Algorithmic assessment of cellular senescence in experimental and clinical specimens.

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

Glass-coated ferromagnetic microwire-induced magnetic hyperthermia for in vitro cancer cell treatment.

Limitations in effectiveness and the invasive nature of current cancer treatment options emphasize the need for further clinical advancements. Among other approaches, targeted hyperthermia is as a new strategy aimed at targeting cancerous cells to improve the efficacy of radiotherapy or cytotoxic drugs. However, the testing of magnetic vehicles has mainly focused on the use of nanoparticles. In this work, Fe77B10Si10C3 glass-coated amorphous magnetic microwires were assessed for the first time as magnetic vehicles with high potential for the localized heating of osteosarcoma cells by means of an AC magnetic field. The results from the in vitro assays performed inside a microfluidic device demonstrated the ability of these magnetic microwires to induce malignant cell death. Exposing the system to different magnetic fields for less than 1 h provoked a reduction up to 89% of the osteosarcoma cell population, whereas healthy myoblastoma cells remained nearly unaffected. The proposed technology demonstrates in vitro the effectiveness of these microwires as vehicles for targeted magnetic hyperthermia.

Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling.

Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAF(V600E), the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAF(V600E)-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAF(V600E)-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.

Sex and age-related differences in perceived, desired and measured percentage body fat among adults.

BACKGROUND: Body image distortion/discrepancy leads to psychological stress, disordered eating and mental and physical disease. To begin to assess body image distortion/discrepancy, we compared perceived, desired and measured percentage body fat in male versus female and college-aged versus non-college aged individuals. In addition, we assessed the acute stress response to body composition measurement. METHODS: Body fat percentage of 15 college aged ('College Students'; CS) (mean = 19 years) and 16 non-college aged ('Non-College Aged Students'; NCS) (mean = 39 years) males and females was assessed with the BodPod Body Composition Tracking System (Life Measurement Instruments, Concord, CA, USA). Participants indicated their perception of body fat and their desired body fat using a somatomorphic matrix. Salivary cortisol, heart rate and blood pressure were also measured. Data were analysed by analysis of variance and alpha was set at 0.05. RESULTS: Mean (SD) percentage body fat of males [15.2% (6.1%)] was significantly lower than that of females [28.4% (6.4%)] (P < 0.0001). Both CS and NCS females perceived their body fat to be lower (5%) than measured body fat and desired their body fat to be lower (12%) than measured (P < 0.05). CS and NCS male participants demonstrated the opposite result; both CS and NCS male populations perceived their body fat to be higher (5%) than measured body fat and desired their body fat to be higher (4%) than measured (P < 0.05). No differences between any groups were observed in heart rate, blood pressure or cortisol response to body fat measurement. CONCLUSIONS: Sex-related but not age-related differences in perceived, desired and measured percentage body fat were observed.

Mitochondrial DNA damage induces apoptosis in senescent cells.

Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV-HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells.

Environmental stress, ageing and glial cell senescence: a novel mechanistic link to Parkinson's disease?

Exposure to environmental toxins is associated with a variety of age-related diseases including cancer and neurodegeneration. For example, in Parkinson's disease (PD), chronic environmental exposure to certain toxins has been linked to the age-related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti-cancer mechanism that occurs in a number of proliferative cell types and causes the arrest of proliferation of cells at risk of malignant transformation following exposure to potentially oncogenic stimuli. With age, senescent cells accumulate and express a senescence-associated secretory phenotype (SASP; that is the robust secretion of many inflammatory cytokines, growth factors and proteases). Whereas cell senescence in peripheral tissues has been causally linked to a number of age-related pathologies, little is known about the induction of cellular senescence and the SASP in the brain. On the basis of recently reported findings, we propose that environmental stressors associated with PD may act in part by eliciting senescence and the SASP within non neuronal glial cells in the ageing brain, thus contributing to the characteristic decline in neuronal integrity that occurs in this disorder.

