Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

Background

Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.

Results

We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.

Conclusions

In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.     

Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence

Background

Altered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence.

Results

Treatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists.

Conclusions

This is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies.

     

Pathogenesis and vertical transmission of a transplacental rat cytomegalovirus

Background

Cytomegalovirus (CMV) congenital infection is the major viral cause of well-documented birth defects in human. Because CMV is species-specific, the main obstacle to developing animal models for congenital infection is the difference in placental architecture, which preludes virus transmission across the placenta. The rat placenta, resembling histologically to that of human, could therefore facilitate the study of CMV congenital infection in human.

Results

In this report, we present clear evidences of the transplacental property of a new rat CMV (RCMV), namely ALL-03, which had been isolated from placenta and uterus of the house rat. Our study signifies the detection of infectious virus, virus particles, viral protein and DNA as well as immune response to demonstrate a natural model of acute CMV infection including the immunocompetent and immunocompromised host associated with or without pregnancy. It is characterized by a full range of CMV related clinical signs; lesions and anatomical virus distribution to uterus, placenta, embryo, fetus, neonate, lung, kidney, spleen, liver and salivary gland of the infected rats in addition to the virus-specific seroconversion. The preference of the virus for different organs mimics the situation in immunocompromised man. Most interestingly, the placenta was observed to be involved in the maternofetal infection and hence confirmed the hypothesis that the RCMV strain ALL-03 is capable to cross the placenta and infect the offsprings congenitally.

Conclusion

The maternal viremia leading to uterine infection which subsequently infecting to the fetus through the placenta is the most likely phenomenon of CMV vertical transmission in our study.     

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Beta blocker use in subjects with type 2 diabetes mellitus and systolic heart failure does not worsen glycaemic control

Background

The decrease and dysfunction of endothelial progenitor cells (EPCs) has been assumed as an important cause/consequence of diabetes mellitus (DM) and its complications, in which the senescence of EPCs induced by hyperglycemia may play an immensurable role. However, the mechanisms of EPCs senescence has not been fully investigated. Recently, ribosomal protein S6 kinase 4 (RSK4), a member of serine/threomine (Ser/Thr) kinase family and p53-related gene, is reported to regulate the replicative and stress-induced senescence of different cells.

Presentation of the hypothesis

These above lead to consideration of an evidence-based hypothesis that RSK4 may serve as a mediator of EPCs senescence in DM.

Testing the hypothesis

EPCs of healthy subjects and DM patients are isolated from peripheral blood and incubated with high glucose (HG). Then, the EPCs senescence would be detected by senescence associated ??-galactosides (SA-??-gal) staining. Meanwhile, the RSK4 expression is assessed by RT-PCR and western blot. Moreover, overexpressing or RNA interfering of RSK4 in EPCs to investigate the relationship between RSK4 expression and the senescence of EPCs are necessary to substantiate this hypothesis. Also, studies on possible upstream and downstream factors of RSK4 would be explored to reveal the RSK4-mediated senescence pathway in EPCs.

Implications of the hypothesis

If proved, this hypothesis will provide another mediator of EPCs senescence, and may establish a novel pathogenesis for DM and further benefit to the management of DM.     

Innate antiviral immunity of human PBLs and immunoregulatory activity of EGb 761

Background

The aim of the studies was to examine the potential immunoregulatory activity of Ginkgo biloba extract (EGb 761) on cytokine production, one of the mechanisms of innate antiviral immunity, by human peripheral blood leukocytes (PBLs) ex vivo.

Methodology

PBLs isolated from healthy blood donors were treated with different, nontoxic concentrations of EGb 761. Levels of different cytokines (TNF-α, IFN-α, IFN-Γ, IL-10 and IL-12), important in innate immunity development, were determined by ELISA.

Results

EGb 761, apart from strengthening of antiviral response, showed a differential impact on cytokine production by human PBLs ex vivo. It decreased the level of TNF-α and IFN-α but strongly increased the level of IFN-γ in PBLs stimulated by vesicular stomatitis virus (VSV) and non-stimulated PBLs. The extract reduced the production of IL-10 and IL-12 by human PBLs. The results were discussed and compared with previously published findings on the activity of the synthetic drug donepezil.

Conclusions

According to the results from the present study and our previous investigations, we report immunoregulatory activity of EGb 761 on different cytokine production by human PBLs ex vivo, which indicates the possibility of using the drug for the treatment of many immune deficiencies or infectious diseases through strengthening of innate immunity reactions.     

Characteristics of primary and immortalized fibroblast cells derived from the miniature and domestic pigs

Background

The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences.

