Antiproliferative and Antimetastatic Properties of 16-Azidomethyl Substituted 3-O-Benzyl Estrone Analogs.

Four diastereomers of 16-azidomethyl substituted 3-O-benzyl estradiol (1-4) and their two estrone analogs (16AABE and 16BABE) were tested for their antiproliferative properties against human gynecological cancer cell lines. The estrones were selected for additional experiments based on their outstanding cell growth-inhibiting activities. Both compounds increased hypodiploid populations of breast cancer cells, and 16AABE elicited cell cycle disturbance as evidenced by flow cytometry. The two analogs substantially increased the rate of tubulin polymerization in vitro. 16AABE and 16BABE inhibited breast cancer cells' migration and invasive ability, as evidenced by wound healing and Boyden chamber assays. Since both estrone analogs exerted remarkable estrogenic activities, as documented by a luciferase reporter gene assay, they can be considered as promising drug candidates for hormone-independent malignancies.

Investigation of Anticancer Properties of Monoterpene-Aminopyrimidine Hybrids on A2780 Ovarian Cancer Cells.

The present study aimed to characterize the antiproliferative and antimetastatic properties of two recently synthesized monoterpene-aminopyrimidine hybrids (1 and 2) on A2780 ovary cancer cells. Both agents exerted a more pronounced cell growth inhibitory action than the reference agent cisplatin, as determined by the MTT assay. Tumor selectivity was assessed using non-cancerous fibroblast cells. Hybrids 1 and 2 induced changes in cell morphology and membrane integrity in A2780 cells, as evidenced by Hoechst 33258-propidium iodide fluorescent staining. Cell cycle analysis by flow cytometry revealed substantial changes in the distribution of A2780 ovarian cancer cells, with an increased rate in the subG1 and G2/M phases, at the expense of the G1 cell population. Moreover, the tested molecules accelerated tubulin polymerization in a cell-free in vitro system. The antimetastatic properties of both tested compounds were investigated by wound healing and Boyden chamber assays after 24 and 48 h of incubation. Treatment with 1 and 2 resulted in time- and concentration-dependent inhibition of migration and invasion of A2780 cancer cells. These results support that the tested agents may be worth of further investigation as promising anticancer drug candidates.

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy.

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems-the only DNA transposons currently employed in clinical trials-during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver.

BACKGROUND: Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS: We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS: We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

HNF1B Loss Exacerbates the Development of Chromophobe Renal Cell Carcinomas.

Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313-26. ©2017 AACR.

8-Oxoguanine DNA glycosylase1-driven DNA repair-A paradoxical role in lung aging.

Age-associated changes in lung structure and function are some of the most important predictors of overall health, cognitive activities and longevity. Common to all aging cells is an increase in oxidatively modified DNA bases, primarily 8-oxo-7,8-dihydroguanine (8-oxoG). It is repaired via DNA base excision repair pathway driven by 8-oxoguanine DNA glycosylase-1 (OGG1-BER), whose role in aging has been the focus of many studies. This study hypothesizes that signaling and consequent gene expression during cellular response to OGG1-BER "wires" senescence/aging processes. To test OGG1-BER was mimicked by repeatedly exposing diploid lung fibroblasts cells and airways of mice to 8-oxoG base. Results showed that repeated exposures led to G1 cell cycle arrest and pre-matured senescence of cultured cells in which over 1000 genes were differentially expressed -86% of them been identical to those in naturally senesced cells. Gene ontology analysis of gene expression displayed biological processes driven by small GTPases, phosphoinositide 3-kinase and mitogen activated kinase cascades both in cultured cells and lungs. These results together, points to a new paradigm about the role of DNA damage and repair by OGG1 in aging and age-associated disease processes.

The PI3K/AKT Pathway and Renal Cell Carcinoma.

The phosphatidylinositol 3 kinase (PI3K)/AKT pathway is genetically targeted in more pathway components and in more tumor types than any other growth factor signaling pathway, and thus is frequently activated as a cancer driver. More importantly, the PI3K/AKT pathway is composed of multiple bifurcating and converging kinase cascades, providing many potential targets for cancer therapy. Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer that is notoriously resistant to traditional chemotherapies or radiotherapies. The PI3K/AKT pathway is modestly mutated but highly activated in RCC, representing a promising drug target. Indeed, PI3K pathway inhibitors of the rapalog family are approved for use in RCC. Recent large-scale integrated analyses of a large number of patients have provided a molecular basis for RCC, reiterating the critical role of the PI3K/AKT pathway in this cancer. In this review, we summarize the genetic alterations of the PI3K/AKT pathway in RCC as indicated in the latest large-scale genome sequencing data, as well as treatments for RCC that target the aberrant activated PI3K/AKT pathway.

