Increased DNA Synthesis Research Articles

Effects of selection for litter size variability on ovarian folliculogenesis, ovarian cell proliferation, apoptosis, and production of regulatory peptides in rabbits

The present study aimed to identify the novel mechanisms regulating rabbit fecundity. For this purpose, the association between litter size variability, fecundity, ovarian morphology, and markers of proliferation, apoptosis, steroidogenesis, and the presence of regulatory proteins were examined in ovarian cells from two rabbit lines divergently selected for low (LL) and high (HL) variability in litter size throughout sixteen generations. Ovaries and uteri were isolated from multiparous non-lactating female rabbits from each line at sixteen generations of selection. One part of the ovary was subjected to histomorphometric analysis of folliculogenesis. From the rest of ovary, ovarian granulosa cells (OGCs) were isolated, cultured, and cell viability, proliferation (accumulation of PCNA and of cyclin B1, and BrdU-positive cells), and apoptosis (accumulation of caspase 3, bax and DNA fragmentation) were evaluated by the Trypan blue exclusion test and BrdU, quantitative immunocytochemistry, and cell death detection assays. Furthermore, OGCs were subjected to proteomic analysis by using the nano HPLC-Chip-MS/MS method. The release of progesterone and oestradiol was measured by ELISA. The LL had more than one kit per litter than the HL (7.6 kits vs. 6.5 kits, p ≤ 0.05). No differences were found in the diameter of primordial and primary ovarian follicles, theca, and granulosa thickness, but the diameter of oocytes in the primary and secondary follicles was higher in the LL than in the HL (88.49 µm in the LL vs. 77.86 µm in the HL for oocytes of primary follicles, p ≤ 0.05; 122.10 µm in the LL vs. 109.87 µm in the HL for oocytes of secondary follicles, p ≤ 0.05). Preovulatory follicles were presented only in the ovaries of the LL. The LL had higher incorporation of BrdU and reduced accumulation of bax within OGCs (0.92% in the LL vs. 0.44% in the HL for incorporation of BrdU, p ≤ 0.05; 41% in the LL vs. 48% in the HL for accumulation of bax, p ≤ 0.05). Ovarian fragments from the LL produced less progesterone and oestradiol than those of the HL (12 ng/mg tissue/day in the LL vs, 45 ng/mg tissue/day in the HL for progesterone, p ≤ 0.05; 11 ng/mg tissue/day in the LL vs. 30 ng/mg tissue/day in the HL for oestradiol, p ≤ 0.05). Besides, the OGCs from the LL produced a higher number of specific regulatory proteins involved in cell differentiation, proliferation, and adhesion than the HL (50 vs 38, p ≤ 0.05). In conclusion, higher prolificacy in the LL line would be caused by: (1) the selection of growing primordial ovarian follicles; (2) better transformation to preovulatory follicles; (3) increased cytoplasmic maturation of oocytes; (4) increased DNA synthesis and decreased cytoplasmic apoptosis in OGCs; (5) changes in ovarian steroidogenesis; and (6) changes in the number of peptides involved in cell differentiation, proliferation, and adhesion. HIGHLIGHTS Assessing fecundity in two divergently rabbit lines for variability in litter size. Fecundity and its variability are related to the diameter and quality of oocytes. Selection and better growth of preovulatory follicles have been related to fecundity.

Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging, we aimed to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question DIPG24 cells were cultured with pluripotent stem cell-derived cortical organoids. We created “mosaic” co-cultures enriched for tumour cell-neuronal cell interactions versus “assembloid” co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This suggested changes in cell interaction with the external environment reflected by decreased gene ontology terms associated with adhesion and extracellular matrix, and increased DNA synthesis and replication, in DIPG24 cells under either co-culture condition. By contrast, the mosaic co-culture was associated with neuron-specific brahma-associated factor (nBAF) complex signalling, a process associated with neuronal maturation. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Acetaminophen overdose-induced acute liver injury can be alleviated by static magnetic field.

