Differential control of the NIMA-related kinases,Nek6 and Nek7, by serum stimulation

Neks (NIMA-related kinases) are mammalian serine/threonine (Ser/Thr) protein kinases structurally related to Aspergillus NIMA (Never in Mitosis, gene A), which plays essential roles in mitotic signaling. Among these kinases, Nek6 and Nek7 are structurally related and constitute a subfamily in the NIMA/Nek family, although their functions still remain almost elusive. In this report, we studied the enzymatic regulation of Nek6 and Nek7 to gain an insight into their cellular functions. Recombinant Nek7 produced in bacteria was active comparably to Nek6; however, the Nek7 activity in mammalian cells was found to be significantly lower than Nek6. Since Nek6 previously has been reported to in vitro phosphorylate p70 ribosomal S6 kinase at Thr412, we examined if Nek6 and Nek7 activities were controlled by the amino acid supplement, which is known to affect the phosphorylation at Thr412, and did not observe any significant effect. However, we unexpectedly found that Nek7 kinase activity was rapidly and efficiently increased by serum deprivation, while Nek6 activity was decreased. This is well consistent with the lower activity of Nek7 in cells under normal growth conditions. In addition, it was suggested that Nek7 activity would be regulated in a cell cycle-dependent manner, although Nek6 was not. These clear differences in enzymatic control between the highly similar kinases, Nek6 and Nek7, suggest their distinct signaling functions in mammalian cells.     

Molecular basis for premature senescence induced by surfactants in normal human cells

Sublethal doses of surfactants as exemplified by NP-40 clearly induce premature senescence in normal human cells. To understand molecular basis for this phenomenon, we tried to suppress it with use of various inhibitors. An inhibitor of p38 of the MAPK family almost completely suppressed growth arrest and morphological changes induced by surfactants; however, other inhibitors tested had no effect. Oleic acid, a weak inducer of premature senescence, was found to suppress the effect of NP-40. Fluorescein-labeled oleic acid rapidly bound to the cell surface, and this binding was clearly blocked by pre-treatment with surfactants, suggesting that surfactants and oleic acid compete for binding to the cell surface. Moderate concentrations of cycloheximide, an inhibitor of protein synthesis, also suppressed the senescent features induced by NP-40. These results suggest that surfactants activate p38 signaling pathway by binding to the cell surface, and induce cellular senescence.     

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.     

Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells

NIMA-related kinase 2 (Nek2), a serine–threonine protein kinase, plays a major role in mitotic progression, including timing of mitotic entry, chromatin condensation, spindle organization, and cytokinesis. Nek2 overexpression results in premature centrosome separation, while kinase death Nek2 mutant expression or Nek2-depleted cells lead to centrosome separation failure. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with Nek2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between Nek2 and TRF1 are far from clear. Here, we show that mitotic aberrations through Nek2 overexpression are likely to require TRF1. Our results demonstrate that Nek2 directly binds and phosphorylates TRF1 through multiple sites on TRF1. Nek2 overexpression in breast cancer cells, MDA-MB-231 and MCF7, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the phenomenon of unaligned chromosomes by Nek2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1-depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA-transfected cells. Interestingly, when exogenous TRF1 was added back in Nek2-overexpressed cells with endogenous TRF1 depletion, cells had re-induced cytokinetic failure. Therefore, we propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability.     

Interaction of Pin1 with Nek6 and characterization of their expression correlation in Chinese hepatocellular carcinoma patients

The peptidyl-prolyl isomerase Pin1 is prevalently overexpressed in human cancers and is regarded as a new diagnostic and therapeutic target. Pin1 interacts with several proteins involved in cell cycle events in a phosphorylation-dependent manner. Among them, NIMA (never in mitosis, gene A) was first identified to interact with Pin1. In this report, we found that Pin1 could interact with Nek6, one of the human NIMA-related kinases (Neks). This interaction was confirmed by GST pull-down assay, which was further confirmed by immunoprecipitation experiments, as well as immunofluorescence colocalization. We further studied Pin1 and Nek6 mRNA level in 40 pairs of hepatocellular carcinoma cases, finding significant correlations between Nek6 and Pin1 mRNA expression levels in these samples.     

Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells

In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated β-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage. [BMB Reports 2014; 47(10): 575-580]     

Persistent DNA damage‐induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well‐known anti‐tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β‐galactosidase activity and enlarged γH2AX foci co‐localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence‐associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour‐promoting behaviour.     