Resistance to Antiangiogenic Therapy Is Associated with an Immunosuppressive Tumor Microenvironment in Metastatic Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer, and antiangiogenic therapy is the current mainstay of treatment. Patients with RCC develop innate or adaptive resistance to antiangiogenic therapy. There is a need to identify biomarkers that predict therapeutic resistance and guide combination therapy. We assessed the interaction between antiangiogenic therapy and the tumor immune microenvironment and determined their impact on clinical outcome. We found that antiangiogenic therapy-treated RCC primary tumors showed increased infiltration of CD4(+) and CD8(+) T lymphocytes, which was inversely related to patient overall survival and progression-free survival. Furthermore, specimens from patients treated with antiangiogenic therapy showed higher infiltration of CD4(+)FOXP3(+) regulatory T cells and enhanced expression of checkpoint ligand programed death-ligand 1 (PD-L1). Both immunosuppressive features were correlated with T-lymphocyte infiltration and were negatively related to patient survival. Treatment of RCC cell lines and RCC xenografts in immunodeficient mice with sunitinib also increased tumor PD-L1 expression. Results from this study indicate that antiangiogenic treatment may both positively and negatively regulate the tumor immune microenvironment. These findings generate hypotheses on resistance mechanisms to antiangiogenic therapy and will guide the development of combination therapy with PD-1/PD-L1-blocking agents.

Genetic and pharmacological strategies to refunctionalize the von Hippel Lindau R167Q mutant protein.

Aberrant von Hippel Lindau (VHL) protein function is the underlying driver of VHL-related diseases, including both sporadic and inherited clear cell renal cell carcinoma (ccRCC). About one third of VHL mutations are missense point mutations, with R167Q being the most common VHL point mutation in hereditary VHL disease. Although it has been studied extensively, the ability of VHL-R167Q to downregulate hypoxia-inducible factor 2α (HIF2α) is still controversial. In addition, the manner in which the mutation contributes to tumorigenesis is not fully understood. No therapeutic approach is available to target VHL-R167Q and similar missense point mutations. We analyzed VHL-R167Q proteostasis and function at normoxia, at hypoxia with different oxygen pressure, and in a xenograft mouse model. We showed that the protein levels of VHL-R167Q dictate its ability to downregulate HIF2α and suppress tumor growth. Strikingly, the proteasome inhibitors bortezomib and carfilzomib, which are currently in clinical use, stabilize VHL-R167Q and increase its ability to downregulate HIF2α. VHL-R167Q binds elongin C and elongin B with considerably less avidity than wild-type VHL does but retains residual capacity to generate a VHL-elongin C-elongin B complex, downregulate HIF2α, and suppress tumorigenesis, which could be rescued by increase of VHL-R167Q levels. Finally, we used in silico approaches and identified other missense VHL mutants in addition to VHL-R167Q that might be rescued by similar strategies. Thus, our studies revealed detailed information describing how VHL-R167Q contributes to tumorigenesis and identified a potential targeted therapy for ccRCC and other VHL-related disease in patients carrying VHL-R167Q or similar missense mutations.

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair.

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.

Agents that stabilize mutated von Hippel-Lindau (VHL) protein: results of a high-throughput screen to identify compounds that modulate VHL proteostasis.

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.

Biochemical identification of a hydroperoxide derivative of the free 8-oxo-7,8-dihydroguanine base.

