Level Of DNA Synthesis Research Articles

GPx3 knockdown inhibits the proliferation and DNA synthesis and enhances the early apoptosis of human spermatogonial stem cells via mediating CXCL10 and cyclin B1.

Spermatogenesis is regulated by genetic and epigenetic factors. However, the genes and signaling pathways mediating human spermatogenesis remain largely unknown. Here, we have for the first time explored the expression, function, and mechanism of glutathione peroxidase 3 (GPx3) in controlling the proliferation and apoptosis of human spermatogonial stem cells (SSCs). We found that GPx3 was expressed in human SSCs. Notably, we revealed that GPx3 knockdown resulted in the decrease in the proliferation, DNA synthesis, and cyclin B1 level in human SSC lines, which possessed the phenotypic features of human primary SSCs. Flow cytometry and TUNEL assays showed that GPx3 silencing led to enhancement of early apoptosis of human SSC line. RNA sequencing was utilized to identify CXCL10 as a target of GPx3 in human SSCs, and notably, both double immunostaining and co-immunoprecipitation (co-IP) demonstrated that there was an association between GPx3 and CXCL10 in these cells. CXCL10-shRNA resulted in the reduction in the proliferation and DNA synthesis of human SSC line and an increase in apoptosis of these cells. Taken together, these results implicate that GPx3 regulates the proliferation, DNA synthesis, and early apoptosis of human SSC line via mediating CXCL10 and cyclin B1. This study, thus, offers a novel insight into the molecular mechanism regulating the fate determinations of human SSCs and human spermatogenesis.

Down-regulation and Clinic-pathological Correlation of SIK-1 and SIK-1-LNC in Non-small Cell Lung Cancer Patients

Non-small-cell lung cancer (NSCLC) is currently the leading cause of mortality cancer. Introducing noninvasive approaches to diagnose NSCLC, especially at an early phase, might improve the disease's prognosis. Long noncoding RNAs (lncRNAs), which are important regulators of the expression genes inside the cells, have been linked to a range of biological processes, such as cancer progression and metastasis, including NSCLC. The present work aims to determine the potential involvement of SIK-1-LNC and SIK-1 in NSCLC pathogenesis and the possible use of these molecules as novel biomarkers or therapeutic targets. In this work, the expression levels of SIK-1-LNC and SIK-1 in 50 pairs of NSCLC tumor and tumor marginal tissues were evaluated. So, after total RNA extraction and complementary DNA synthesis, the SIK-1-LNC and SIK-1 expression levels were evaluated by real-time PCR. In the study groups, clinical and pathological characteristics of the NSCLC patients were also examined. Our findings showed that tumor samples had much lower levels of SIK-1 and SIK-1-LNC expression than tumor margin samples. SIK-1-LNC expression was correlated with SIK-1 levels in NSCLC samples. Interestingly, both stage and lymph node metastasis features of the tumor were associated significantly with SIK-1 and SIK-1-LNC expression levels. A ROC curve analysis indicated a biomarker index of 0.69 and 0.74 for SIK-1 and SIK-1-LNC, respectively. Collectively, our study emphasized the role of SIK-1-LNC and SIK-1 downregulation in NSCLC oncogenesis. Additionally, SIK-1 and SIK-1-LNC, particularly the latter, have shown remarkable potential to be utilized as new NSCLC biomarkers and therapeutic targets.

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication

Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1–38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.

MTHFR C677T and A1298C Polymorphisms in Breast Cancer, Gliomas and Gastric Cancer: A Review.

