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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2011

Blood Gene Expression Profiling of Breast Cancer Survivors Experiencing Fibrosis

Purpose: To extend knowledge on the mechanisms and pathways involved in maintenance of radiation-induced fibrosis (RIF) by performing gene expression profiling of whole blood from breast cancer (BC) survivors with and without fibrosis 3-7 years after end of radiotherapy treatment. Methods and Materials: Gene expression profiles from blood were obtained for 254 BC survivors derived from a cohort of survivors, treated with adjuvant radiotherapy for breast cancer 3-7 years earlier. Analyses of transcriptional differences in blood gene expression between BC survivors with fibrosis (n = 31) and BC survivors without fibrosis (n = 223) were performed using R version 2.8.0 and tools from the Bioconductor project. Gene sets extracted through a literature search on fibrosis and breast cancer were subsequently used in gene set enrichment analysis. Results: Substantial differences in blood gene expression between BC survivors with and without fibrosis were observed, and 87 differentially expressed genes were identified through linear analysis. Transforming growth factor-{beta}1 signaling was identified as the most significant gene set, showing a down-regulation of most of the core genes, together with up-regulation of a transcriptional activator of the inhibitor of fibrinolysis, Plasminogen activator inhibitor 1 in the BC survivors with fibrosis. Conclusion: Transforming growth factor-{beta}1 signaling was found down-regulated during the maintenance phase of fibrosis as opposed to the up-regulation reported during the early, initiating phase of fibrosis. Hence, once the fibrotic tissue has developed, the maintenance phase might rather involve a deregulation of fibrinolysis and altered degradation of extracellular matrix components.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2021

VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

The Influenza A Virus Endoribonuclease PA-X Usurps Host mRNA Processing Machinery to Limit Host Gene Expression

Often, many viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, yet the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the B100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2013

Gene expression profiles in irradiated cancer cells

Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B

DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein–DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. Additionally, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. Our results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Finally, although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Differential Gene Expression in Host Ubiquitination Processes in Childhood Malarial Anemia

Background: Malaria remains one of the leading global causes of childhood morbidity and mortality. In holoendemic Plasmodium falciparum transmission regions, such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dl] is the primary form of severe disease. Ubiquitination is essential for regulating intracellular processes involved in innate and adaptive immunity. Although dysregulation in ubiquitin molecular processes is central to the pathogenesis of multiple human diseases, the expression patterns of ubiquitination genes in SMA remain unexplored. Methods: To examine the role of the ubiquitination processes in pathogenesis of SMA, differential gene expression profiles were determined in Kenyan children ( n = 44, aged <48 mos) with either mild malarial anemia (M l MA; Hb ≥9.0 g/dl; n = 23) or SMA (Hb <6.0 g/dl; n = 21) using the Qiagen Human Ubiquitination Pathway RT 2 Profiler PCR Array containing a set of 84 human ubiquitination genes. Results: In children with SMA, 10 genes were down-regulated ( BRCC3 , FBXO3 , MARCH5 , RFWD2 , SMURF2 , UBA6 , UBE2A , UBE2D1 , UBE2L3 , UBR1 ), and five genes were up-regulated ( MDM2 , PARK2 , STUB1 , UBE2E3 , UBE2M ). Enrichment analyses revealed Ubiquitin-Proteasomal Proteolysis as the top disrupted process, along with altered sub-networks involved in proteasomal, protein, and ubiquitin-dependent catabolic processes. Conclusion: Collectively, these novel results show that protein coding genes of the ubiquitination processes are involved in the pathogenesis of SMA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Effect of cisplatin on the clock genes expression in the liver, heart and kidney

Highlights: • Cisplatin-induced nephrotoxicity showed a circadian rhythm. • Cisplatin altered the clock genes expression in a tissue- and gene-specific manner. • Dbp was consistently suppressed in peripheral tissues after cisplatin treatment. Cisplatin is a platinum-based chemotherapy drug that is widely used to treat various types of malignancies. Although the involvement of circadian clock in cisplatin metabolism and excretion has been reported, the effect of cisplatin on circadian rhythm remains unclear. In the present study, we investigated the effects of cisplatin on clock genes expression in mouse peripheral tissues. Cisplatin induced severe nephrotoxicity, as revealed by the significant increase of blood urea nitrogen and serum creatinine levels. Moreover, cisplatin circadian time-dependently induced p21 expression in the liver, heart and kidney, with the highest increase during the dark phase. In addition, cisplatin altered the clock genes expression in the liver, heart and kidney in a tissue- and gene-specific manner. Interesting, the expression of D site of the albumin promoter binding protein (Dbp), a gene involved in detoxification and drug metabolism, was consistently suppressed in the liver, heart and kidney after cisplatin treatment, implying a role of DBP in the toxicity of cisplatin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Leptin modulates gene expression in the heart, cardiomyocytes and the adipose tissue thus mitigating LPS-induced damage

Highlights: • LPS enhanced expression of the leptin gene in mouse tissues and cardiomyocytes. • Leptin downregulated pro-inflammatory genes in vivo and in cardiomyocytes after LPS treatment. • Leptin upregulated expression of antioxidant genes in vivo and in cardiomyocytes after LPS treatment. • Leptin reduced ROS levels in cardiomyocytes after LPS treatment. • Leptin acts to counteract LPS-induced damage in the heart and cardiomyocytes by modulating gene expression. • Anti-inflammatory effects of leptin in transgenic aMUPA mice overexpressing leptin surpassed that of wild type mice. Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1β in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2017

