyte/macrophage proliferation compared with controls. CONCLUSION: These data provide a plausible mechanism for accelerated atherogenesis in susceptible congenic mice, involving decreased expression of Cdkn2a and increased proliferation of monocyte/macrophages, with possible relevance to the 9p21 human locus.

Overexpression of the cell cycle inhibitor p16INK4a promotes a prothrombotic phenotype following vascular injury in mice.

OBJECTIVE: Age-associated cellular senescence is thought to promote vascular dysfunction. p16(INK4a) is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16(INK4a) overexpression to venous thrombosis. METHODS AND RESULTS: Mice overexpressing p16(INK4a) were studied with 4 different vascular injury models: (1) ferric chloride (FeCl(3)) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl(3) and vascular ligation to examine thrombus resolution; and (4) lipopolysaccharide administration to initiate inflammation-induced vascular dysfunction. p16(INK4a) transgenic mice had accelerated occlusion times (13.1 ± 0.4 minutes) compared with normal controls (19.7 ± 1.1 minutes) in the FeCl(3) model and 12.7 ± 2.0 and 18.6 ± 1.9 minutes, respectively in the Rose Bengal model. Moreover, overexpression of p16(INK4a) delayed thrombus resolution compared with normal controls. In response to lipopolysaccharide treatment, the p16(INK4a) transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography assays. Finally, bone marrow transplantation studies suggested increased p16(INK4a) expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16(INK4a) expression in venous thrombosis. CONCLUSIONS: Venous thrombosis is augmented by overexpression of the cellular senescence protein p16(INK4a).

Expression of p16(INK4a) prevents cancer and promotes aging in lymphocytes.

Previous authors have suggested that tumor suppressor expression promotes aging while preventing cancer, but direct experimental support for this cancer-aging hypothesis has been elusive. Here, by using somatic, tissue-specific inactivation of the p16(INK4a) tumor suppressor in murine T- or B-lymphoid progenitors, we report that ablation of p16(INK4a) can either rescue aging or promote cancer in a lineage-specific manner. Deletion of p16(INK4a) in the T lineage ameliorated several aging phenotypes, including thymic involution, decreased production of naive T cells, reduction in homeostatic T-cell proliferation, and attenuation of antigen-specific immune responses. Increased T-cell neoplasia was not observed with somatic p16(INK4a) inactivation in T cells. In contrast, B lineage-specific ablation of p16(INK4a) was associated with a markedly increased incidence of systemic, high-grade B-cell neoplasms, which limited studies of the effects of somatic p16(INK4a) ablation on B-cell aging. Together, these data show that expression of p16(INK4a) can promote aging and prevent cancer in related lymphoid progeny of a common stem cell.

p38MAPK controls expression of multiple cell cycle inhibitors and islet proliferation with advancing age.

Aging is a complex organismal process that is controlled by genetic, environmental, and behavioral factors. Accumulating evidence supports a role for different cell cycle inhibitors in mammalian aging. Little is known, however, about the upstream signals that induce their expression. Here, we explore the role of p38MAPK by generating a dominant-negative allele (p38(AF)) in which activating phosphorylation sites Thr180 and Tyr182 are mutated. Heterozygous p38(AF) mice show a marked attenuation of p38-dependent signaling and age-induced expression of multiple cell cycle inhibitors in different organs, including pancreatic islets. As a result, aged p38(AF/+) mice show enhanced proliferation and regeneration of islets when compared to wild-type littermates. We further find an age-related reduction in expression of the p38-specific phosphatase Wip1. Wip1-deficient mice demonstrate decreased islet proliferation, while Wip1 overexpression rescues aging-related decline in proliferation and regenerative capacity. We propose that modulation of p38MAPK activity may provide new avenues for treating certain age-related degenerative diseases.

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

Expression of p16Ink4a compensates for p18Ink4c loss in cyclin-dependent kinase 4/6-dependent tumors and tissues.

Cell cycle progression from G(1) to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and cdk4/6 activity in vivo. Mouse embryo fibroblasts (MEF) lacking p16(Ink4a) and p18(Ink4c) showed similar growth kinetics as wild-type MEFs despite increased cdk4 activity. In vivo, germline deficiency of p16(Ink4a) and p18(Ink4c) resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16(Ink4a-/-);p18(Ink4c-/-) mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18(Ink4c-/-) mice and in MEFs from p16(Ink4a-/-), p18(Ink4c-/-), or p16(Ink4a-/-);p18(Ink4c-/-) mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4 deficiency was associated with enhanced proliferation (i.e., islets, pituitary, and B lymphocytes) but had no effect on proliferation in other tissues such as the small bowel. These data suggest that p16(Ink4a) and p18(Ink4c) coordinately regulate the in vivo catalytic activity of cdk4/6 in specific compartments of adult mice.

RNA expression analysis of formalin-fixed paraffin-embedded tumors.

RNA expression analysis is an important tool in cancer research, but a limitation has been the requirement for high-quality RNA, generally derived from frozen samples. Such tumor sets are often small and lack clinical annotation, whereas formalin-fixed paraffin-embedded (FFPE) materials are abundant. Although RT-PCR-based methods from FFPE samples are finding clinical application, genome-wide microarray analysis has proven difficult. Here, we report expression profiling on RNA from 157 FFPE tumors. RNA was extracted from 2- to 8-year-old FFPE or frozen tumors of known and unknown histologies. Total RNA was analyzed, reverse-transcribed and used for the synthesis of labeled aRNA after two rounds of amplification. Labeled aRNA was hybridized to a 3'-based 22K spot oligonucleotide arrays, and compared to a labeled reference by two-color microarray analysis. After normalization, gene expression profiles were compared by unsupervised hierarchical clustering. Using this approach, at least 24% of unselected FFPE samples produced RNA of sufficient quality for microarray analysis. From our initial studies, we determined criteria based on spectrophotometric analyses and a novel TaqMan-based assay to predict which samples were of sufficient quality for microarray analysis before hybridization. These criteria were validated on an independent set of tumors with a 100% success rate (20 of 20). Unsupervised analysis of informative gene expression profiles distinguished tumor type and subtype, and identified tumor tissue of origin in three unclassified carcinomas. Although only a minority of FFPE blocks could be analyzed, we show that informative RNA expression analysis can be derived from selected FFPE samples.