Acetaminophen (APAP), the most frequently used mild analgesic and antipyretic drug worldwide, is implicated in causing 46% of all acute liver failures in the USA and between 40% and 70% in Europe. The predominant pharmacological intervention approved for mitigating such overdose is the antioxidant N-acetylcysteine (NAC); however, its efficacy is limited in cases of advanced liver injury or when administered at a late stage. In the current study, we discovered that treatment with a moderate intensity static magnetic field (SMF) notably reduced the mortality rate in mice subjected to high-dose APAP from 40% to 0%, proving effective at both the initial liver injury stage and the subsequent recovery stage. During the early phase of liver injury, SMF markedly reduced APAP-induced oxidative stress, free radicals, and liver damage, resulting in a reduction in multiple oxidative stress markers and an increase in the antioxidant glutathione (GSH). During the later stage of liver recovery, application of vertically downward SMF increased DNA synthesis and hepatocyte proliferation. Moreover, the combination of NAC and SMF significantly mitigated liver damage induced by high-dose APAP and increased liver recovery, even 24 h post overdose, when the effectiveness of NAC alone substantially declines. Overall, this study provides a non-invasive non-pharmaceutical tool that offers dual benefits in the injury and repair stages following APAP overdose. Of note, this tool can work as an alternative to or in combination with NAC to prevent or minimize liver damage induced by APAP, and potentially other toxic overdoses.

Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro.

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro.

High-content analysis of testicular toxicity of BPA and its selected analogs in mouse spermatogonial, Sertoli cells, and Leydig cells revealed BPAF induced unique multinucleation phenotype associated with the increased DNA synthesis

Bisphenol A is an endocrine disruptor that has been shown to have testicular toxicity in animal models. Its structural analog, including bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) have been introduced to the market as BPA alternatives. Previously, we developed high-content analysis (HCA) assays and applied machine learning to compare the testicular toxicity of BPA and its analogs in spermatogonial cells and testicular cell co-culture models. There are diverse cell populations in the testis to support spermatogenesis, but their cell type-specific toxicities are still not clear. The purpose of this study is to examine the selective toxicity of BPA, BPS), BPAF, and TBBPA on these testicular cells, including Sertoli cells, Leydig cells, and spermatogonia cells. We developed a high-content image-based single-cell analysis and measured a broad spectrum of adverse endpoints related to the development of reproductive toxicology, including cell number, nuclear morphology, DNA synthesis, cell cycle progression, early DNA damage response, cytoskeleton structure, DNA methylation status, and autophagy. We introduced an HCA index and spectrum to reveal multiple HCA parameters and observed distinct toxicity profiling of BPA and its analogs among three testicular types. The HCA spectrum shows the dynamic, chemical-specific, dose-dependent changes of each HCA parameter. Each chemical displayed a unique dose-dependent profile within each type of cell. All three types of cells showed the highest response to BPAF at 10 μM across all endpoints measured. BPAF targeted spermatogonial cell (C18) more significantly at 5 μM. BPS more likely targeted Sertoli cell (TM4) and Leydig cell (TM3) and less at spermatogonia cells. TBBPA targeted spermatogonia, Sertoli cells, and less at TM3 cells. BPA is mainly targeted at TM4, followed by TM3 cells, and less at spermatogonial cells. Most importantly, we observed that BPAF induced a dose-dependent increase in spermatogonia cells, not in Sertoli and Leydig cells. In summary, our current HCA assays revealed the cell-type-specific toxicities of BPA and its analogs in different testicular cells. Multinucleation induced by BPAF, along with increased DNA damage and synthesis at low doses, could possibly have a profound long-term effect on reproductive systems.

Bioactive Oligopeptides from Ginseng (Panax ginseng Meyer) Suppress Oxidative Stress-Induced Senescence in Fibroblasts via NAD+/SIRT1/PGC-1α Signaling Pathway.