STAT3 can serve as a hit in the process of malignant transformation of primary cells.

The transcription factor signal transducer and activator of transcription 3 (STAT3) acts downstream of many pro-oncogenic signals, including cytokines, growth factors and oncogenes, and is accordingly constitutively active in a wide variety of tumors that often become addicted to it. Moreover, STAT3 is a key player in mediating inflammation-driven tumorigenesis, where its aberrant continuous activation is typically triggered by local or systemic production of the pro-inflammatory cytokine IL-6. We recently showed that mouse embryonic fibroblasts (MEFs) derived from STAT3C k/in mice, which express physiological levels of the constitutively active mutant STAT3C, display features of transformed cells such as increased proliferation, resistance to apoptosis and senescence, and aerobic glycolysis. Here, we show that pre-existing constitutively active STAT3 is sufficient to prime primary MEFs for malignant transformation upon spontaneous immortalization. Transformation is strictly STAT3-dependent and correlates with high resistance to apoptosis and enhanced expression of anti-apoptotic/pro-survival genes. Additionally, hypoxia inducible factor (HIF)-1α level is elevated by twofold and contributes to STAT3 oncogenic activity by supporting high rates of aerobic glycolysis. Thus, constitutively active STAT3, an accepted essential factor for tumor growth/progression, can also act as a first hit in multistep carcinogenesis; this ability to predispose cells to malignant transformation may be particularly relevant in the pro-oncogenic niche represented by chronically inflamed tissues.

Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells.

Cancer cells often acquire a constitutively active nuclear factor-kappaB (NF-kappaB) program to promote survival, proliferation and metastatic potential by mechanisms that remain largely unknown. Extending observations from an immunologic setting, we demonstrate that microRNA-146a and microRNA-146b (miR-146a/b) when expressed in the highly metastatic human breast cancer cell line MDA-MB-231 function to negatively regulate NF-kappaB activity. Lentiviral-mediated expression of miR-146a/b significantly downregulated interleukin (IL)-1 receptor-associated kinase and TNF receptor-associated factor 6, two key adaptor/scaffold proteins in the IL-1 and Toll-like receptor signaling pathway, known to positively regulate NF-kappaB activity. Impaired NF-kappaB activity was evident from reduced phosphorylation of the NF-kappaB inhibitor IkappaBalpha, reduced NF-kappaB DNA-binding activity and suppressed expression of the NF-kappaB target genes IL-8, IL-6 and matrix metalloproteinase-9. Functionally, miR-146a/b-expressing MDA-MB-231 cells showed markedly impaired invasion and migration capacity relative to control cells. These findings implicate miR-146a/b as a negative regulator of constitutive NF-kappaB activity in a breast cancer setting and suggest that modulating miR-146a/b levels has therapeutic potential to suppress breast cancer metastases.

Catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines.

Proinflammatory cytokines act at receptors in the CNS to alter physiological and behavioral responses. Exposure to stressors increases both peripheral and central proinflammatory cytokines, yet the mechanism(s) of induction remain unknown. Experiments here examined the role of catecholamines in the in vivo induction of proinflammatory cytokines following tailshock stress. Rats were pretreated i.p. with 2.0 mg/kg prazosin (alpha1-adrenoceptor antagonist), 10.0 mg/kg propranolol (beta-adrenoceptor antagonist), or 5.0 mg/kg labetalol (alpha1- and beta-adrenoceptor antagonist) 30 min prior to tailshock exposure and plasma interleukin-1beta (IL-1beta) and IL-6, along with tissue interleukin-1beta from the hypothalamus, hippocampus, and pituitary were measured immediately following stressor termination. Prazosin attenuated stress-induced plasma IL-1beta and IL-6, but had no effect on tissue IL-1beta levels, while propranolol attenuated plasma IL-6 and blocked tissue IL-1beta elevation, and labetalol, which cannot cross the blood-brain barrier, attenuated plasma IL-1beta and IL-6, blocked pituitary IL-1beta, but had no effect on central tissue IL-1beta levels. Furthermore, administration of 50.0 mg/kg N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride, a neurotoxin that lesions neural projections from the locus coeruleus, prevented stress-induced elevation in hippocampal IL-1beta, a region highly innervated by the locus coeruleus, but had no effect on hypothalamic IL-1beta, a region that receives few locus coeruleus projections. Finally, i.p. injection of 5.0 mg/kg isoproterenol (beta-adrenoceptor agonist) was sufficient to induce circulating IL-1 and IL-6, and tissue IL-1beta. These data suggest catecholamines play an important role in the induction of stress-induced proinflammatory cytokines and that beta-adrenoceptors are critical for tissue IL-1beta induction, while both alpha- and beta-adrenoceptors contribute to the induction of plasma cytokines.