Simultaneous treatment with camptothecin and valproic acid suppresses induction of Bcl-XL and promotes apoptosis of MCF-7 breast cancer cells

Camptothecin derivatives have been widely used for chemotherapy in patients with various cancers, but intrinsic and acquired drug resistance is major drawback to be overcome. In the present study, we demonstrated that simultaneous treatment with camptothecin and valproic acid induced apoptosis of MCF-7 cells, whereas neither agent alone could efficiently induce apoptosis. This induction of apoptosis was associated with loss of the mitochondrial membrane potential and was caspase dependent. Further investigation showed that concurrent treatment modulated the expression of pro-apoptotic and anti-apoptotic genes. Bcl-X_L expression was induced in MCF-7 cells treated with camptothecin alone, but not in cells treated simultaneously with camptothecin and valproic acid. Ectopic overexpression of Bcl-X_L in MCF-7 cells completely suppressed the induction of apoptosis, even with simultaneous treatment. On the other hand, efficient induction of apoptosis was achieved by treatment with camptothecin and Bcl-X_L inactivation (using siRNA or BH3 mimetic). The cytotoxic effect of camptothecin combined with valproic acid was more than additive for MCF-7 cells. Taken together, our results suggest that simultaneous administration of camptothecin and valproic acid might be useful for anticancer therapy.     

Hyperosmolarity induced by high glucose promotes senescence in human glomerular mesangial cells

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes.     

Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage

Cellular senescence is an important phenomenon in decreased cellular function. Recently, it was shown that cellular senescence is induced in proliferating cells within a short period of time by oxidative stresses. This phenomenon is known as premature senescence. However, it is still unknown whether premature senescence can be also induced in cardiomyocytes. The aim of the present study was to investigate whether a senescence-like phenotype can be induced in cardiomyocytes by oxidative stress. In cardiomyocytes obtained from aged rats (24 months of age), the staining for senescence-associated beta-galactosidase increased significantly and the protein or RNA levels of cyclin-dependent kinase inhibitors increased compared to those of young rats. Decreased cardiac troponin I phosphorylation and telomerase activity were also observed in aged cardiomyocytes. Treatment of cultured neonatal rat cardiomyocytes with a low concentration of doxorubicin (DOX) (10(-7) mol L(-1)) did not induce apoptosis but did induce oxidative stress, which was confirmed by 2',7'-dichlorofluorescin diacetate staining. In DOX-treated neonatal cardiomyocytes, increased positive staining for senescence-associated beta-galactosidase, cdk-I expression, decreased cardiac troponin I phosphorylation, and decreased telomerase activity were observed, as aged cardiomyocytes. Alterations in mRNA expression typically seen in aged cells were observed in DOX-treated neonatal cardiomyocytes. We also found that promyelocytic leukemia protein and acetylated p53, key proteins involved in stress-induced premature senescence in proliferating cells, were associated with cellular alterations of senescence in DOX-treated cardiomyocytes. In conclusion, cardiomyocytes treated with DOX showed characteristic changes similar to cardiomyocytes of aged rats. promyelocytic leukemia-related p53 acetylation may be an underlying mechanism of senescence-like alterations in cardiomyocytes. These findings indicate a novel mechanism of myocardial dysfunction induced by oxidative stress.     

FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2

Human mesenchymal stem cells (hMSCs) are able to both self-replicate and differentiate into a variety of cell types. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro, but its mechanisms have not been clarified yet. In this study, we investigated whether cellular senescence was involved in the stimulation of hMSCs growth by FGF-2 and the expression levels of transforming growth factor-beta1 and -beta2 (TGF-betas). Because hMSCs were induced cellular senescence due to long-term culture, FGF-2 decreased the percentage of senescent cells and suppressed G1 cell growth arrest through the suppression of p21(Cip1), p53, and p16(INK4a) mRNA expression levels. Furthermore, the levels of TGF-betas mRNA expression in hMSCs were increased by long-term culture, but FGF-2 suppressed the increase of TGF-beta2 mRNA expression due to long-term culture. These results suggest that FGF-2 suppresses the hMSCs cellular senescence dependent on the length of culture through down-regulation of TGF-beta2 expression.     

Model of the developing tumorigenic phenotype in mammalian cells and the roles of sustained stress and replicative senescence

The molecular mechanisms that drive mammalian cells to the development of cancer are the subject of intense biochemical, genetic and medical studies. But for the present, there is no comprehensive model that might serve as a general framework for the interpretation of experimental data. This paper is an attempt to create a conceptual model of the mechanism of the developing tumorigenic phenotype in mammalian cells, defined as having high genomic instability and proliferative activity. The basic statement in the model is that mutations acquired by tumor cells are not caused directly by external DNA damaging agents, but instead are produced by the cell itself as an output of a Mutator Response similar to the bacterial "SOS response" and characterized by the initiation of error-prone cell cycle progression and an elevated rate of mutation. This response may be induced in arrested mammalian cells by intracellular and extracellular proliferative signals combined with blocked apoptosis. The mutant cells originated by this response are subjected to natural selection via apoptosis and turnover. This selection process favors the survival of cells with high proliferative activity and the suppression of apoptosis resulting in the long run in the appearance of immortalized cells with high proliferative activity. Either a sustained stressful environment accompanied by continuing apoptotic cell death, or replicative senescence, provides conditions suitable for activation of the Mutator Response, namely the emergence of arrested cells with blocked apoptosis and the induction of proliferative signal. It also accelerates the selection process by providing continuing cell turnover. The proposed mechanism is described at the level of involved metabolic pathways and proteins and substantiated by the related experimental data available in the literature.     