The physicochemical properties and multiple bioactive effects of ginseng oligopeptides (GOPs), plant-derived small molecule bioactive peptides, suggest a positive influence on health span and longevity. Given this, cellular senescence is the initiating factor and key mechanism of aging in the organism, and thus the current study sought to explore the effects of GOPs on H2O2-induced cellular senescence and its potential mechanisms. Senescence was induced in mouse embryonic fibroblasts NIH/3T3 by 4 h of exposure to 200 µM H2O2 and confirmed using CCK-8 assay and Western blot analyses of p16INK4A and p21Waf1/Cip1 after 24 h of growth medium administration with or without GOPs supplementation (25, 50, and 100 µg/mL). We found that GOPs delayed oxidative stress-induced NIH/3T3 senescence by inhibiting the G1 phase arrest, increasing DNA synthesis in the S phase, decreasing the relative protein expression of p16INK4A and p21Waf1/Cip1, promoting cell viability, protecting DNA, and enhancing telomerase (TE) activity. Further investigation revealed that the increase in antioxidative capacity and anti-inflammation capacity might form the basis for the retarding of the senescence effects of GOPs. Furthermore, GOPs supplementation significantly improved mitochondrial function and mitochondrial biogenesis via the NAD+/SIRT1/PGC-1𝛼 pathway. These findings indicate that GOPs may have a positive effect on health span and lifespan extension via combating cellular senescence, oxidative stress, and inflammation, as well as modulating longevity regulating pathway NAD+/SIRT1/PGC-1𝛼.

Characterization of resistant MCF-7 breast cancer cells developed by repeated cycles of photodynamic therapy.

Breast cancer mainly affects women and causes a severe global threat to health. It is often managed and treated with surgery, chemotherapy, immunotherapy, and radiation therapy. Generally, chemotherapy as a treatment option is often opposed by responsive tumor relapse and development of resistance, a significant setback of current treatment. Photodynamic therapy (PDT) offers a promising modality that can treat cancer by combining a photosensitizer and laser irradiation in the presence of oxygen. However, one problem of PDT in treating breast cancer is the apparition of the resistant cell population. Thus, we aimed for stepwise selection and characterization of MCF-7 cells resistant to PDT with a sulfonated zinc phthalocyanine (ZnPcS4) photosensitizer. The wild-type MCF-7 was exposed to successive cycles of ZnPcS4-PDT, and 10resistant populations were finally obtained. In wild-type and parental cells, we analyzed the cell morphology (light microscopy), cell cycle (BrdU staining), cell viability (MTT assay), antioxidant activity (superoxide dismutase measurement), and immunofluorescence expression of resistant p-glycoprotein (P-gp). The results indicate that resistant cells showed a mesenchymal cell phenotype, few differences in antioxidant activity, an increased DNA synthesis, and more expression of P-gp than the wild-type parental cells. These distinctive features of resistant cells can provide insight into the emergence of MCF-7 cell resistance to PDT, which was necessary to design the best therapeutic procedure for improved efficacy.

Rat prostate tumors induce DNA synthesis in remote organs

Advanced cancers induce systemic responses. However, if such systemic changes occur already when aggressive tumors are small, have not been thoroughly characterized. Here, we examined how localized prostate cancers of different sizes and metastatic potential affected DNA synthesis in the rest of the prostate and in various remote organs. Non-metastatic Dunning R-3327 G (G) tumor cells, metastatic MatLyLu (MLL) tumor cells, or vehicle were injected into the prostate of immunocompetent rats. All animals received daily injections of Bromodeoxyuridine (BrdU), to label cells/daughter cells with active DNA synthesis. Equal sized G- and MLL-tumors, similarly increased BrdU-labeling in the prostate, lymph nodes and liver compared to tumor-free controls. Prior to metastasis, MLL-tumors also increased BrdU-labeling in bone marrow and lungs compared to animals with G-tumors or controls. In animals with MLL-tumors, BrdU-labeling in prostate, lungs, brown adipose tissue and skeletal muscles increased in a tumor-size-dependent way. Furthermore, MLL-tumors induced increased signs of DNA damage (γH2AX staining) and accumulation of CD68 + macrophages in the lungs. In conclusion, small localized prostate cancers increased DNA synthesis in several remote tissues in a tumor type- and size-dependent way. This may suggest the possibility for early diagnosis of aggressive prostate cancer by examining tumor-induced effects in other tissues.