Physical activity alters the brain Hsp72 and IL-1beta responses to peripheral E. coli challenge.

Physically active rats have facilitated heat shock protein 72 (Hsp72) responses after stressor exposure in both brain and peripheral tissues compared with sedentary rats. This study verifies that physically active animals do not have elevated Hsp72 levels compared with sedentary animals in the hypothalamus, pituitary, or dorsal vagal complex. We then examined whether 1) physically active rats respond more efficiently than sedentary rats to a bacterial challenge; 2) peripheral immune challenge elicits brain induction of Hsp72; 3) this induction is facilitated by prior freewheel running; and 4) Hsp72 upregulation produced by peripheral immune challenge results in a commensurate decrease in the proinflammatory cytokine IL-1beta. Adult male Fischer 344 rats were housed with either a mobile or locked running wheel. Six weeks later, rats were injected intraperitoneally with saline or Escherichia coli and killed 30 min, 2.5 h, 6 h, and 24 h later. Serum endotoxin and IL-1beta, and peritoneal fluid endotoxin and E. coli colony-forming units (CFUs) were measured. Hsp72 and IL-1beta were measured in hypothalamus, pituitary, and dorsal vagal complex. The results were that physically active rats had a faster reduction in endotoxin and E. coli CFUs and lower levels of circulating endotoxin and cytokines compared with sedentary rats. E. coli challenge elicited significantly greater time-dependent increases of both Hsp72 and IL-1beta in hypothalamus, pituitary, and dorsal vagal complex of physically active animals but not sedentary animals. Contrary to our hypothesis, increases in Hsp72 were positively correlated with IL-1beta. This study extends our findings that physical activity facilitates stress-induced Hsp72 to include immunological stressors such as bacterial challenge and suggests that brain Hsp72 and IL-1beta responses to peripheral immune challenge may contribute to exercise-mediated resistance to long-term sickness.

Splenic norepinephrine depletion following acute stress suppresses in vivo antibody response.

Exposure to an intense acute stressor immediately following immunization leads to a reduction in anti-KLH IgM, IgG, and IgG2a, but not IgG1. Stress also depletes splenic norepinephrine (NE) content. Immunization during pharmacological (alpha-methyl-p-tyrosine) or stress-induced splenic NE depletion results in antibody suppression similar to that found in rats immunized prior to stressor exposure. Prevention of splenic NE depletion during stress by tyrosine, but not pharmacological elevation (mirtazapine) of NE, resulted in normal antibody responses. These data support the hypothesis that splenic NE depletion is necessary and sufficient for stress-induced suppression of antibody to a T-cell dependent antigen.

Circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response produced by peripheral E. coli challenge.

Peripheral administration of a variety of inflammatory stimuli, such as endotoxin or cytokines, induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, few data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus the present studies examined whether subcutaneously administered Escherichia coli can activate sickness responses and whether circulating endotoxin and/or proinflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 x 10(7), 2.5 x 10(8), 2.5 x 10(9) colony-forming units) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days after the injection. A second set of groups of animals were killed 3, 6, 12, 18, 24, and 48 h after the injection, and blood samples and brains were collected. Injections dose dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 h after the injection. In addition, E. coli significantly increased serum interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha and brain IL-1beta levels beginning at the 6-h time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 h after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 h after injection. Cytokines were elevated at the inflammation site but were not detectable in the serum or brain at 2 and 4 h. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain, and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.

