Luciferase Reporter Gene Assay System Research Articles

Functional analysis of protein interactions using coupled bi-fluorescence complementation/GFP nanobody techniques.

Transcription factors (TFs) form homo- or hetero-dimeric DNA binding complexes along with associated co-regulators that can have transcriptional repressor or activator functions. Defining the specific composition of the complexes is therefore key to understanding their biological role. Here, we utilized bimolecular fluorescence complementation (BiFC) to visualize the formation of defined TF dimers and associated co-regulators derived from the activator protein-1 (AP-1) and myocyte enhancer factor 2 (MEF2) families. Firstly, BiFC signals were observed in cells co-expressing TFs tagged with complimentary combinations of the split fluorescent protein, demonstrating the engineered formation ofdefined dimer complexes. Next, we applied this approach and determined that defined AP-1 dimers localized at discrete sub-nuclear locations. Subsequently, a combination of BiFC coupled with GFP binding peptide (GBP)-nanotrap allowed observation of protein-protein interactions between a co-regulator, HDAC4, and defined BiFC-MEF2 engineered dimers. To determine transactivation properties of defined TF dimers in a cellular system, the Gal4-DNA binding domain fused to GBP was utilized to assess the transcriptional properties of the BiFC-TF dimers using a generically applicable Gal4/UAS luciferase reporter gene assay system. Here, we report efficacy of a BiFC/GBP-nanobody approach that allows engineering, visualization, and functional analysis of defined TF dimers.

Silencing circular RNA hsa_circABCC1 inhibits osteosarcoma progression through down-regulating HDAC4 via sponging miR-591.

Circular RNA (circRNA) has been shown to play an important regulatory role in the development of various cancers, including osteosarcoma (OS). However, the role of circRNA ABCC1 (circABCC1) in OS was still poorly understood. The aim of our study was to investigate the role of circABCC1 in OS progression and its potential molecular mechanisms. The expression of circABCC1, microRNA-591 (miR-591) and histone deacetylase 4 (HDAC4) in OS tissues or cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) analyses. In vitro experiments, the viability, proliferation, apoptosis, migration, invasion and autophagy of U2OS and HOS cells were assessed in vitro using cell counting kit-8 (CCK-8) assay, 5-ethynyl-29-deoxyuridine (EdU) assay, flow cytometry (FCM) assay, transwell migration and invasion assays (transwell) and WB assay, respectively. Interactions between circABCC1 and miR-591, miR-591 and HDAC4 were confirmed using a dual luciferase reporter gene assay system. The oncogenic role of circABCC1 in OS in vivo was examined by establishing a tumor xenograft model. CircABCC1 was significantly elevated in OS tissues (about 3.1-folds) and cells (U2OS (about 2.1-folds) and HOS (about 2.8-folds)) compared with the control (p < .05). Silencing of circABCC1 significantly reduced the viability and proliferation, promoted apoptosis, impaired migration and invasion, and increased autophagy of U2OS and HOS cells (p < .05). In addition, miR-591 was confirmed to be a target of circABCC1, exerting an opposite effect to circABCC1 (p < .05). MiR-591 attenuation in U2OS and HOS cells was able to reply to the inhibition of cell proliferation, migration and invasion as well as promotion of cell apoptosis and autophagy mediated by silencing circABCC1 (p < .05). HDAC4 was verified to be the target gene of miR-591 in U2OS and HOS cells and was regulated by the circABCC1/miR-591 axis (p < .05), and restoration of HDAC4 levels in U2OS and HOS cells was able to restore the altered cellular function caused by silencing circABCC1 (p < .05). In addition, knockdown of circABCC1 attenuated tumor growth in vivo (p < .05). Silencing of circABCC1 inhibits osteosarcoma progression by attenuating HDAC4 expression through sponging miR-591.

Silencing of long noncoding RNA LEF1-AS1 prevents the progression of hepatocellular carcinoma via the crosstalk with microRNA-136-5p/WNK1.

Long noncoding RNAs (lncRNAs) have been recognized as cancer-associated biological molecules, favoring hepatocellular carcinoma (HCC) progression. This study was conducted to elucidate the effects lncRNA lymphoid enhancer-binding Factor 1 antisense RNA (LEF1-AS1) on the pathological development of HCC, along with the crosstalk involving microRNA-136-5p (miR-136-5p) and with-no-K (lysine) kinase 1 (WNK1). The study recruited primary HCC tissues and their corresponding nonneoplastic liver tissues. The gain- and loss-of-function studies were performed in HCC cells HuH-7 and tumor xenografts in nude mice. The dual luciferase reporter gene assay system, RNA pull-down, and radioimmunoprecipitation assays were applied to detect their interactions among lncRNA LEF1-AS1, miR-136-5p, and WNK1. 5-Ethynyl-2'-deoxyuridine staining, scratch test, Transwell assays, and in vitro tube formation assays were conducted to examine HCC cell proliferation, migration, and invasion and HUVEC angiogenesis. HCC tissues and cells contained high lncRNA LEF1-AS1 expression. LncRNA LEF1-AS1 upregulation triggered markedly increased HCC cell proliferation, migration, and invasion and human umbilical vein endothelial cell angiogenesis. In vivo silencing lncRNA LEF1-AS1 resulted in reduced tumor cell vitality and matrix metalloproteinase-9 and the vascular endothelial growth factor expression. Additionally, the role of lncRNA LEF1-AS1 was found to be largely dependent on WNK1. Association of lncRNA LEF1-AS1 with WNK1 blocked the inhibitory effect of miR-136-5p on WNK1, which was confirmed by in vivo experiments. Altogether, our results revealed an important role of lncRNA LEF1-AS1 in regulating the HCC progression by regulating WNK1, providing a potential biomarker for the therapeutic modalities regarding HCC.

Effects of MiR-107 on The Chemo-drug Sensitivity of Breast Cancer Cells.

BackgroundA growing body of evidence indicates that aberrant expression of miR-107 plays a core role in cancers. This study aims to demonstrate the function of miR-107 and its roles in chemo-drug resistance in breast cancer cells.MethodologyCCK-8 assays were carried out to test the effect of miR-107 mimics on the proliferation of MCF-7 cells. The apoptosis level of each group was detected by flow cytometry. miR-107 level, mRNA levels of Bcl-2/Bax and TRIAP1 were detected by quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis. Protein levels of Bcl-2/Bax, p-Akt/Akt in MCF-7 cells were detected by using Western Blot. Lastly, the dual luciferase reporter gene assay system was used to confirm interaction between miR-107 and its target gene TRIAP1.ResultsCCK-8 assays indicated that miR-107 mimics augmented Taxol-induced cell viability inhibition. Flow cytometry showed that miR-107 mimics augmented Taxol-induced elevation of cell apoptosis. qRT-PCR analysis revealed that miR-107 mimics inhibited the mRNA expression of Bcl-2 and induced the mRNA level of Bax. Western Blotting indicated that miR-107 mimics inhibited the expression of proteins Bcl-2 and p-Akt, and induced the expression of Bax, while showing no significant effects on Akt. The relative luciferase activity revealed that oncogene TRIAP1 is a potential target gene of miR-107.ConclusionsmiR-107 plays a role in regulating chemo-drug sensitivity in mammary cancer cell by targeting TRIAP1.

Sevoflurane inhibits migration and invasion of colorectal cancer cells by regulating microRNA-34a/ADAM10 axis.

Sevoflurane is frequently used volatile anesthetic in cancer surgery. It has been suggested that treatment with sevoflurane could suppress migration and invasion of several human cancer cells in vitro. However, the effects of sevoflurane on colorectal cancer (CRC) remains largely unclear. In this study, CRC HCT116 and SW480 cells were treated by various concentrations of sevoflurane. MTT assay and Transwell assay were applied to evaluate the cell viability, migration and invasion abilities of CRC cell lines, respectively. Real-time quantitative PCR (RT-qPCR) was used to examine the expression level of miR-34a, and western blot assay was employed to detect the protein level of ADAM10. The target interaction between miR-34a and ADAM10 was verified through bioinformatics analysis, luciferase reporter gene assay system. We found Aberrant inhibitory effects induced by sevoflurane on the cell viability, migration and invasion abilities of HCT116 and SW480 cells in a dose-dependent manner were observed. Up-regulation of miR-34a strikingly suppressed the cell proliferation, migration and invasion abilities of the two cell lines. Sevoflurane could facilitate the miR-34a expression and its suppressor effects on CRC cells was reversed by pre-treatment with miR-34a inhibitors. ADAM10 was identified as a downstream gene of miR-34a, and down-regulated by miR-34a. Overexpression of ADAM10 reverted both miR-34a and sevoflurane-induced repression in the cell proliferation, migration and invasion abilities of CRC cells. Our data showed Sevoflurane inhibits the migration and invasion of colorectal cancer cells by regulating microRNA-34a/ADAM10 axis.

The effect and mechanism of miR-210 in down-regulating the autophagy of lung cancer cells

This project aims to investigate the roles of miR-210 in autophagy of lung cancer cells and the related mechanism. The expressions of miR-210 and ATG7 in 30 cancer tissues and the adjacent tissues in patients with lung cancer were compared using RT-qPCR methods, Western Blot assay was carried out to test the expression of ATG7 in protein. Moreover, the dual luciferase reporter gene assay system was used to confirm ATG7 is a target gene of miR-210. Furthermore, lung cancer cell line A549 was transfected with either miR-210 mimics or inhibitors and RT-qPCR methods was used to detect the expression of miR-210 and ATG7. Next, MTT assay was used to examine the effect of miR-210 on the growth of the lung cancer cells, and finally, the expression of autophagy related genes, ATG7, LC3-II/LC3-I and Beclin-1 were detected by Western Blot and ICC assay. We observed that miR-210 was significantly increased and ATG7 was markedly decreased in cancer tissue of patients with lung cancer compared with normal tissue. Moreover, results of dual luciferase reporter assay indicated that ATG7 is a direct target of miR-210. Next, transfection of miR-210 mimics in lung cancer cells induced significant increase in cell proliferation, and transfection of miR-210 inhibitors lead to inhibited cell proliferation. Furthermore, over-expression of miR-210 induced marked decrease in the expression of ATG7, LC3-II/LC3-I and Beclin-1, while transfection of miR-210 inhibitors induced significant increase in the expression of ATG7, LC3-II/LC3-I and beclin-1. Our results suggested that miR-210 plays a great role in autophagy of lung cancer cell by targeting ATG7.

Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D₁ Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay.

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

Cyanidin-3-glucoside Alleviates 4-Hydroxyhexenal-Induced NLRP3 Inflammasome Activation via JNK-c-Jun/AP-1 Pathway in Human Retinal Pigment Epithelial Cells.

Recently, the NLRP3 inflammasome activation in the eyes has been known to be associated with the pathogenesis of age-related macular degeneration. The aim of this study was to investigate the protective effects of cyanidin-3-glucoside (C3G), an important anthocyanin with great potential for preventing eye diseases, against 4-hydroxyhexenal- (HHE-) induced inflammatory damages in human retinal pigment epithelial cells, ARPE-19. We noticed that C3G pretreatment to the ARPE-19 cells rescued HHE-induced antiproliferative effects. Cell apoptosis ratio induced by HHE was also decreased by C3G, measured by flow cytometry. The activation of NLRP3 inflammasome induced by HHE was found with increases of caspase-1 activity, proinflammatory cytokine releases (IL-1β and IL-18), and NLRP3 inflammasome-related gene expressions (NLRP3, IL-1β, IL-18, and caspase-1). The C3G showed potent inhibitive effects on these NLRP3 inflammasome activation hallmarks induced by HHE. Moreover, we noticed that the C3G's pretreatment leads to a delayed and a decreased JNK activation in HHE-challenged ARPE-19 cells. Finally, using a luciferase reporter gene assay system, we demonstrated that HHE-induced activation protein- (AP-) 1 transcription activity was abolished by C3G pretreatment in a dose-dependent manner. Taken together, these data showed that HHE leads to inflammatory damages to ARPE-19 cells while C3G has great protective effects, highlighting future potential applications of C3G against AMD-associated inflammation.

The cloning and activity of human Hes1 gene promoter.

The aim of the current study was to obtain and analyze the activity of the human Hes1 gene promoter. The genomic DNA of human HeLa cell was used as template, polymerase chain reaction (PCR) was used to amplify the 5' end sequence of Hes1 gene and then the amplified segment was connected to pMD18‑T vector. Subsequently, double enzyme digestion was used for identification and the sequence was detected; the promoter with the correct sequence was inserted into pGL3‑Basic, and the sequence was identified by double enzyme digestion. The recombinant DNA with correct sequence was transiently transfected into cervical cancer cells, and the dual luciferase reporter gene assay system was used to detect the activity of the promoter. The results demonstrated that the human Hes1 gene promoter amplified by PCR was the same as that of the sequence in the gene bank, and the dual luciferase reporter gene assay system demonstrated that there was promoter activity in cervical cancer cells. In conclusion, the Hes1 luciferase reporter recombinant vector was successfully established and transfected into HeLa cells to verify that it has promoter activity, and the core area of the promoter has several tumor‑promoting and tumor suppressor genes. This provides a basis for understanding the regulatory mechanism of Hes1 transcription and translation.