Folate (vitamin B9) is found in some water-soluble foods or as a synthetic form of folic acid and is involved in many essential biochemical processes. Dietary folate is converted into tetrahydrofolate, a vital methyl donor for most methylation reactions, including DNA methylation. 5,10-methylene tetrahydrofolate reductase (MTHFR) is a critical enzyme in the folate metabolism pathway that converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, which produces a methyl donor for the remethylation of homocysteine to methionine. MTHFR polymorphisms result in reduced enzyme activity and altered levels of DNA methylation and synthesis. MTHFR polymorphisms have been linked to increased risks of several pathologies, including cancer. Breast cancer, gliomas and gastric cancer are highly heterogeneous and aggressive diseases associated with high mortality rates. The impact of MTHFR polymorphisms on these tumors remains controversial in the literature. This review discusses the relationship between the MTHFR C677T and A1298C polymorphisms and the increased risk of breast cancer, gliomas, and gastric cancer. Additionally, we highlight the relevance of ethnic and dietary aspects of population-based studies and histological stratification of highly heterogeneous tumors. Finally, this review discusses these aspects as potential factors responsible for the controversial literature concerning MTHFR polymorphisms.

Relative DNA Methylation and Demethylation Efficiencies during Postnatal Liver Development Regulate Hepatitis B Virus Biosynthesis.

Hepatitis B virus (HBV) transcription and replication increase progressively throughout postnatal liver development with maximal viral biosynthesis occurring at around 4 weeks of age in the HBV transgenic mouse model of chronic infection. Increasing viral biosynthesis is associated with a corresponding progressive loss of DNA methylation. The loss of DNA methylation is associated with increasing levels of 5-hydroxymethylcytosine (5hmC) residues which correlate with increased liver-enriched pioneer transcription factor Forkhead box protein A (FoxA) RNA levels, a rapid decline in postnatal liver DNA methyltransferase (Dnmt) transcripts, and a very modest reduction in ten-eleven translocation (Tet) methylcytosine dioxygenase expression. These observations are consistent with the suggestion that the balance between active HBV DNA methylation and demethylation is regulated by FoxA recruitment of Tet in the presence of declining Dnmt activity. These changes lead to demethylation of the viral genome during hepatocyte maturation with associated increases in viral biosynthesis. Consequently, manipulation of the relative activities of these two counterbalancing processes might permit the specific silencing of HBV gene expression with the loss of viral biosynthesis and the resolution of chronic HBV infections.IMPORTANCE HBV biosynthesis begins at birth and increases during early postnatal liver development in the HBV transgenic mouse model of chronic infection. The levels of viral RNA and DNA synthesis correlate with pioneer transcription factor FoxA transcript plus Tet methylcytosine dioxygenase-generated 5hmC abundance but inversely with Dnmt transcript levels and HBV DNA methylation. Together, these findings suggest that HBV DNA methylation during neonatal liver development is actively modulated by the relative contributions of FoxA-recruited Tet-mediated DNA demethylation and Dnmt-mediated DNA methylation activities. This mode of gene regulation, mediated by the loss of DNA methylation at hepatocyte-specific viral and cellular promoters, likely contributes to hepatocyte maturation during liver development in addition to the postnatal activation of HBV transcription and replication.

Roles of Interleukin-1 Receptor Antagonist in Prostate Cancer Progression.

Background: Inflammation is known to promote tumor formation and progression; however, we found a natural anti-inflammatory factor, interleukin (IL)-1 receptor antagonist (IL1RN), in a mouse transgenic adenocarcinoma of the mouse prostate (TRAMP)-C1-derived tumor microenvironment (TME). We sought to characterize the functions of the IL1RN-secreting cells in the TME. Methods: We compared tumors collected from two syngeneic mouse models and isolated tumor-infiltrating leukocytes (TILs) with different cluster of differentiation 11b (CD11b) statuses. We examined the proliferation functions of the TILs and the IL1RN using several approaches, including a colony-formation assay and DNA synthesis levels. Results: We demonstrated that CD11b-deficient TILs (TILs/CD11b−) secreted the IL1RN and promoted proliferation by analyzing conditioned media. In addition to mouse TRAMP-C1, proliferation functions of the IL1RN were confirmed in several human castration-resistant prostate cancer (CRPC) cell lines and one normal epithelial cell line. The androgen-sensitive lymph node carcinoma of the prostate (LNCaP) cell line showed cytotoxic responses to IL1β treatment and androgen-dependent regulation of IL-1 receptor type 1 (IL1R1), while the C4-2 CRPC cell line did not. IL1RN rescued LNCaP cells from the cytotoxic effects of IL1β/IL1R1 signaling. Conclusions: Our results support TILs/CD11b− cells being able to protect androgen-dependent cells from inflammatory damage and promote the malignant progression of prostate cancers partly through the IL1RN in the TME.

