A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r=0.8486, p<0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25,207.3+/-6548.6, H2O2 52,487.4+/-16,284.9, p<0.05; X-Gal: control 41.31+/-7.0%, H2O2 92.97+/-2.8%, p<0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture.     

Retracted: HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.     

PTEN status switches cell fate between premature senescence and apoptosis in glioma exposed to ionizing radiation

Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) has frequently been observed in human gliomas, conferring AKT activation and resistance to ionizing radiation (IR) and drug treatments. Recent reports have shown that PTEN loss or AKT activation induces premature senescence, but many details regarding this effect remain obscure. In this study, we tested whether the status of PTEN determined fate of the cell by examining PTEN-deficient U87, U251, and U373, and PTEN-proficient LN18 and LN428 glioma cells after exposure to IR. These cells exhibited different cellular responses, senescence or apoptosis, depending on the PTEN status. We further observed that PTEN-deficient U87 cells with high levels of both AKT activation and intracellular reactive oxygen species (ROS) underwent senescence, whereas PTEN-proficient LN18 cells entered apoptosis. ROS were indispensable for inducing senescence in PTEN-deficient cells, but not for apoptosis in PTEN-proficient cells. Furthermore, transfection with wild-type (wt) PTEN or AKT small interfering RNA induced a change from premature senescence to apoptosis and depletion of p53 or p21 prevented IR-induced premature senescence in U87 cells. Our data indicate that PTEN acts as a pivotal determinant of cell fate, regarding senescence and apoptosis in IR-exposed glioma cells. We conclude that premature senescence could have a compensatory role for apoptosis in the absence of the tumor suppressor PTEN through the AKT/ROS/p53/p21 signaling pathway.     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Genetics of melanoma progression: the rise and fall of cell senescence

There are many links between cell senescence and the genetics of melanoma, meaning both familial susceptibility and somatic–genetic changes in sporadic melanoma. For example, CDKN2A, the best‐known melanoma susceptibility gene, encodes two effectors of cell senescence, while other familial melanoma genes are related to telomeres and their maintenance. This article aimed to analyze our current knowledge of the genetic or epigenetic driver changes necessary to generate a cutaneous metastatic melanoma, the commonest order in which these occur, and the relation of these changes to the biology and pathology of melanoma progression. Emphasis is laid on the role of cell senescence and the escape from senescence leading to cellular immortality, the ability to divide indefinitely.     

ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.     

Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice

A premature senescence and death 128 (psd128) mutant was isolated from an ethyl methane sulfonate‐induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six‐leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence‐related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map‐based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA‐type ATPase with 809 amino‐acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype.     

Autophagy is a key factor in maintaining the regenerative capacity of muscle stem cells by promoting quiescence and preventing senescence

Autophagy, a highly regulated cellular degradation and recycling process, can occur constitutively at a basal level, and plays an essential role in many aspects of cell physiology. A recently published study (see the related punctum in Autophagy, Vol. 12, No. 4) suggests that basal autophagy is also important for maintaining the regenerative capacity of muscle stem cells, and that the decline of autophagy with aging is the cause of entry into senescence from quiescence in satellite cells.     

A novel method for autophagy detection in primary cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8⁺ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

MCM7 silencing promotes cutaneous melanoma cell autophagy and apoptosis by inactivating the AKT1/mTOR signaling pathway

Cutaneous melanoma (CM) has become a major public health concern. Studies illustrate that minichromosome maintenance protein 7 (MCM7) participate in various diseases including skin disease. Our study aimed to study the effects of MCM7 silencing on CM cell autophagy and apoptosis by modulating the AKT threonine kinase 1 (AKT1)/mechanistic target of rapamycin kinase (mTOR) signaling pathway. Initially, microarray analysis was used to screen the CM-related gene expression data as well as differentially expressed genes. Subsequently, MCM7 expression vector and lentivirus RNA used for MCM7 silencing (LV-shRNA-MCM7) were constructed, and these vectors, dimethyl sulfoxide (DMSO) and AKT activator SC79 were then introduced into CM cell line SK-MEL-2 to validate the role of MCM7 in cell autophagy, viability, apoptosis, cell cycle, migration, and invasion. To further investigate the regulatory mechanisms of MCM7 in CM progress, the expression of MCM7, AKT1, mTOR, cyclin D1, as well as autophagy and apoptosis relative factors, such as LC3B, SOD2, DJ-1, p62, Bcl-2, Bax, and caspase-3 in melanoma cells was determined. MCM7 might mediate the AKT1/mTOR signaling pathway to influence the progress of melanoma. MCM7 silencing contributed to the increased expression of Bax, capase-3, and autophagy-related genes (LC3B, SOD2, and DJ-1), but decreased the expression of Bcl-2, which suggested that MCM7 silencing promoted autophagy and cell apoptosis. At the same time, MCM7 silencing also attenuated cell viability, invasion, and migration, and reduced the cyclin D1 expression and protein levels of p-AKT1 and p-mTOR. Taken together, MCM7 silencing inhibited CM via inactivation of the AKT1/mTOR signaling pathway.     

Radiosensitization in human breast carcinoma cells by thymoquinone: role of cell cycle and apoptosis

TQ (thymoquinone), the bioactive constituent of black seed (Nigella sativa), has been shown to inhibit the growth of various human cancers both in vitro and in vivo. This study reports the radiosensitizing effect of TQ on human breast carcinoma cells (MCF7 and T47D). TQ in combination with single dose of ionizing radiation (2.5 Gy) was found to exert supra-additive cytotoxic effects on both the carcinomas as measured by cell proliferation and colony-formation assays. Annexin V binding and FACS analysis revealed the role of enhanced apoptosis and cell cycle modulation in the mechanism of TQ-mediated radiosensitization, thus supporting TQ as an adjuvant for preclinical testing in cancer chemo-radiotherapy.     

Role of autophagy in inhibiting the proliferation of A549 cells by type III interferon

Interferons (IFNs) have anti‐viral and anti‐tumour effects. Type III interferon, as a member of the recently discovered interferon family, has been proved to inhibit tumour proliferation and promote the apoptosis of various tumour cells. However, whether type III IFN could inhibit the proliferation of lung cancer was not clear. In this study, we found that interferon λ (IFN λ) could inhibit the proliferation of A549 cells and induce autophagy and apoptosis of A549 cells. IFN λ could promote the expression of autophagy gene Beclin1 and interfere the expression of autophagy gene Beclin1 with small interfering RNA, thus inhibiting the effect of type III interferon on anti‐proliferation and promoting apoptosis of lung cancer cell. These results suggested that IFN λ could inhibit the proliferation of A549 cells by activating autophagy pathway, and IFN λ might be one of the potential therapeutic drugs for lung cancer.     

Combined MTOR and autophagy inhibition: Phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma

The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models. This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients. In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma. The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%). An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly. Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss. No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease. The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo. Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ. This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity. Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted.