Base excision repair but not DNA double‐strand break repair is impaired in aged human adipose‐derived stem cells

The decline in DNA repair capacity contributes to the age‐associated decrease in genome integrity in somatic cells of different species. However, due to the lack of clinical samples and appropriate tools for studying DNA repair, whether and how age‐associated changes in DNA repair result in a loss of genome integrity of human adult stem cells remains incompletely characterized. Here, we isolated 20 eyelid adipose‐derived stem cell (ADSC) lines from healthy individuals (young: 10 donors with ages ranging 17–25 years; old: 10 donors with ages ranging 50–59 years). Using these cell lines, we systematically compared the efficiency of base excision repair (BER) and two DNA double‐strand break (DSB) repair pathways—nonhomologous end joining (NHEJ) and homologous recombination (HR)—between the young and old groups. Surprisingly, we found that the efficiency of BER but not NHEJ or HR is impaired in aged human ADSCs, which is in contrast to previous findings that DSB repair declines with age in human fibroblasts. We also demonstrated that BER efficiency is negatively associated with tail moment, which reflects a loss of genome integrity in human ADSCs. Mechanistic studies indicated that at the protein level XRCC1, but not other BER factors, exhibited age‐associated decline. Overexpression of XRCC1 reversed the decline of BER efficiency and genome integrity, indicating that XRCC1 is a potential therapeutic target for stabilizing genomes in aged ADSCs.     

An in vitro study of Ocimum sanctum as a chemotherapeutic agent on oral cancer cell-line

Oral squamous cell carcinoma (OSCC) is the most commom cancer in the world. If remain untreated for several years, it may be fatal. Hence, it is important to prevent and treat OSCC at an early stage. In this study the effect of aqueous and dry leaves extract of Ocimum sanctum was observed on Ca9-22 cell line, which is an OSCC cell line. For this, Ca9-22 cell line was cultured and maintained. After 24 h, the cells were treated with aqueous and dry leaves extract of Ocimum sanctum plant. Viability of the cancerous cells were studied by 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and neutral red uptake (NRU) assay. Minimum inhibitory concentrations (MIC), lethal concentration₂₅ (LC₂₅), lethal concentration₅₀ (LC₅₀) and highest permissive concentration (HPC) was calculated by probit computational method. Experimentally, the MIC value was 5 mg/L, whereas the HPC was 30 mg/L of the plant extract in aqueous state. For the dry extract the MIC was 5 mg/L whereas the HPC was 35 mg/L for both MTT and NRU assays. For MTT assay LC values: 7.41 (LC₂₅), 14.79 (LC₅₀) and 26.91 mg/L (LC₇₅) for aqueous extract and 12.58 (LC₂₅), 20.89 (LC₅₀), 29.51 mg/L (LC₇₅) for dry extract. For NRU assay LC values were 10.23 (LC₂₅), 14.79 (LC₅₀) and 20.89 mg/L (LC₇₅) aqueous extract, and 16.59 (LC₂₅), 23.44 (LC₅₀), 30.19 mg/L (LC₇₅) dry extract of the plant. From the above study it was concluded that, Ocimum sanctum have anti-cancerous activity. It can further be used for therapeutic purposes.     

Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro

Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase β in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

The tumor suppressor Lgl1 regulates front-rear polarity of migrating cells

Cell migration is a highly integrated, multistep process that plays an important role in physiological and pathological processes. The migrating cell is highly polarized, with complex regulatory pathways that integrate its component processes spatially and temporally.¹ Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back. Science 2003; 302:1704-9; PMID:14657486; http://dx.doi.org/10.1126/science.1092053[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] The Drosophila tumor suppressor, Lethal (2) giant larvae (Lgl), regulates apical-basal polarity in epithelia and asymmetric cell division.² Etienne-Manneville S. Polarity proteins in migration and invasion. Oncogene 2008; 27:6970-80; PMID:19029938; http://dx.doi.org/10.1038/onc.2008.347[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] But little is known about the role of Lgl in establishing cell polarity in migrating cells. Recently, we showed that the mammalian Lgl1 interacts directly with non-muscle myosin IIA (NMIIA), inhibiting its ability to assemble into filaments in vitro.³ Dahan I, Yearim A, Touboul Y, Ravid S. The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration. Mol Biol Cell 2012; 23:591-601; PMID:22219375; http://dx.doi.org/10.1091/mbc.E11-01-0015[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Lgl1 also regulates the cellular localization of NMIIA, the maturation of focal adhesions, and cell migration.³ Dahan I, Yearim A, Touboul Y, Ravid S. The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration. Mol Biol Cell 2012; 23:591-601; PMID:22219375; http://dx.doi.org/10.1091/mbc.E11-01-0015[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] We further showed that phosphorylation of Lgl1 by aPKCζ prevents its interaction with NMIIA and is important for Lgl1 and acto-NMII cytoskeleton cellular organization.⁴ Dahan I, Petrov D, Cohen-Kfir E, Ravid S. The tumor suppressor Lgl1 forms discrete complexes with NMII-A and Par6α-aPKCζ that are affected by Lgl1 phosphorylation. J Cell Sci 2014; 127:295-304; PMID:24213535; http://dx.doi.org/10.1242/jcs.127357[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Lgl is a critical downstream target of the Par6-aPKC cell polarity complex; we showed that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6-aPKCζ in different cellular compartments.⁴ Dahan I, Petrov D, Cohen-Kfir E, Ravid S. The tumor suppressor Lgl1 forms discrete complexes with NMII-A and Par6α-aPKCζ that are affected by Lgl1 phosphorylation. J Cell Sci 2014; 127:295-304; PMID:24213535; http://dx.doi.org/10.1242/jcs.127357[Crossref], [PubMed], [Web of Science ®] , [Google Scholar] We further showed that aPKCζ and NMIIA compete to bind directly to Lgl1 through the same domain. These data provide new insights into the role of Lgl1, NMIIA, and Par6-aPKCζ in establishing front-rear polarity in migrating cells. In this commentary, I discuss the role of Lgl1 in the regulation of the acto-NMII cytoskeleton and its regulation by the Par6-aPKCζ polarity complex, and how Lgl1 activity may contribute to the establishment of front-rear polarity in migrating cells.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Association between the XRCC3 T241M polymorphism and risk of cancer: Evidence from 157 case–control studies

The T241M polymorphism in the X-ray cross-complementing group 3 (XRCC3) had been implicated in cancer susceptibility. The previous published data on the association between XRCC3 T241M polymorphism and cancer risk remained controversial. Hence, we performed a meta-analysis to investigate the association between cancer susceptibility and XRCC3 T241M (61,861 cases and 84,584 controls from 157 studies) polymorphism in different inheritance models. We used odds ratios with 95% confidence intervals to assess the strength of the association. Overall, significantly increased cancer risk was observed in any genetic model (dominant model: odds ration [OR] = 1.07, 95% confidence interval [CI] = 1.00–1.13; recessive model: OR = 1.15, 95% CI = 1.08–1.23; additive model: OR = 1.17, 95% CI = 1.08–1.28) when all eligible studies were pooled into the meta-analysis. In further stratified and sensitivity analyses, the elevated risk remained for subgroups of bladder cancer and breast cancer, especially in Caucasians. In addition, significantly decreased lung cancer risk was also observed. In summary, this meta-analysis suggests the participation of XRCC3 T241M in the susceptibility for bladder cancer and breast cancer, especially in Caucasians, and XRCC3 T241M polymorphism is associated with decreased lung cancer risk. Moreover, our work also points out the importance of new studies for T241M association in some cancer types, such as gastric cancer, colorectal cancer, and melanoma skin cancer, where at least some of the covariates responsible for heterogeneity could be controlled, to obtain a more conclusive understanding about the function of the XRCC3 polymorphism in cancer development.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Tagging SNPs in the ERCC4 gene are associated with gastric cancer risk

ERCC4 plays an essential role in the nucleotide excision repair (NER) pathway, which is involved in the removal of a wide variety of DNA lesions. To determine whether the ERCC4 tagging SNPs (tSNPs) are associated with risk of gastric cancer, we conducted a hospital-based case-control study of 350 cases and 468 cancer-free controls. In the logistic regression (LR) analysis, we found a significantly decreased risk of gastric cancer associated with the rs744154 GC/CC genotypes [adjusted odds ratio (OR) = 0.56, 95% confidence interval (CI) = 0.42–0.75, false discovery rate (FDR) P = 0.003] compared with the wild-type GG genotype. Haplotype-based association study revealed that the CGC haplotype that containing the rs744154 C allele can decrease the risk of gastric cancer compared with the most common haplotype GGT (adjusted OR = 0.61, 95% CI = 0.46–0.81). Using the multifactor dimensionality reduction (MDR) analysis, we identified that the SNP rs744154 and smoking status were the best two predictive factors for gastric cancer with a testing accuracy of 55.76% and a perfect cross-validation consistency (CVC) of 10 (P = 0.001). Furthermore, the smokers with the rs744154 GC/CC genotypes showed a decreased risk of gastric cancer (adjusted OR = 0.55, 95% CI = 0.35–0.85) compared with the smokers with the GG genotype using multivariate LR analysis. The above findings consistently suggested that genetic variants in the ERCC4 gene may play a protective role in the etiology of gastric cancer, even in the smokers.