Nitidine chloride induces S phase cell cycle arrest and mitochondria-dependent apoptosis in HaCaT cells and ameliorates skin lesions in psoriasis-like mouse models

Psoriasis is a common dermatosis causing considerable inconvenience to 4% of the general population. Traditional psoriasis treatments often cause side effects, drug resistance and complications, necessitating development of safer and more effective treatments. In this study, we screened over 600 natural compounds to identify viability inhibitors of human HaCaT keratinocytes cultured in vitro. The results showed that nitidine chloride was a highly effective inhibitor. Further studies revealed that nitidine chloride inhibited HaCaT proliferation and induced S phase cell cycle arrest; these effects were associated with reduced DNA synthesis, decreased Ki67, cyclin A, and cyclin D1 levels, and increased p53 protein expression. Nitidine chloride also significantly downregulated bcl-2 and upregulated bax, cleaved caspase-9 and cleaved caspase-3. Mechanistic studies revealed that nitidine chloride-induced apoptosis involved the c-Jun N-terminal kinase (JNK) pathway. More importantly, in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)- and imiquimod (IMQ)-induced epidermal hyperplasia and inflammation models, nitidine chloride inhibited topical edema in mouse ear and back skin, substantially reducing tissue thickness and weight. In some cases, nitidine chloride also ameliorated conditions caused by TPA and IMQ, such as angiogenesis and infiltration of large numbers of inflammatory cells around blood vessels. Additionally, nitidine chloride inhibited the expression of various proinflammatory cytokines in the two animal models. In conclusion, our results are the first to demonstrate that nitidine chloride inhibits the proliferation of HaCaT cells, induces apoptosis partly via the JNK signaling pathway in vitro and ameliorates skin lesions and inflammation in vivo, making it an appropriate candidate for psoriasis treatment.

Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391

pol VICE391 (RumAʹ2B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous “SOS-induced” mutagenesis than the related E. coli pol V (UmuDʹ2C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to increased levels of DNA synthesis compared to UmuC.We have generated a steric gate pol VICE391 variant (pol VICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol VICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome.Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol VICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol VICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.

Aedes aegypti antiviral adaptive response against DENV-2

Priming is the conceptual term defining memory phenomenon in innate immune response. Numerous examples of enhanced secondary immune response have been described in diverse taxa of invertebrates; which naturally lacks memory response. In mosquitoes, a previous non-lethal challenge with some specific pathogens modify their immune response against the same microorganism; developing an improved antimicrobial reaction. In this work, we explore the ability of Aedes aegypti to mount a higher antiviral response upon a second oral DENV challenge. When previously challenged with inactive virus, we observed that the posterior infection showed a diminished number of DENV infectious particles in midguts and carcasses. In challenged tissues, we detected higher de novo midgut DNA synthesis than control group, as determined by DNA incorporation of 5-bromo-2-deoxyuridine. We demonstrated that inactive DENV particle are capable to induce DNA synthesis levels comparable to infective DENV. We considered the Drosophila melanogaster hindsight and Delta-Notch mosquitoes orthologues as potential de novo DNA synthesis pathway components (as observed in fly oocyte development and midgut tissue renewal). We showed that Aedes aegypti hindsight transcript relative expression levels were higher than control during DENV infection and inactive DENV particle alimentation. Also, Aedes aegypti second challenge with active DENV induced higher hindsight, Delta and Notch transcriptions in the primed mosquitoes (compared with the primary infection levels). Considering that the mosquito de novo DNA synthesis is concomitant to viral particle reduction, this finding opens a new perspective on the mechanisms underlying the vector antiviral immune response and the effector molecules involved.