Results

The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype.

Conclusion

These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro.     

Longer uncommon polyamines have a stronger defense gene-induction activity and a higher suppressing activity of Cucumber mosaic virus multiplication compared to that of spermine in Arabidopsis thaliana

Key message

Our work suggests that long chain polyamines and their derivatives are potential chemicals to control viral pathogens for crop production.

Abstract

Previously we showed that two tetraamines, spermine (Spm) and thermospermine (T-Spm), induce the expression of a subset of defense-related genes and repress proliferation of Cucumber mosaic virus (CMV) in Arabidopsis. Here we tested whether the longer uncommon polyamines (LUPAs) such as caldopentamine, caldohexamine, homocaldopentamine and homocaldohexamine have such the activity. LUPAs had higher gene induction activity than Spm and T-Spm. Interestingly the genes induced by LUPAs could be classified into two groups: the one group was most responsive to caldohexamine while the other one was most responsive to homocaldopentamine. In both the cases, the inducing activity was dose-dependent. LUPAs caused local cell death and repressed CMV multiplication more efficiently as compared to Spm. LUPAs inhibited the viral multiplication of not only avirulent CMV but also of virulent CMV in a dose-dependent manner. Furthermore, LUPAs can activate the systemic acquired resistance against CMV more efficiently as compared to Spm. When Arabidopsis leaves were incubated with LUPAs, the putative polyamine oxidase (PAO)-mediated catabolites were detected even though the conversion rate was very low. In addition, we found that LUPAs induced the expression of three NADPH oxidase genes (rbohC, rbohE and rbohH) among ten isoforms. Taken together, we propose that LUPAs activate two alternative reactive oxygen species evoked pathways, a PAO-mediated one and an NADPH-oxidase-mediated one, which lead to induce defense-related genes and restrict CMV multiplication.     

Attenuation of TORC1 signaling delays replicative and oncogenic RAS-induced senescence

Numerous stimuli, including oncogenic signaling, DNA damage or eroded telomeres trigger proliferative arrest, termed cellular senescence. Accumulating evidence suggests that cellular senescence is a potent barrier to tumorigenesis in vivo, however oncogene induced senescence can also promote cellular transformation.^1,2 Several oncogenes, whose overexpression results in cellular senescence, converge on the TOR (target of rapamycin) pathway. We therefore examined whether attenuation of TOR results in delay or reversal of cellular senescence. By using primary human fibroblasts undergoing either replicative or oncogenic RAS-induced senescence, we demonstrated that senescence can be delayed, and some aspects of senescence can be reversed by inhibition of TOR, using either the TOR inhibitor rapamycin or by depletion of TORC1 (TOR Complex 1). Depletion of TORC2 fails to affect the course of replicative or RAS-induced senescence. Overexpression of REDD1 (Regulated in DNA Damage Response and Development), a negative regulator of TORC1, delays the onset of replicative senescence. These results indicate that TORC1 is an integral component of the signaling pathway that mediates cellular senescence.     

Dynamic expression of synemin isoforms in mouse embryonic stem cells and neural derivatives

Background

Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence.

Results

In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice.

Conclusion

Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence.     

Molecular network profiling of U373MG human glioblastoma cells following induction of apoptosis by novel marine-derived anti-cancer 1,2,3,4-tetrahydroisoquinoline alkaloids

Background

Glioblastoma is the most aggressive form of brain tumors showing resistance to treatment with various chemotherapeutic agents. The most effective way to eradicate glioblastoma requires the concurrent inhibition of multiple signaling pathways and target molecules involved in the progression of glioblastoma. Recently, we obtained a series of 1,2,3,4-tetrahydroisoquinoline alkaloids with potent anti-cancer activities, including ecteinascidin-770 (ET-770; the compound 1a) and renieramycin M (RM; the compound 2a) from Thai marine invertebrates, together with a 2’-N-4”-pyridinecarbonyl derivative of ET-770 (the compound 3). We attempted to characterize the molecular pathways responsible for cytotoxic effects of these compounds on a human glioblastoma cell line U373MG.

Methods

We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics.

Results

All of these compounds induced apoptosis of U373MG cells at nanomolar concentrations. The compound 3 reduced the expression of 417 genes and elevated the levels of 84 genes, while ET-770 downregulated 426 genes and upregulated 45 genes. RM decreased the expression of 274 genes and increased the expression of 9 genes. The set of 196 downregulated genes and 6 upregulated genes showed an overlap among all the compounds, suggesting an existence of the common pathways involved in induction of apoptosis. We identified the ErbB (EGFR) signaling pathway as one of the common pathways enriched in the set of downregulated genes, composed of PTK2, AKT3, and GSK3B serving as key molecules that regulate cell movement and the nervous system development. Furthermore, a GSK3B-specific inhibitor induced apoptosis of U373MG cells, supporting an anti-apoptotic role of GSK3B.