Stem cells and the rate of living.

The "rate-of-living theory" is an ancient explanation of longevity which holds that aging occurs due to the exhaustion of some finite substance-breaths, heartbeats, etc. While this theory as originally conceived has been debunked, new work (Ruzankina et al. [2007], in this issue of Cell Stem Cell) suggests that mammals in fact do have a finite number of stem cell replications per life.

p16INK4a induces an age-dependent decline in islet regenerative potential.

The p16INK4a tumour suppressor accumulates in many tissues as a function of advancing age. p16INK4a is an effector of senescence and a potent inhibitor of the proliferative kinase Cdk4 (ref. 6), which is essential for pancreatic beta-cell proliferation in adult mammals. Here we show that p16INK4a constrains islet proliferation and regeneration in an age-dependent manner. Expression of the p16INK4a transcript is enriched in purified islets compared with the exocrine pancreas, and islet-specific expression of p16INK4a, but not other cyclin-dependent kinase inhibitors, increases markedly with ageing. To determine the physiological significance of p16INK4a accumulation on islet function, we assessed the impact of p16INK4a deficiency and overexpression with increasing age and in the regenerative response after exposure to a specific beta-cell toxin. Transgenic mice that overexpress p16INK4a to a degree seen with ageing demonstrated decreased islet proliferation. Similarly, islet proliferation was unaffected by p16INK4a deficiency in young mice, but was relatively increased in p16(INK4a)-deficient old mice. Survival after toxin-mediated ablation of beta-cells, which requires islet proliferation, declined with advancing age; however, mice lacking p16INK4a demonstrated enhanced islet proliferation and survival after beta-cell ablation. These genetic data support the view that an age-induced increase of p16INK4a expression limits the regenerative capacity of beta-cells with ageing.

Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing.

Mammalian ageing is associated with reduced regenerative capacity in tissues that contain stem cells. It has been proposed that this is at least partially caused by the senescence of progenitors with age; however, it has not yet been tested whether genes associated with senescence functionally contribute to physiological declines in progenitor activity. Here we show that progenitor proliferation in the subventricular zone and neurogenesis in the olfactory bulb, as well as multipotent progenitor frequency and self-renewal potential, all decline with age in the mouse forebrain. These declines in progenitor frequency and function correlate with increased expression of p16INK4a, which encodes a cyclin-dependent kinase inhibitor linked to senescence. Ageing p16INK4a-deficient mice showed a significantly smaller decline in subventricular zone proliferation, olfactory bulb neurogenesis, and the frequency and self-renewal potential of multipotent progenitors. p16INK4a deficiency did not detectably affect progenitor function in the dentate gyrus or enteric nervous system, indicating regional differences in the response of neural progenitors to increased p16INK4a expression during ageing. Declining subventricular zone progenitor function and olfactory bulb neurogenesis during ageing are thus caused partly by increasing p16INK4a expression.

Ink4a/Arf expression is a biomarker of aging.

The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16INK4a and Arf, which are principal mediators of cellular senescence. To study the links between senescence and aging in vivo, we examined Ink4a/Arf expression in rodent models of aging. We show that expression of p16INK4a and Arf markedly increases in almost all rodent tissues with advancing age, while there is little or no change in the expression of other related cell cycle inhibitors. The increase in expression is restricted to well-defined compartments within each organ studied and occurs in both epithelial and stromal cells of diverse lineages. The age-associated increase in expression of p16INK4a and Arf is attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlates with diminished expression of an in vivo marker of senescence, as well as decreased pathology of those organs. Last, the age-related increase in Ink4a/Arf expression can be independently attributed to the expression of Ets-1, a known p16INK4a transcriptional activator, as well as unknown Ink4a/Arf coregulatory molecules. These data suggest that expression of the Ink4a/Arf tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.

Identification of direct p73 target genes combining DNA microarray and chromatin immunoprecipitation analyses.

The newly discovered p53 family member, p73, has a striking homology to p53 in both sequence and modular structure. Ectopic expression of p73 promotes transcription of p53 target genes and recapitulates the most characterized p53 biological effects such as growth arrest, apoptosis, and differentiation. Unlike p53-deficient mice that develop normally but are subject to spontaneous tumor formation, p73-deficient mice exhibit severe defects in the development of central nervous system and suffer from inflammation but are not prone to tumor development. These phenotypes suggest different biological activities mediated by p53 and p73 that might reflect activation of specific sets of target genes. Here, we have analyzed the gene expression profile of H1299 cells after p73alpha or p53 activation using oligonucleotide microarrays capable of detecting approximately 11,000 mRNA species. Our results indicate that p73alpha and p53 activate both common and distinct groups of genes. We found 141 and 320 genes whose expression is modulated by p73alpha and p53, respectively. p73alpha up-regulates 85 genes, whereas p53 induces 153 genes, of which 27 are in common with p73alpha. Functional classification of these genes reveals that they are involved in many aspects of cell function ranging from cell cycle and apoptosis to DNA repair. Furthermore, we report that some of the up-regulated genes are directly activated by p73alpha or p53.

A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.