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans.

Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection.

Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy.

Assessment of the proliferation status of glioblastoma cell and tumour tissue after nanoplatinum treatment.

Glioblastoma is one of the most frequent primary brain tumours of the central nervous system, with a poor survival time. With inefficient chemotherapy, it is urgent to develop new strategies for tumour therapy. The present approach is based on the inhibition of cell proliferation using platinum nanoparticles (NP-Pt). The aim of the study was to evaluate and compare the antiproliferative properties of NP-Pt and cisplatin against U87 and U118 glioma cell lines and U87 tumour tissue. NP-Pt and cisplatin were incubated with U87 and U118 glioma cells or administered directly into glioma tumour tissue. Cell morphology, the level of DNA synthesis, the migration of cells, protein expression levels of proliferating cell nuclear antigen (PCNA) and the level of DNA oxidation in glioma tumours were investigated. The results showed that NP-Pt treatment of U87 and U118 glioma cells decreased the level of DNA synthesis and the migration of cancer cells but also downregulated the level of PCNA protein expression in tumour tissue. Furthermore, NP-Pt caused oxidative DNA damage in tumour tissue to a higher degree than cisplatin. Consequently, NP-Pt can be considered as an effective inhibitor of glioblastoma tumour cell proliferation. However, the mechanism of action and potential side effects need to be elucidated further.

Immune activation effects of Eurotium cristatum on T cells through NF-κB signaling pathways in humans

ABSTRACTThe immunization effect of Eurotium cristatum isolated from Fuzhuan brick-tea was evaluated. Ethyl acetate extraction (E2) had an increasing effect on T cell subset proportions. And then four compounds (physcion, echinulin, catenarin and emodin) were isolated from E. cristatum. The immunization effect of echinulin was observed through determining T cell subsets’ percentage, DNA synthesis and cytokine levels. It suggested that the immunization effect of compounds might work in relation to its structure. In addition, echinulin accelerated protein of p65, IκBα upregulation and nuclear translocation of p65 by the NF-κB signaling pathway. The result showed that the immunization effect of echinulin is closely related to the activation of NF-κB activity. Meanwhile echinulin increased transcript levels of p65 and IκBα in T cells at 200 μM (middle dosages group). It demonstrated that the immunization effection of echinulin on T cells is enhanced by activating NF-κB signaling pathway.

Ascending Aortic Constriction Promotes Cardiomyocyte Proliferation in Neonatal Rats.

Adult heart suffering from increased workload will undergo myocardial hypertrophy, subsequent cardiomyocyte (CM) death, and eventually heart failure. However, the effect of increasing afterload on the neonatal heart remains unknown. We performed ascending aortic constriction (AAC) in neonatal rats 8-12 hours after birth (P0, P indicates postpartum). Seven days after surgery, in vivo heart function was evaluated using cardiac ultrasonography. Haematoxylineosin and Masson staining were used to assess CM diameter and collagen deposition. Moreover, expression of both EdU and Ki67 were evaluated to determine DNA synthesis levels, and pH3 and aurora B as markers for mitosis in CMs. CM isolation was performed by heart perfusion at P0, P3, P5, and P7, respectively. CM number on P0 was 1.01 ± 0.29 × 106. We found that CM cell cycle activation was significantly increased among constricted hearts, as demonstrated by increased Ki67, EdU, pH3, and aurora B positive cells/1000 CMs. At day 7 (P7), constriction group hearts manifested increased wall thickness (0.55 ± 0.05 mm versus 0.85 ± 0.10 mm, P < 0.01, n = 6), and improved hemodynamics as well as left ventricular ejection fraction (65.5 ± 3.7% versus 77.7 ± 4.8%, P < 0.01, n = 6). Of note, the population of CMs was also markedly increased in the constriction group (2.92 ± 0.27 × 106 versus 3.41 ± 0.40 × 106, P < 0.05, n = 6). In summary, we found that during the first week after birth significant numbers of neonatal CMs can reenter the cell cycle. Ascending aortic constriction promotes neonatal rat CM proliferation resulting in 16.7% more CMs in the heart.