High-performance liquid chromatographic analysis for the determination of a novel polymer-bound camptothecin derivative (MAG-camptothecin) and free camptothecin in human plasma

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C₈ analytical column, using a mixture of acetonitrile–25 mM phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.     

Intracellular localisation studies of doxorubicin and Victoria Blue BO in EMT6-S and EMT6-R cells using confocal microscopy

The subcellular localisation of doxorubicin and Victoria Blue BO (VBBO) in a murine mammary tumour cell line EMT6-S, and the resistant sub-lineEMT6-R was studied, using confocal microscopy, in order to investigate their sites of action. In cells treated with doxorubicin (10 μ M) for 90 min, the pattern of intracellular drug distribution differed between the two cell lines. Doxorubicin was found to localise mainly in the nucleus of the sensitive cell line, whereas weak fluorescence was observed in the cytoplasm of the resistant cells, in a punctuate pattern, with no nuclear involvement. The drug also appeared to be effluxed more rapidly by the resistant cell line. The accumulation of doxorubicin at various time intervals over 1h in EMT6-S cells showed that the drug clearly interacted with both the plasma membrane and the nucleus. In contrast to doxorubicin, the intracellular distribution of VBBO in both EMT6-S and EMT6-R was similar, VBBO was clearly localised throughout the cytoplasm, in a punctuate pattern, which may be consistent with the widespread distribution of mitochondria. A more apical pattern of accumulation was noted in the EMT6-R cell line. No interaction with the plasma membrane was evident. These results indicate that the main modes of action for the two drugs differ markedly, suggesting involvement of both the membrane and the nucleus in the case of doxorubicin, but mitochondrial involvement for VBBO. This revised version was published online in August 2006 with corrections to the Cover Date.     

YAP prevents premature senescence of astrocytes and cognitive decline of Alzheimer's disease through regulating CDK6 signaling

Senescent astrocytes accumulate with aging and contribute to brain dysfunction and diseases such as Alzheimer''s disease (AD), however, the mechanisms underlying the senescence of astrocytes during aging remain unclear. In the present study, we found that Yes‐associated Protein (YAP) was downregulated and inactivated in hippocampal astrocytes of aging mice and AD model mice, as well as in D‐galactose and paraquat‐induced senescent astrocytes, in a Hippo pathway‐dependent manner. Conditional knockout of YAP in astrocytes significantly promoted premature senescence of astrocytes, including reduction of cell proliferation, hypertrophic morphology, increase in senescence‐associated β‐galactosidase activity, and upregulation of several senescence‐associated genes such as p16, p53 and NF‐κB, and downregulation of Lamin B1. Further exploration of the underlying mechanism revealed that the expression of cyclin‐dependent kinase 6 (CDK6) was decreased in YAP knockout astrocytes in vivo and in vitro, and ectopic overexpression of CDK6 partially rescued YAP knockout‐induced senescence of astrocytes. Finally, activation of YAP signaling by XMU‐MP‐1 (an inhibitor of Hippo kinase MST1/2) partially rescued the senescence of astrocytes and improved the cognitive function of AD model mice and aging mice. Taken together, our studies identified unrecognized functions of YAP‐CDK6 pathway in preventing astrocytic senescence in vitro and in vivo, which may provide further insights and new targets for delaying brain aging and aging‐related neurodegenerative diseases such as AD.     

Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress‐induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic‐based approach, here we identify TrxR1 as a caveolar membrane‐resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1‐binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin‐binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative‐stress‐mediated activation of the p53/p21^Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1‐mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21^Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.     

Ultrastructural alterations of the myocardium induced by doxorubicin

Morphologic changes in Doxorubicin (DXR)-induced cardiomyopathy are characterized by marked dilatation of the sarcoplasmic reticulum (SR). DXR was administered to New Zealand White rabbits for 5 or 8 weeks and the three-dimensional structure of the sarcotubular system in cardiac muscle cells from each rabbit was examined under a field-emission type scanning electron microscope (SEM) after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure. Five weeks after the initial injection of DXR, partial dilatation of the SR and damaged mitochondria with lysis of cristae were observed three-dimensionally. After 8 weeks, the three-dimensional structure of the SR showed extensive spherical ballooning which could be seen clearly in bold relief. Thus, we could directly visualize structural alterations of the sarcotubular system in DXR-induced cardiomyopathy using the SEM.     

Induction of senescence by adenosine suppressing the growth of lung cancer cells

Extracellular adenosine is well reported to suppress tumor growth by induction of apoptosis. However, in this study we found that adenosine treatment results in cellular senescence in A549 lung cancer cells both in vitro and in vivo; adenosine induces cell cycle arrest and senescence in a p53/p21 dependent manner; adenosine elevates the level of phosphor-γH2AX, pCHK2 and pBRCA1, the markers for prolonged DNA damage response which are likely responsible for initiating the cellular senescence. Our study first demonstrates that adenosine suppresses growth of cancer cells by inducing senescence and provides additional evidence that adenosine could act as an effective anticancer agent for targeted cancer therapy.