Pleiotrophin Expression and Actions in Pancreatic β-Cells.

Pleiotrophin (PTN) is a heparin-binding cytokine that is widely expressed during early development and increases in maternal circulation during pregnancy.Aged PTN-deficient mice exhibit insulin resistance, suggesting a role in metabolic control. The objectives of this study were to determine if PTN is expressed in mouse pancreatic β-cells in young vs. adult animals, and its effects on DNA synthesis, β-cell gene expression and glucose-stimulated insulin secretion (GSIS). The Ptn gene was expressed in isolated fractions of young mouse β-cells, especially within immature β-cells with low glucose transporter 2 expression. Expression was retained in the adult pancreas but did not significantly change during pregnancy. PTN and its receptor, phosphotyrosine phosphatase-β/ζ, were also expressed in the proliferative INS1E β-cell line. Fluorescence immunohistochemistry showed that PTN peptide was present in islets of Langerhans in adult mice, associated predominantly with β-cells. The percentage of β-cells staining for PTN did not alter during mouse pregnancy, but intense staining was seen during β-cell regeneration in young mice following depletion of β-cells with streptozotocin. Incubation of INS1E cells with PTN resulted in an increased DNA synthesis as measured by Ki67 localization and increased expression of Pdx1 and insulin. However, both DNA synthesis and GSIS were not altered by PTN in isolated adult mouse islets. The findings show that Ptn is expressed in mouse β-cells in young and adult life and could potentially contribute to adaptive increases in β-cell mass during early life or pregnancy.

H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma.To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM.We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM.To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c‐Myc using 10058‐F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM.Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. DisclosuresNo relevant conflicts of interest to declare.

Abstract P325: The Role Of Runx1 In Cardiomyocyte Cell Cycle And Ploidy

Adult mammalian cardiomyocytes (CMs) are thought to be post-mitotic and therefore unable to regenerate the myocardium after injury. In recent years various studies have shown that the adult mammalian CM is capable of a small amount of proliferation, potentially restricted to a subset of CMs. One such study demonstrated that having greater percentages of the rare mononuclear diploid cardiomyocyte (MNDCM) is associated with improved outcomes after myocardial infarction (MI). An accompanying genome-wide association analysis identified genetic loci associated with the frequency of the MNDCM population. One candidate to come out of this screen was Runx1. Concurrently, RUNX1 captured the attention of cardiac regeneration researchers due to its increased presence in disease states, with some suggesting it may be a marker for dedifferentiation (fetal gene induction). One recent study demonstrated improved calcium handling and decreased eccentric hypertrophy following RUNX1 ablation after injury, perhaps corroborating the idea that RUNX1 is involved with CM dedifferentiation. We hypothesize that Runx1 influences dedifferentiation in CMs, impacting ploidy, as well as CM cell cycle activity and post-MI outcomes. We found that CM-specific overexpression (OE) of Runx1 results in a doubling of the MNDCM population, thereby validating its influence on the population. Via multiple contexts including postnatal development and adult injury, knocking out Runx1 decreases DNA synthesis while Runx1 OE increases DNA synthesis. Furthermore, an initial analysis of RNAseq data demonstrates that RUNX1 OE in a neonatal mouse hearts demonstrated differential expression in genes related to cardiac conduction, contraction, heart development, regeneration, and regulation of cell differentiation . After MI in the adult mouse heart, the effects of Runx1 OE resulted in transient benefits which included increased cell cycle activity and preservation of function. These data suggest that Runx1 is not simply a marker of CM dedifferentiation, but also a regulator of the process including cell cycle activation. Ongoing work will tease apart this role in more detail and could establish RUNX1 as a prominent therapeutic target for mitigating effects of cardiac injury.