Chamber-specific differences in human cardiac fibroblast proliferation and responsiveness toward simvastatin.

Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. A chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear whether similar differences exist in human cardiac fibroblasts (HCFs) and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than threefold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with a higher sensitivity toward inhibition compared with ventricular fibroblasts. The DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 μM) that bypasses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Chamber-specific differences exist in the human heart because atrial fibroblasts have a higher proliferative capacity and are more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury.

178. Effect of Heme Oxygenase-1 Overexpression on DNA Synthesis of Adult Cardiac Progenitor Cells Under Hypoxia

Although the studies in animals and humans have shown that the beneficial effects of adult c-kit+ cardiac progenitor cells (CPCs) on left ventricle function and remodeling post-myocardial infarction persist for several weeks and up to 1 year after CPC administration, the vast majority (>95%) of transplanted CPCs in animal studies die or vanish shortly after cell administration. Additionally, the less blood flow in the scar tissue after heart attack results in the oxygen reduced to 1 - 2% (hypoxia) which makes the cardiac microenvironment inadequate for cell DNA synthesis, thereby implying that gene manipulations on the transplanted CPCs that limit cell death under hypoxia may enhance the efficacy (and thus clinical applicability) of cell therapy. One of the most powerful cytoprotective proteins known is heme oxygenase-1 (HO-1). To test the hypothesis that HO-1 gene may improve newly synthesized DNA level in CPCs under hypoxia, adult CPCs were isolated and sorted from wild-type (WT), HO-1 transgenic (TG) or knockout (KO) mouse hearts. The c-kit positivity in all live CPCs was 60 - 65% detected by FACS with specific monoclonal anti-c-kit antibody in which Mast cells were used as positive control and CHO cells as negative control. HO-1 protein overexpression in HO-1TG CPCs was confirmed by FACS and immunoblotting (2.8-fold greater than that in WT CPCs); in contrast, HO-1KO CPCs had no HO-1. A hypoxia chamber system containing 1% of O2, 5% of CO2, and 94% of nitrogen was employed to mimic the damaged environment in the cardiac scar tissue. Furthermore, Bromodeoxyuridine (BrdU), an analog of thymidine that can be incorporated into the newly synthesized DNA of replicating cells in S-phase, was used to determine DNA synthesis levels in CPCs. Cells were cultured for 16 h and then labeled with BrdU for 30 min before BrdU immunofluorescent staining. Under normal oxygen tension (21% of O2), HO-1TG CPCs exhibited an significant increase in nuclear BrdU intensity relative to either WT or HO-1KO CPCs (+88% vs WT group, and +63% vs HO-1KO group), indicating that the new DNA synthesis in HO-1TG CPCs are very active during cell mitosis as compared to WT or HO-1KO CPCs. Under hypoxic condition (1% of O2), although new DNA synthesis in S-phase in all of the groups was much less active, HO-1TG CPCs showed even greater differences (+2.4-fold vs WT, +2.0-fold vs HO-1KO) in the extent of BrdU nuclear incorporation as compared with WT or HO-1KO CPC group (FigFig), providing a strong evidence that HO-1 gene plays an important role in protecting CPCs under hypoxia via promotion of new DNA synthesis in BrdU-labeled cell mitosis. These results demonstrate that HO-1 gene promotes new DNA synthesis during mitoses in adult CPCs not only under normal oxygen condition but also in the hypoxia environment, therefore HO-1TG CPCs may provide higher survival rate than WT and HO-1KO CPCs. The data support the hypothesis that HO-1 gene improves the survival and reparative ability of adult CPCs via enhancing newly synthesized DNA in replicating cells in S-phase under hypoxia. Thus, the adult CPCs carried HO-1 gene as a new approach may have potentially clinical application to increase the efficacy of cell therapy for patients with heart attack.View Large Image | Download PowerPoint Slide