8-Oxo-7,8-dihydroguanine is one the most abundant base lesions in pro- and eukaryotic DNA. In mammalian cells, it is excised by the 8-oxoguanine DNA glycosylase (OGG1) during DNA base-excision repair, and the generated free 8-oxoG base is one of the DNA-derived biomarkers of oxidative stress in biological samples. The modification of 8-oxoG in the context of nucleoside and DNA has been the subject of many studies; however, the oxidative transformation of the free 8-oxoG base has not been described. By using biochemical and cell biological assays, we show that in the presence of molecular oxygen, the free 8-oxoG base transforms to a highly reactive hydroperoxide (8-oxoG*). Specifically, 8-oxoG* oxidizes Amplex red to resorufin, H(2)DCF to DCF, Fe(2+) to Fe(3+), and GSH to GSSG. This property of 8-oxoG* was diminished by treatment with catalase and glutathione peroxidase, but not superoxide dismutase. 8-OxoG* formation was prevented by reducing agents or nitrogen atmosphere. Its addition to CM-H(2)DCF-DA-loaded cells rapidly increased intracellular DCF fluorescence. There were no such properties observed for 8-oxodeoxyguanosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2'-deoxyguanosine, guanine, adenine, guanosine, and 8-hydroxyadenine. These data imply that a free 8-oxoG base is more susceptible to oxidation than is its nucleoside form and, consequently, it stands as unique among intact and oxidatively modified purines.

Effects of Colostrinin on gene expression-transcriptomal network analysis.

Colostrinin (CLN) is a uniform mixture of low-molecular weight proline-rich polypeptides isolated from the mother's first milk, colostrum. Exposure of cells to CLN decreases intracellular levels of reactive oxygen species by regulating glutathione metabolism and modulating activities of antioxidant enzymes and mitochondrial function. It also inhibits beta amyloid-induced apoptosis and induces neurite outgrowth of pheochromocytoma cells. Administration of CLN to Alzheimer's disease patients has resulted in a stabilizing effect on cognitive function. We analyzed CLN-induced gene expression changes using high-density oligonucleotide arrays and transcriptomal network analysis. We found that CLN elicited highly complex and multiphasic changes in the gene expression profile of treated cells. CLN treatment affected a total of 58 molecular networks, 27 of which contained at least 10 differentially expressed genes. Here we present CLN-modulated gene networks as potential underlying molecular mechanisms leading to the reported effects of CLN on cellular oxidative state, chemokine and cytokine production, and cell differentiation, as well as on pathological processes like allergy, asthma, Alzheimer's, and other neurological diseases. Based on our results, we also predict possible modulatory effects of CLN on adipocytokine gene networks that play a crucial role in the pathobiology of diabetes, cardiovascular disorders, obesity, and inflammation. Taken together, CLN-altered gene expression networks presented here provide the molecular basis for previously described biological phenomena and predict potential fields of application for CLN in the prevention and treatment of diseases.

Real-time reverse transcription-polymerase chain reaction detection of Newcastle disease virus using light upon extension fluorogenic primers.

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10(+1.2) egg infectious dose (EID)(50)/0.2 ml and 10(+2.2) EID(50)/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.

Colostrinin decreases spontaneous and induced mutation frequencies at the hprt locus in Chinese hamster V79 cells.

Colostrinin (CLN), a uniform mixture of low-molecular weight, proline-rich polypeptides, induces neurite outgrowth of pheochromocytoma cells, extends the lifespan of diploid fibroblast cells, inhibits beta amyloid-induced apoptosis and resulted in improved cognitive function when administered to Alzheimer's patients. Here we investigated CLN's antimutagenic activity in cells stressed oxidatively or exposed to chemical or physical agents. Our data show that CLN did not alter cell cycle kinetics and cloning efficiency, while it inhibited the development of spontaneous mutations at the coding region of the hypoxanthine phosphoribosyl-transferase (hprt) gene in Chinese hamster V79 cells. In a dose-dependent manner, CLN lowered reactive oxygen species (ROS)-induced frequency of cells resistant to 6-thioguanine (6-TG) to nearly background level. Likewise, CLN decreased the frequency of methyl methanesulfonate- or mitomycin C-induced mutations in V79 cells. Notably, CLN (at 100, 250, and 500 ng per ml concentrations) decreased UVA-induced mutation frequency, while only the highest dose of CLN also decreased significantly the number of UVB-induced 6-TG-resistant mutant cells. Similar results were obtained using cell cultures of human origin. Overall, our data show that CLN significantly lowers the mutation frequency that develops spontaneously or is induced by ROS, chemical and physical agents. CLN itself has no mutagenic activity. Therefore, CLN may be used in human therapies systemically and/or locally for the prevention of diseases associated with sequence alterations in genomic and mitochondrial DNA.

Delayed vaccine virus replication in chickens vaccinated subcutaneously with an immune complex infectious bursal disease vaccine: quantification of vaccine virus by real-time polymerase chain reaction.