Integrating epithelial cancer, aging stroma and cellular senescence.

Cancer incidence rises exponentially with age in humans and many other mammalian species. At least two critical changes are essential in order for cancer to develop: an accumulation of oncogenic mutations and a permissive tissue environment in which mutant cells can survive, proliferate, and express their neoplastic phenotype. Increasing evidence suggests that the rise in cancer with age results from a synergy between the accumulation of mutations and age-related, pro-oncogenic changes in the tissue milieu. Most age-related cancers derive from epithelial cells. Epithelial tissues are supported by a stroma, which is composed of extracellular matrix and several cell types. The stroma is essential for the function of the epithelium, and is maintained, remodeled and repaired by fibroblasts. One age-related change that occurs in epithelial tissues is the accumulation of senescent cells. Cellular senescence is a potent tumor suppressive mechanism that irreversibly arrests proliferation in response to damage or stimuli that put cells at risk for neoplastic transformation. Senescent cells, particularly senescent stromal fibroblasts, secrete factors that can disrupt tissue architecture and/or stimulate neighboring cells to proliferate. We suggest that senescent cells can create a tissue environment that synergizes with oncogenic mutations to promote the progression of age-related cancers. Recent evidence lends support to this idea, and suggests that cellular senescence may be an example of evolutionary antagonistic pleiotropy.

Role of extracellular HSP72 in acute stress-induced potentiation of innate immunity in active rats.

Acute stress can compromise acquired, and potentiate innate, immunity. Recent evidence suggests that the impact of stress on measures of immunity can be modulated by the physical activity status of the organism and that extracellular heat shock protein 72 (eHSP72) contributes to the activation of innate immunity produced by stress. Therefore, this study investigated whether physical activity status would impact the immunologically enhancing effects of stressor exposure [inescapable tail-shock stress (IS)] on innate immunity and whether changes in eHSP72 responses could play a role. Adult, male Fischer 344 rats lived with mobile (physically active) or immobile (sedentary) running wheels. After 6 wk, rats were exposed to IS or to no stress. Immediately after IS, all rats were injected subcutaneously with live Escherichia coli. Inflammation was assessed daily, and plasma eHSP72 was measured at various time points. Rats exposed to IS resolved their inflammation faster than nonstressed rats, but the beneficial impact of stress on recovery was greater in physically active rats. All rats had equal increases in circulating eHSP72 after IS. Splenocytes harvested from a separate cohort of nonstressed rats were cultured with eHSP72, and nitric oxide and cytokines were measured. Physically active rats responded to eHSP72 stimulation in vitro with a greater nitric oxide and cytokine response than sedentary rats. Thus physically active rats both recover faster than sedentary rats after bacterial challenge + IS exposure and demonstrate potentiated cellular responses to eHSP72 activation that could be important for bacterial recovery.

C. elegans clk-2, a gene that limits life span, encodes a telomere length regulator similar to yeast telomere binding protein Tel2p.

An important quest in modern biology is to identify genes involved in aging. Model organisms such as the nematode Caenorhabditis elegans are particularly useful in this regard. The C. elegans genome has been sequenced [1], and single gene mutations that extend adult life span have been identified [2]. Among these longevity-controlling loci are four apparently unrelated genes that belong to the clk family. In mammals, telomere length and structure can influence cellular, and possibly organismal, aging. Here, we show that clk-2 encodes a regulator of telomere length in C. elegans.

Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.