Effects of Ionizing Radiation on Murine Gene Expression in Skin and Bone

Long duration spaceflight causes a negative calcium balance and reduces bone density in astronauts. The potential for exposure to space radiation to contribute to lasting decrements in bone mass is not yet understood. Sustained changes to bone mass have a relatively long latency for development, however skin is a radiation sensitive organ and changes in skin gene expression may serve as an early radiation biomarker of exposures and may correlate with adverse effects on skeletal tissue. Previous studies have shown that FGF18 gene expression levels of hair follicles collected from astronauts on the ISS rose over time. In the hair follicle, FGF18 signaling mediates radioresistance in the telogen by arresting the cell cycle, and FGF18 has the potential to function as a radioprotector. In bone, FGF18 appears to regulate cell proliferation and differentiation positively during osteogenesis and negatively during chondrogenesis. Cellular defense responses to radiation are shared by a variety of organs, hence in this study, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We have examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (TBI). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and TBI (0.5 Gy Fe-56 600 MeV/n and 0.5 Gy H-1 150 MeV/n). Animals were euthanized one and 11 days post-IR. Statistical analysis was performed via a Student's ttest. In skin samples one day after IR, skin expression of FGF18 was significantly greater (3.8X) than sham-irradiated controls (3.8X), but did not differ 11 days post TBI. Expression levels of other radiation related genes (Nfe2l2, Trp53, Cdkn1a, FoxO3, Gadd45g, SOD1), was not different due to TBI at either time point. In bone (femora) TBI significantly increased (3.8X) expression of the pro-bone resorption cytokine, MCP-1, one day after TBI. FGF18 expression in skin and MCP- 1 expression in bone were found to be positively correlated (P less than 0.002, r=0.8779). Further, microcomputed tomography analysis of tibae from these animals showed reduced fractional cancellous bone volume (-21.7%) at 11 days post exposure. These results suggest that early radiation induced changes in FGF18 gene expression in skin may have value for predicting subsequent loss of cancellous bone mass.

space radiation↗
DOE OSTI2014

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Exon disruptive variants in Populus trichocarpa associated with wood properties exhibit distinct gene expression patterns

Abstract Forest trees may harbor naturally occurring exon disruptive variants (DVs) in their gene sequences, which potentially impact important ecological and economic phenotypic traits. However, the abundance and molecular regulation of these variants remain largely unexplored. Here, 24,420 DVs were identified by screening 1014Populus trichocarpafull genomes. The identified DVs were predominantly heterozygous with allelic frequencies below 5% (only 26% of DVs had frequencies greater than 5%). Using common garden‐grown trees, DVs were assessed for gene expression variation in the developing xylem, revealing that their gene expression can be significantly altered, particularly for homozygous DVs (in the range of 27%–38% of cases depending on the studied common garden). DVs were further investigated for their correlations with 13 wood quality traits, revealing that, among the 148 discovered DV associations, 15 correlated with more than one wood property and six genes had more than one DV in their coding sequences associated with wood traits. Approximately one‐third of DVs correlated with wood property variation also showed significant gene expression variation, confirming their non‐spurious impact. These findings offer potential avenues for targeted introduction of homozygous mutations using tree biotechnology, and while the exact mechanisms by which DVs may directly influence wood formation remain to be unraveled, this study lays the groundwork for further investigation.

Genetics & Heredity↗
DOE OSTI2018

RNA interference in vivo in Schistosoma japonicum: Establishing and optimization of RNAi mediated suppression of gene expression by long dsRNA in the intra-mammalian life stages of worms

Schistosomes are parasitic platyhelminths that threaten over 600 million people globally. In recent years, RNA interference (RNAi) has been widely used as a molecular tool in research into the genomic function of parasites. We aim to develop effective protocols for application of RNAi technology in the intra-mammalian life stages of Schistosoma japonicum. In this work, the expression of the parasite gene encoding cathepsin B1 (SjCB1) was targeted by exposing the worms to 10 μg of long dsRNA dissolved in 0.1 ml of 0.7% NaCl injected into the tail vein of infected mice. This method was effective and specific for eliciting SjCB1 gene suppression in both male and female adult worms in vivo (>79.4% in male and >91.5% in female knockdown relative to control). In 60 cercaria infected mice, RNAi suppression of gene expression was best achieved by using 10 μg of target dsRNA for at least 4 days. The recommended procedure for interference producing long-term suppression was an injection of dsRNA on the first day of infection with booster injections administered every 4 days for up to 26 days. Long-term suppression of three published functional genes (peroxiredoxin-1, mago nashi, insulin receptor) in S. japonicum provided more information about the role of the expression of these genes in producing particular phenotypes. The protocols described here may be more convenient, economical and applicable, than currently available technology and have contributed to the observation of more phenotypes during worm development from schistosomula to adult. These approaches may promote and facilitate further studies into functional schistosome genomics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and suppressions of oxidative stress and cell proliferation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Genetic Selection for Context-Dependent Stochastic Phenotypes: Sp1 and TATA Mutations Increase Phenotypic Noise in HIV-1 Gene Expression

The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tattransactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly ‘switch’ between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify ‘Switching’ phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Oxidative stress/reactive metabolite gene expression signature in rat liver detects idiosyncratic hepatotoxicants

Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds—chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates. - Highlights: • 28 of 97 drugs gave a positive OS/RM gene expression signature in rat liver. • The specificity of the signature for human idiosyncratic hepatotoxicants was 98%. • The sensitivity of the signature for human idiosyncratic hepatotoxicants was 75%. • The signature can help eliminate hepatotoxicants from drug development.

60 APPLIED LIFE SCIENCES↗