Cimicifugamide from Cimicifuga rhizomes functions as a nonselective β-AR agonist for cardiac and sudorific effects

Cimicifuga rhizomes (CR) are used in the treatment of respiratory and cardiovascular diseases in traditional Chinese medicine, but their key effective components and mechanism of action have not yet been reported. In this study, the cardiac, antipyretic and sudorific effects of CR were evaluated using the toad heart failure in vitro model and mice fever and sweating in vivo models. Moreover, the UPLC/Q-TOF-MS-integrated β2-AR luciferase reporter gene assay system was used to screen the bioactive ingredients from CR extract, and the activity of this ingredient were verified using the above-mentioned in vitro and vivo models. Our results showed that CR had anti-heart failure, antipyretic and sweating effects, which could be antagonized by propranolol. On the other hand, cimicifugamide was screened as β2-AR agonist from CR and cimicifugamide could activate β1, 2-ARs more significantly than β3-AR in β-ARs selectivity assessment. The results not only revealed the key effective components and mechanism of CR in traditional use but also supplied a characteristic complementary ingredient for quality control of CR.

MiRNA-100 promotes proliferation of human leukemia cells HL-60 by targeting carboxy-terminal domain small phosphatase-like protein

Objective To investigate the effect of miRNA-100 on the proliferation of human leukemia cells HL-60, and to explore the mechanism of this action. Methods The bioinformatics software and database were applied to predict and analyze target genes of miRNA-100. The vector contained the target gene 3'UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miRNA-100 mimics and target gene wild- type or mutant plasmid into HEK-293T cells. HL-60 cells were transfected with miRNA-100 mimics or anti-miRNA-100. After transfection, Western blot was applied to validate the expression of carboxy-terminal domain small phosphatase-like protein (CTDSPL), and the viability of HL-60 was measured by using cell counting kit (CCK-8) assay at 24 h, 48 h, 72 h, 96 h. Results Online software predicted that CTDSPL was likely to be the target gene of miRNA-100.Dual luciferase reporter gene assay system showed that miRNA-100 could significantly suppress the activity of reporter gene containing CTDSPL 3'-UTR which decreased by about 57.1%(P=0.000 7). Western blot showed that the expression of CTDSPL was increased after being transfected with miRNA-100 antisense oligonucleotides and decreased after being transfected with miRNA-100 mimics.At the same time, the growth rate of cells treated with miRNA-100 mimics or CTDSPL miRNA-100 was increased compared with that in control by CCK-8 test(P<0.05). Conclusions CTDSPL is a downstream target gene of miRNA-100.miRNA-100 can promote leukemia cell proliferation by inhibiting the expression of CTDSPL. Key words: miRNA-100; Carboxy-terminal domain small phosphatase-like protein; Proliferation

Comparative evaluation of different cultivars of Flos Chrysanthemi by an anti-inflammatory-based NF-κB reporter gene assay coupled to UPLC-Q/TOF MS with PCA and ANN

Ethnopharmacological relevanceFlos Chrysanthemi (FC), a commonly used traditional Chinese medicine, has five major cultivars (“Boju”, “Chuju”, “Gongju”, “Hangbaiju” and “Huaiju”) from different sources. However, the active constituents of these cultivars have not been studied or characterized with respect to their bioactivity, which is a serious problem when considering quality and safety. Aim of the studyTo evaluate the differences among the five cultivars of FC, and to establish a method for the standardization and quality control of FC related to its bioactivity. Materials and methodsIn this study, the different ingredients in five cultivars of FC were identified by UPLC-Q/TOF and PCA, and the anti-inflammatory ingredients of FC were predicted and screened by artificial neural network (ANN) and an NF-κB luciferase reporter gene assay system. Using this comprehensive method, we successfully screened the anti-inflammatory markers of different cultivars of FC. ResultsNineteen marker ingredients were confirmed to contribute strongly to the cluster, and eleven compounds in the five cultivars of FC were found to exert potential anti-inflammatory effects. Among these compounds, the NF-κB inhibitor activity of apigenin-7-O-6″-malonyl-glucoside, luteolin-7-O-rutinoside, quercetin-7-O-galactoside, quercetin-3-O-glucoside, apigenin-7-O-rutinoside and apigenin-7-O-glucoside were first reported here. Chlorogenic acid, luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid and luteolin were confirmed to be the most important anti-inflammatory marker ingredients useful for the quality control of FC. ConclusionsThe proposed efficient and systematic method is helpful for the standardization and quality control of FC. Moreover, this comprehensive strategy may prove to be a powerful technique for the rapid establishment of quality control procedures related to bioactivity for other herbal samples and foods.

Brain-derived neurotrophic factor is a novel target gene of the has-miR-183/96/182 cluster in retinal pigment epithelial cells following visible light exposure.