Epstein-Barr virus noncoding small RNA (EBER1) induces cell proliferation by up-regulating cellular mitochondrial activity and calcium influx

Epstein-Barr virus encoded RNAs (EBER1 and EBER2) are two non-polyadenylated, non-protein coding small RNAs expressed at high levels in all forms of EBV latent infections. Although not directly involved in cell transformation, a number of studies have reported that these RNAs may be involved in cell proliferation. However, which of the two EBERs play a major role in this process and the mechanisms involved remains unknown. The aim of this study was to investigate the role and mechanism of EBER1-induced cell proliferation. Using stably transfected EBER1 cell lines, and multiple methodologies, we show that EBER1 transfected epithelial, B and T cell lines proliferate at a higher rate, have higher metabolic activity and increased DNA synthesis. The mitochondrial number and activity was also observed to be higher in the EBER1 transfected cells. Moreover, cytochrome c activity and store operated calcium entry (SOCE) were potentiated in the EBER1 expressing cells. Finally, the genes associated with cell proliferation were also observed to be up-regulated in the EBER1 transfected cells. Taken together, our data has unravelled the role of mitochondria and cellular calcium pathway that appear to be involved in EBER1 induced cell proliferation of EBV infected cells.

121-OR: Activation of FoxO1 Induces Akt Phosphorylation and Mimics the Metabolic Functions of Insulin without Proliferative Effects on 3T3-L1 Adipocytes

Objective: Insulin promotes glucose uptake, lipogenesis, cell proliferation and cell growth mainly via Akt phosphorylation. FoxO1 is a substrate of Akt, which is inactivated when phosphorylated by Akt. Via this mechanism, FoxO1 antagonizes insulin. On the other hand, constitutive activation of FoxO1 reportedly enhances Akt phosphorylation. Thus, FoxO1 activation, via Akt phosphorylation, may mimic the functions of insulin. Methods: We overexpressed constitutively-active FoxO1 (CA-FoxO1) and FoxO1Δ256, a dominant-negative mutant, in 3T3-L1 adipocytes by adenoviruses and investigated the effects of these proteins on insulin’s functions. Results: CA-FoxO1 enhanced Akt phosphorylation, glucose uptake (275%, P&amp;lt;0.01), GLUT4 translocation and lipogenesis (176%, P&amp;lt;0.05) in the non-insulin stimulated state, while DNA synthesis was decreased (44%, P&amp;lt;0.01) in the insulin-stimulated state. Surprisingly, FoxO1Δ256 also enhanced Akt phosphorylation, glucose uptake (154%, P&amp;lt;0.05) and lipogenesis (268%, P&amp;lt;0.01) in the basal state. In contrast to CA-FoxO1, FoxO1Δ256 increased DNA synthesis (196%, P&amp;lt;0.01). FoxO1Δ256 did not exert a dominant negative action even on gene expressions related to adipocyte differentiation, de novo lipogenesis and cell cycle regulation. Akt phosphorylation was more strongly induced by CA-FoxO1 than by 3 nM insulin, while PI 3-kinase activation by FoxO1 was weaker than that induced by 3 nM insulin, indicating the mechanism of Akt phosphorylation by FoxO1 to mainly be downstream from PI 3-kinase. Expressions of IR-β, p110α, Rictor and TRB3 were not significantly affected. Conclusion: Activation of FoxO1 selectively mimics the metabolic functions of insulin, without its proliferative effects. Contrary to previously reported results, FoxO1Δ256 does not always exert a dominant negative action. Disclosure T. Ohno: None. H. Ono: None. K. Takeda: None. K. Yokote: None.

Role of organophosphorous pesticides and acetylcholine in breast carcinogenesis.

Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carcinogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas. The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c-myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.

GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage.

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-β such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.