The distribution of the immune complex vaccine virus for infectious bursal disease (IBD) in tissue was examined and the viral loads of the organs were quantitatively compared. One-day-old specific pathogen free (SPF) and maternally immune broiler chickens were injected subcutaneously with the vaccine. Lymphoid and non-lymphoid tissues were collected at various time intervals during the experiment to test for infectious bursal disease virus (IBDV)-RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR). Only the bursa of Fabricius was found to be positive with unusually long viral persistence in the broiler group. The positive bursa samples were further investigated by using real-time PCR coupled with a TaqMan probe. The highest amounts of the virus were detected at its first appearance in the bursa: on day 14 post vaccination (PV) in the SPF chickens and on day 17 and day 21 PV in the maternally immune broiler group. The virus then gradually cleared, most likely due to the arallel appearance of the active immune response indicated by seroconversion.

[Study of pathogenetic role of Epstein-Barr virus in Hungarian patients with B-cell non-Hodgkin lymphomas]. / Az Epstein-Barr vírus patogenetikai szerepének tanulmányozása magyarországi B-sejtes non-Hodgkin lymphomás betegekben.

INTRODUCTION: The Epstein-Barr virus is a member of herpesvirus family. It plays an important role in the etiopathogenesis of Burkitt lymphoma, other B-cell non-Hodgkin lymphomas, nasopharyngeal carcinoma, X chromosome-linked lymphoproliferative disease, Hodgkin lymphomas and a part of T-cell lymphomas. It has been suggested that association of Epstein-Barr virus with lymphomas and its pathological significance in disease varies in different geographical areas. The aim of our study was to reveal the role of Epstein-Barr virus in B-cell non-Hodgkin lymphomas diagnosed in Hungary. The authors asked: A) What is the frequency of the presence of virus genome in the biopsy specimen from B-cell non-Hodgkin patients? B) Which types of virus latency can be observed? PATIENTS AND METHODS: Genomic DNA and EBER-specific RNA of Epstein-Barr virus in paraffin-embedded specimens were detected by polymerase chain reaction and in situ hybridisation, respectively. RESULTS: Out of 36 B-cell non-Hodgkin lymphoma cases, 16 (45%) were found to be positive for Epstein-Barr virus by the above methods. Expression of latency genes, nuclear antigene-2 and latent membrane protein-1 was studied by immunohistochemical technique. The 16 virus-positive non-Hodgkin lymphoma cases showed Epstein-Barr virus latency types II. (37%) or III. (63%). CONCLUSIONS: The authors data suggest that Epstein-Barr virus may be associated with the development of B-cell non-Hodgkin lymphomas in Hungarian patients.

[Detection of trisomy 8 and monosomy 7 in chronic granulocytic leukemia and myelodysplastic syndrome by cytogenetic analysis and fluorescence in situ hybridization]. / A citogenetikai vizsgálat és fluoreszcencia in situ hibridizáció párhuzamos alkalmazásának jelentósége krónikus granulocytás leukaemiában és myelodysplasiás szindrómában a 8-as triszómia és a 7-es monoszómia kimutatására.

INTRODUCTION AND AIMS: In this retrospective study the authors studied how interphase fluorescence in situ hybridization can be applied to the diagnosis of numerical chromosomal anomalies failed to be identified by cytogenetic analysis. METHODS: Thirty-four patients, 27 with chronic granulocytic leukemia and seven with myelodysplastic syndrome, were studied in order to identify disease-specific aberrations, trisomy 8 and monosomy 7 using both traditional cytogenetic analysis and fluorescence in situ hybridization on interphase bone marrow cells. Using alphoid-satellite centromeric specific probes, trisomy 8 indicating the progression of the disease and poor prognosis in chronic granulocytic leukemia as well as monosomy 7 in myelodysplastic syndrome were identified. RESULTS: In 21 of 34 cases both methods led to the same results. In 13 patients fluorescence in situ hybridization making possible to examine a great number of cells, provided more information about the gain and loss of chromosomes above and clarified uncertain cytogenetic results. CONCLUSIONS: In both hematological malignancies, studied by the authors, fluorescence in situ hybridization proved a useful and sensitive method to determine chromosomes unrecognized or not accurately identified by the traditional cytogenetic analysis and to define the ratio of pathological cells. At the same time the results confirm that conventional cytogenetic analysis is still essential in diagnosing the genetic alterations of malignant cells and point at the chromosomes that are worth further studying by other methods.