Abstract POSTER-BIOL-1302: Sucrose nonfermenting 1-related kinase (SNRK) expression and function in vitro in ovarian cancer cells

Abstract Purpose of study: Lipid metabolism and transport have recently been identified as potential targets for the treatment of cancers where adipocytes are a major component of the tumor microenvironment. Ovarian cancer metastases have a clear predilection for the omentum, which is primarily comprised of adipocytes. Studies have shown that human omental adipocytes promote migration and invasion of ovarian cancer cells and that adipocyte–ovarian cancer cell coculture promotes tumor growth and induces lipolysis in adipocytes and β-oxidation in cancer cells. SNRK is a serine/threonine kinase with sequence similarity to AMP-activated protein kinase (AMPK). AMPK proteins play a central role in cellular energy regulation. Active AMPK restricts cell proliferation, cell growth, and metabolic substrate synthesis, and increases ATP-generating processes, such as glucose and fatty acid oxidation. It has also been suggested that SNRK suppresses adipocyte inflammation via mTORC1 which is a pathway known to be aberrant in ovarian cancer. SNRK has been shown to reduce colon cancer cell proliferation and DNA synthesis and SNRK levels are decreased in human colon cancer tissue. Stable knockdown of SNRK increases in vitro colon cancer cell tumorigenicity indicating that SNRK is a regulator of cell proliferation. Because of the role SNRK appears to play in cell proliferation and metabolism and the link between SNRK and mTORC1 signaling, the goal of this study is to describe expression of SNRK in ovarian cancer cell lines and ovarian tissue from mice. Experimental Procedures: The ovarian cancer cell lines A2780 and SKOV3ip1 as well as a normal surface epithelial cell line IOSE, were grown in culture media and then immunostained for SNRK, as well as CC3 (apoptosis) and PH3 (proliferation). To assess the expression of SNRK in mice, ovarian tissue from wild type mice were harvested and immunostained for SNRK. Summary of data: Cell line immunostaining demonstrated differential expression of SNRK in both ovarian cancer cell lines and the benign ovarian epithelial cell line along with PH3 staining. There was no CC3 staining seen in any of the cell lines. The mouse ovarian tissue shows SNRK staining in the nuclei of follicular cells but no clear staining in the epithelial or stromal cells. Conclusions: Our results show the presence of SNRK in both benign and malignant epithelial ovarian cell lines as well as in benign mouse ovarian tissue. We plan to further explore SNRK expression levels in ovarian cancer by quantifying SNRK mRNA and protein levels in ovarian cancer cell lines as well as in human benign and malignant ovarian tissue. We also plan to knockdown SNRK expression in ovarian cancer cell lines to determine effects on cell proliferation, migration, and invasion as well as changes in proteins involved in proliferation and metabolism. Citation Format: Erin Bishop, Stephanie Cossette, Vakeel Padmanabhan, Ramani Ramchandran. Sucrose nonfermenting 1-related kinase (SNRK) expression and function in vitro in ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1302.

Investigation of brain tumors using (18)F-fluorobutyl ethacrynic amide and its metabolite with positron emission tomography.