Natriuretic peptides and Forkhead O transcription factors act in a cooperative manner to promote cardiomyocyte cell cycle re-entry in the postnatal mouse heart

BackgroundCardiomyocytes proliferate rapidly during fetal life but lose their ability of proliferation soon after birth. However, before terminal withdrawal from the cell cycle, cardiomyocytes undergo another round of cell cycle during early postnatal life in mice. While a transient wave of increased DNA synthesis in cardiomyocyte has been observed in postnatal mouse hearts, the molecular mechanisms describing cardiomyocyte cell cycle re-entry remain poorly understood. Atrial and B-type natriuretic peptides (ANP and BNP) are abundantly expressed in embryonic heart ventricles. After birth, the expression of both genes is strongly reduced in the ventricular myocardium. Forkhead O (FOXO) transcription factors are expressed in both embryonic and postnatal heart ventricles. Their transcriptional activity negatively affects cardiomyocyte proliferation. Upon phosphorylation, FOXO is translocated to the cytoplasm and is transcriptionally inactive. Despite these important findings, it remains largely unknown whether natriuretic peptides and FOXO cooperatively play a role in regulating cardiomyocyte cell cycle activity during early postnatal life.ResultsWe observed that the expression of ANP and BNP and the level of phosphorylated FOXO were transiently increased in the postnatal mouse heart ventricles, which coincided with the burst of cardiomyocyte cell cycle re-entry during early postnatal life in mice. Cell culture studies showed that ANP/BNP signaling and FOXO cooperatively promoted cell cycle activity in neonatal mouse cardiomyocytes. The enhanced cell cycle activity observed in combined treatment of ANP/BNP and dominant-negative FOXO (DN-FOXO), which can bind FOXO recognition sites on DNA but cannot activate transcription, was primarily mediated through natriuretic peptide receptor 3 (Npr3). In mice, simultaneous application of ANP and DN-FOXO in postnatal hearts reactivated cell cycle in cardiomyocytes, resulting in reduced scar formation after experimental myocardial infarction.ConclusionsOur data demonstrate the cooperative effects of natriuretic peptide and DN-FOXO on promoting cardiomyocyte cell cycle activity and mouse cardiac repair and regeneration after injury.

THAP11 Functions as a Tumor Suppressor in Gastric Cancer through Regulating c-Myc Signaling Pathways

We aim to investigate the role of THAP11 (thanatos-associated protein11) in gastric cancer and its regulation mechanisms. THAP11 expression was analyzed in 51 pairs of GC tissues and the corresponding paracancerous tissues by qRT-PCR and Western blot. After THAP11 was overexpressed or knocked-down, cell proliferation, cell cycle, and apoptosis were detected in MKN-45 cells. We found that THAP11 was significantly downregulated in GC tissues and GC cell lines. Functionally, THAP11 overexpression markedly inhibited cell growth, induced G1/G0 cell-cycle arrest, and promoted cell apoptosis of MKN-45 cells, while silencing of THAP11 led to increased cell growth, increased DNA synthesis, and inhibited apoptosis. In addition, THAP11 negatively regulated the expression of c-Myc, decreased cyclinD1 protein, and increased p27 and p21 protein levels. We also found cell growth suppression induced by THAP11 was rescued by c-Myc overexpression, further confirming that THAP11 suppresses gastric cancer cell growth via the c-Myc pathway. THAP11 acts as a cell growth suppressor and exerts its role possibly through negatively regulating c-Myc pathway in gastric cancer.

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication.

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

355-OR: Effects of a Six-Week Intervention with Glucagon-Like Peptide-1 Analogue on Pancreatic Volume, Edema, and DNA Synthesis in Obese Men