Conclusion

Molecular network analysis is a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets.     

Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis

Background

Exposure to vanadium pentoxide (V₂O₅) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V₂O₅ in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V₂O₅-induced bronchitis.

Methods

Normal human lung fibroblasts were exposed to V₂O₅ in a time course experiment. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Selected genes that were significantly changed in the microarray experiment were validated by RT-PCR.

Results

V₂O₅ altered more than 1,400 genes, of which ~300 were induced while >1,100 genes were suppressed. Gene ontology categories (GO) categories unique to induced genes included inflammatory response and immune response, while GO catogories unique to suppressed genes included ubiquitin cycle and cell cycle. A dozen genes were validated by RT-PCR, including growth factors (HBEGF, VEGF, CTGF), chemokines (IL8, CXCL9, CXCL10), oxidative stress response genes (SOD2, PIPOX, OXR1), and DNA-binding proteins (GAS1, STAT1).

Conclusion

Our study identified a variety of genes that could play pivotal roles in inflammation, fibrosis and repair during V₂O₅-induced bronchitis. The induction of genes that mediate inflammation and immune responses, as well as suppression of genes involved in growth arrest appear to be important to the lung fibrotic reaction to V₂O₅.     

Sequence and functional analyses of Haemophilus spp. genomic islands

Background

A major part of horizontal gene transfer that contributes to the diversification and adaptation of bacteria is facilitated by genomic islands. The evolution of these islands is poorly understood. Some progress was made with the identification of a set of phylogenetically related genomic islands among the Proteobacteria, recognized from the investigation of the evolutionary origins of a Haemophilus influenzae antibiotic resistance island, namely ICEHin1056. More clarity comes from this comparative analysis of seven complete sequences of the ICEHin1056 genomic island subfamily.

Results

These genomic islands have core and accessory genes in approximately equal proportion, with none demonstrating recent acquisition from other islands. The number of variable sites within core genes is similar to that found in the host bacteria. Furthermore, the GC content of the core genes is similar to that of the host bacteria (38% to 40%). Most of the core gene content is formed by the syntenic type IV secretion system dependent conjugative module and replicative module. GC content and lack of variable sites indicate that the antibiotic resistance genes were acquired relatively recently. An analysis of conjugation efficiency and antibiotic susceptibility demonstrates that phenotypic expression of genomic island-borne genes differs between different hosts.

Conclusion

Genomic islands of the ICEHin1056 subfamily have a longstanding relationship with H. influenzae and H. parainfluenzae and are co-evolving as semi-autonomous genomes within the 'supragenomes' of their host species. They have promoted bacterial diversity and adaptation through becoming efficient vectors of antibiotic resistance by the recent acquisition of antibiotic resistance transposons.     

p16(INK4a) translation suppressed by miR-24

Background

Expression of the tumor suppressor p16^INK4a increases during aging and replicative senescence.

Methodology/Principal Findings

Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3′-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites.

Conclusions/Significance

Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.     

Nutrient partitioning and grain yield of TaNAM-RNAi wheat under abiotic stress

Aims

Decreased expression of TaNAM genes by RNAi results in delayed senescence and decreased grain protein, iron, and zinc concentrations. Here, we determined whether NAM expression level alters onset of senescence under stress conditions, whether delayed senescence in the TaNAM-RNAi line resulted in improved tolerance to post-anthesis abiotic stress, and determined the effects of post-anthesis abiotic stress on N and mineral remobilization and partitioning to grain.

Methods

Greenhouse-grown WT and TaNAM-RNAi wheat were characterized in two studies:three levels of N fertility or water limitation during grain fill. Studies were conducted under both optimal and heat stress temperatures. Senescence onset was determined by monitoring flag leaf chlorophyll.

Results

Under optimal tempertures, TaNAM-RNAi plants had a yield advantage at lower N. TaNAM-RNAi plants had delayed senescence relative to the WT and lower grain protein and mineral concentrations, N remobilization efficiency, and partitioning of N and most minerals to grain.

Conclusions

Nutritional quality of TaNAM-RNAi grain was consistently lower than WT. Delayed senescence of TaNAM-RNAi plants provided a yield advantage under optimal temperatures but not under water or heat stress. Discovery of specific NAM protein targets may allow separation of the delayed senescence and nutrient partitioning traits, which could be used for improvement of wheat.