To date, imaging of malignant glioma remains challenging. In positron emission tomography-related diagnostic imaging, differential tumor uptake of 3′-deoxy-3′-[18F] fluorothymidine ([18F]FLT) has been shown to reflect the levels of cell proliferation and DNA synthesis. However, additional biomarkers for tumors are urgently required. Aberrant levels of glutathione transferase (GST) activity have been hypothesized to constitute such a novel diagnostic marker. Here, a C6 rat glioma tumor model was used to assess the ability of the positron emission tomography tracers, [18F]FLT and 18F-fluorobutyl ethacrynic amide ([18F]FBuEA), to indicate reactive oxygen species-induced stress responses as well as detoxification-related processes in tumors. Using a GST activity assay, we were able to demonstrate that FBuEA is more readily catalyzed by GST-π than by GST-α. Furthermore, we showed that FBuEA-GS, a metabolite of FBuEA, elicits greater cytotoxicity in tumor cells than in normal fibroblast cells. Finally, in vitro and in vivo investigation of radiotracer distribution of [18F]FBuEA and [18F] FBuEA-GS revealed preferential accumulation in C6 glioma tumor cells over normal fibroblast cells for [18F]FBuEA-GS but not for [18F]FBuEA.

Downregulation of miR-610 promotes proliferation and tumorigenicity and activates Wnt/β-catenin signaling in human hepatocellular carcinoma.

BackgroundWnt/β-catenin signaling pathway plays important roles in human cancer progression. Better understanding the mechanism underlying regulation of Wnt/β-catenin signaling pathway might provide novel therapeutic targets for cancer treatment.MethodsmiR-610 expression levels in hepatocellular carcinoma (HCC) cell lines, HCC tissues and 76 archived HCC specimens were determined using real-time PCR. Cell viability was measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. The level of DNA synthesis was determined by BrdU incorporation assay. Flow cytometry analysis was used to analyze cell cycle progression. The cells proliferation and tumorigenesis were determined by colony formation and anchorage-independent growth assays in vitro, and by xenograft tumors in vivo. Luciferase assay and micro-ribonucleoprotein complex immunoprecipitation assay were used to confirm the association of the targeted mRNAs with miR-610.ResultsmiR-610 was downregulated in human HCC cells and tissues, and correlated with HCC progression and patient survival. Inhibition of miR-610 promoted, but overexpression of miR-610 reduced, HCC cell proliferation and tumorigenicity both in vitro and in vivo. Furthermore, we found that inhibiting miR-610 activated, but overexpressing miR-610 decreased, the Wnt/β-catenin activity through directly suppressing lipoprotein receptor-related protein 6 (LRP6) and transducin β–like protein 1 (TBL1X). The in vitro analysis was consistent with the inverse correlation detected between miR-610 levels with expression of LRP6 and TBL1X in a cohort of human HCC samples.ConclusionsOur results indicate that miR-610 downregulation plays essential roles in HCC progression and reduced miR-610 is correlated with Wnt/β-catenin signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-261) contains supplementary material, which is available to authorized users.

Treatment options beyond IFNα and NUCs for chronic HBV infection: expectations for tomorrow.

Chronic hepatitis B virus (HBV) infection may progress to cirrhosis, hepatocellular carcinoma (HCC) and end-stage liver failure with time. Interruption of this process can only be achieved through effective antiviral treatment. This approach has so far involved the use of immunomodulators such as pegylated interferon alpha (Peg-IFNα) for a finite period of up to a year and nucleos-(t)ide analogues (NUCs) for treatment over much longer periods of time. The latter act by suppressing HBV replication at the level of DNA synthesis by inhibiting the viral reverse transcriptase/DNA polymerase and causing premature termination of DNA synthesis. The ideal treatment end point is loss of HBsAg in both HBeAg+ve and HBeAg-ve patients following monotherapy. This, however, is only achievable in a minority of patients. Secondary outcomes are durable HBeAg loss and seroconversion to anti-HBe, which occur in about 18-30% of HBeAg+ve patients depending on the antiviral used, and sustained suppression of HBV-DNA accompanied by biochemical normalization and histological improvement in non-HBeAg+ve seroconverting and HBeAg-ve patients. There is therefore a need for additional direct-acting antivirals (DAAs) targeting different stages of the life cycle of the virus, as well as immunotherapeutic approaches. Such developments may pave the way for their use either alone or more likely in combination in the fight against chronic HBV infection. Such drugs or approaches, which are currently undergoing preclinical or clinical testing, are the subject of this review.