Plasma concentrations of the two pancreatic enzymes, amylase and lipase, are increased within the normal physiological range after initiation of glucagon-like peptide-1 receptor agonist (GLP-1RA) treatment. An association between the use of GLR-1RA and incidence of acute pancreatitis or pancreatic cancer has not been found - neither in rodent studies nor in preclinical studies or in the large cardiovascular outcome trials with GLP-1RAs. The increased concentrations of pancreatic enzymes have been suggested to be explained by increased DNA or protein synthesis found in rodent and acinar cell studies. However, whether this translates into humans is unknown. We therefore investigated the effect of liraglutide, a GLP-1RA, on pancreatic volume, edema and DNA synthesis reflected by 18F-flourothymidine (FLT)-uptake using state-of-the-art positron emission tomography (PET)-magnetic resonance imaging (MRI) before initiation, after four weeks during titration of liraglutide and after six weeks during steady state on maximum dose of liraglutide 3.0 mg in 14 obese men (age 38 ± 3 years, BMI 32 ± 1 kg/m2) without diabetes. Plasma concentrations of amylase and lipase were assessed in parallel. Amylase (+7 U/L [95% confidence intervals, 3 - 11] p&amp;lt;0.01) and lipase (+19 U/L [7-30] p&amp;lt;0.01) increased during liraglutide treatment while body weight decreased (-4.5 kg [-5,9 - -3,1] p&amp;lt;0.01). Pancreatic volume did not change from baseline to steady state of treatment (+2.1 cm3 [-10 - 14] p=0.72]) and no signs of change in cellular infiltration of the pancreas were found (p=0.22). During titration of liraglutide, FLT-uptake increased (+0.09 g/mL [0.000 - 0.17], p=0.05). In conclusion, six weeks of treatment with liraglutide did not affect pancreatic volume, edema or cellularity in obese individuals. Increased DNA synthesis reflected by FLT-uptake in the pancreas seen after four weeks of liraglutide treatment may be responsible for the increase in pancreatic enzymes. Disclosure M.S. Svane: None. H.H. Johannesen: None. C. Martinussen: None. K.N. Bojsen-Moller: None. M.L. Hansen: None. A.E. Hansen: None. C.F. Deacon: Employee; Spouse/Partner; Merck &amp; Co., Inc. Stock/Shareholder; Spouse/Partner; Merck &amp; Co., Inc. Other Relationship; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck &amp; Co., Inc., Novartis AG, Novo Nordisk A/S. B. Hartmann: None. S.H. Keller: None. T.L. Klausen: None. A. Loft: None. A. Kjaer: None. S. Madsbad: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; Boehringer Ingelheim International GmbH, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk A/S. J.O. Löfgren: None. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp &amp; Dohme Corp.

Premature satellite cell activation before injury accelerates myogenesis and disrupts neuromuscular junction maturation in regenerating muscle.

Satellite cell (SC) activation, mediated by nitric oxide (NO), is essential to myogenic repair, whereas myotube function requires innervation. Semaphorin (Sema) 3A, a neuro-chemorepellent, is thought to regulate axon guidance to neuromuscular junctions (NMJs) during myotube differentiation. We tested whether "premature" SC activation (SC activation before injury) by a NO donor (isosorbide dinitrate) would disrupt early myogenesis and/or NMJs. Adult muscle was examined during regeneration in two models of injury: myotoxic cardiotoxin (CTX) and traumatic crush (CR) (n = 4-5/group). Premature SC activation was confirmed by increased DNA synthesis by SCs immediately in pretreated mice after CTX injury. Myotubes grew faster after CTX than after CR; growth was accelerated by pretreatment. NMJ maturation, classified by silver histochemistry (neurites) and acetylcholinesterase (AchE), and α-bungarotoxin staining (Ach receptors, AchRs) were delayed by pretreatment, consistent with a day 6 rise in the denervation marker γ-AchR. With pretreatment, S100B from terminal Schwann cells (TSCs) increased 10- to 20-fold at days 0 and 10 after CTX and doubled 6 days after CR. Premature SC activation disrupted motoneuritogenesis 8-10 days post-CTX, as pretreatment reduced colocalization of pre- and postsynaptic NMJ features and increased Sema3A-65. Premature SC activation before injury both accelerated myogenic repair and disrupted NMJ remodeling and maturation, possibly by reducing Sema3A neuro-repulsion and altering S100B. This interpretation extends the model of Sema3A-mediated motoneuritogenesis during muscle regeneration. Manipulating the timing and type of Sema3A by brief NO effects on SCs suggests an important role for TSCs and Sema3A-65 processing in axon guidance and NMJ restoration during muscle repair.