Mammalian cells can respond to damage or stress by entering a state of arrested growth and altered function termed cellular senescence. Several lines of evidence suggest that the senescence response suppresses tumorigenesis. Cellular senescence is also thought to contribute to aging, but the mechanism is not well understood. We show that senescent human fibroblasts stimulate premalignant and malignant, but not normal, epithelial cells to proliferate in culture and form tumors in mice. In culture, the growth stimulation was evident when senescent cells comprised only 10% of the fibroblast population and was equally robust whether senescence was induced by replicative exhaustion, oncogenic RAS, p14(ARF), or hydrogen peroxide. Moreover, it was due at least in part to soluble and insoluble factors secreted by senescent cells. In mice, senescent, much more than presenescent, fibroblasts caused premalignant and malignant epithelial cells to form tumors. Our findings suggest that, although cellular senescence suppresses tumorigenesis early in life, it may promote cancer in aged organisms, suggesting it is an example of evolutionary antagonistic pleiotropy.

Cellular senescence, cancer and aging: the telomere connection.

Telomeres are the repetitive DNA sequences and specialized proteins that form the distinctive structure that caps the ends of linear chromosomes. Telomeres allow cells to distinguish the chromosome ends from double strand DNA breaks. The telomeric structure prevents the degradation or fusion of chromosome ends, and thus is essential for maintaining the integrity and stability of eukaryotic genomes. In addition, and perhaps less widely appreciated, telomeres may also indirectly influence gene expression. The length, structure and organization of telomeres are regulated by a host of telomere-associated proteins, and can be influenced by basic cellular processes such as cell proliferation, differentiation, and DNA damage. In mammalian cells, telomere length and/or telomere structure have been linked to both cancer and aging. Here, we briefly review what is known about mammalian telomeres and the proteins that associate with them, and discuss the cellular and organismal consequences of telomere dysfunction and the evidence that cells with dysfunctional telomeres can contribute to cancer and aging phenotypes.

The human melanocyte: a model system to study the complexity of cellular aging and transformation in non-fibroblastic cells.

The melanocyte is a neural crest-derived cell that localizes in humans to several organs including the epidermis, eye, inner ear and leptomeninges. In the skin, melanocytes synthesize and transfer melanin pigments to surrounding keratinocytes, leading to skin pigmentation and protection against solar exposure. We have investigated the process of replicative senescence and accompanying irreversible cell cycle arrest, in melanocytes in culture. As was found in other cell types, progressive telomere shortening appears to trigger replicative senescence in normal melanocytes. In addition, senescence is associated with increased binding of the cyclin-dependent kinase inhibitor (CDK-I) p16(INK4a) to CDK4, down-regulation of cyclin E protein levels (and consequent loss of cyclin E/CDK2 activity), underphosphorylation of the retinoblastoma protein RB and subsequent increased levels of E2F4-RB repressive complexes. In contrast to fibroblasts, however, the CDK-Is p21(Waf-1) and p27(Kip-1) are also down-regulated. These changes appear to be important for replicative senescence because they do not occur in melanocytes that overexpress the catalytic subunit of the enzyme telomerase (hTERT), or in melanomas, which are tumors that originate from melanocytes or melanoblasts. In contrast to unmodified melanocytes, hTERT overexpressing (telomerized) melanocytes displayed telomerase activity, stable telomere lengths and an extended replicative life span. However, telomerized melanocytes show changes in cell cycle regulatory proteins, including increased levels of cyclin E, p21(Waf-1) and p27(Kip-1). Cyclin E, p21(Waf-1) and p27(Kip-1) are also elevated in many primary melanomas, whereas p16(INK4a) is mutated or deleted in many invasive and metastatic melanomas. Thus, the molecular mechanisms leading to melanocyte senescence and transformation differ significantly from fibroblasts. This suggests that different cell types may use different strategies to halt the cell cycle in response to telomere attrition and thus prevent replicative immortality.

Cellular senescence as a tumor-suppressor mechanism.

Organisms with renewable tissues had to evolve mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. Recent findings have revealed the complexities of the senescence phenotype and unexpected possible consequences for the organism.