Light-induced retinal injury is clinically and experimentally well-documented. It may be categorized into three types: Photothermal, photomechanical and photochemical injuries. To date, the variation in the hsa-miR-183/96/182 cluster and its potential target genes in human primary retinal pigment epithelial (RPE) cells, following visible light exposure, has not been reported. In the present study, RPE cells were exposed to 4 h of constant visible light. The expression of the hsa-miR-183/96/182 cluster was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and its potential target genes were investigated. Additionally, hsa-miR-183, hsa-miR-96, hsa-miR-182 and has-miR-183/96/182 mimics were designed and synthesized in vitro, and transfected into the RPE cells. Subsequently, the expression of brain-derived neurotrophic factor (BDNF) mRNA and protein was measured, using RT-qPCR and western blotting, respectively. The regulation of miRNAs to the BDNF gene were then validated using a dual luciferase reporter gene assay system. The expression of hsa-miR-183, hsa-miR-96 and hsa-miR-182 significantly increased in RPE cells following 4 h of visible light exposure, compared with RPE cells that had been exposed to dark conditions (P<0.01). Following RPE cell transfection with mimics, BDNF mRNA and protein expression in the RPE cells was significantly downregulated compared with control RPE cells (P<0.05, P<0.01, respectively). Similarly, the ratio of Renilla luciferase/firefly luciferase significantly decreased in the RPE cells of the mimic + wild type (WT) group compared with cells of the psiCHECK(TM)-2 (a vector lacking the sequence of the BDNF gene), wild type and mimic + mutation groups (P<0.05, P<0.01). The present study suggests that BDNF is a target gene of the has-miR-183-96-182 cluster in RPE cells. The present study suggests an underlying protective mechanism against retinal light injury and may provide a novel target for the prevention and treatment of light-induced retinal injury.

MiR-125b promotes proliferation of human acute myeloid leukemia cells by targeting Bak1

To investigate miR- 125b regulation mechanism by identifying miR-125b target genes and its function in acute myeloid leukemia (AML). The bioinformatics software and database were applied to predict and analyze target genes of miR-125b. The vector contained the target gene 3'-UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miR-125b mimics and target gene wild-type or mutant plasmid into HEK-293T cells. Further in leukemia cell lines NB4 and HL-60, the protein level of target gene was measured by Western blot after overexpression miR-125b. Finally, the viabilities of NB4 and HL-60 cells were measured by CCK-8 assay at 24 h, 48 h, 72 h, 96 h after electroporation. Bcl-2-antagonist/killer 1 (Bak1), a pro-apoptotic gene, was a target gene of miR-125b by software predicts. Reporter vector containing the 3'-UTR Bak1 wild and mutation sites were co-transfected with small molecule analogues of miR-125b in HEK-293T cells. Dual luciferase reporter gene assay system showed that miR-125b significantly suppressesthe reporter gene activity containing Bak1 3'-UTR by about 53.8% (P<0.05), but it didn't suppresses the reporter gene activity containing 3'-UTR Bak1 mutation. Western blot showed that miR-125b mimics significantly down-regulated the expression of Bak1 in human leukemia cell lines NB4 and HL-60. Meanwhile, the growth rate of cells treated with miR-125b obviously increased compared with that in control by CCK-8 test (P<0.05). Our findings strongly indicated that BAK1 was a downstream target gene of miR-125b, and miR-125b promoted proliferation in human AML cells at least partially by targeting Bak1, so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.

Butyrate-rich Colonic Microenvironment Is a Relevant Selection Factor for Metabolically Adapted Tumor Cells

The short chain fatty acid (SCFA) butyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.

Abstract B91: Induction of RXR transcriptional activity and consequent upregulation of p21 by 3-amino-6-(3-aminopropyl)-5,6-dihydro-5, 11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride in MCF-7 breast cancer cells

Abstract Retinoid X receptor (RXR) is known to play various roles in embryogenesis, metabolic processes, differentiation, and apoptosis. Recently, RXR has been studied as a target for cancer chemoprevention and treatment. To discover potential cancer chemopreventive agents acting through RXRs, we developed a cell line-based RXR response element (RXRE)-luciferase reporter gene assay system. Following extensive screening, 3-amino-6-(3aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities in a dose-dependent manner. Subsequently, the substance was found to inhibit the proliferation of MCF-7 breast cancer cells in a concentration-dependent manner. Prompted by these results, we examined the underlying mechanism of AM6-36 anti-proliferative activity using MCF-cells as a model. In brief, at lower concentrations, G2/M arrest was observed, and S phase arrest was enhanced at higher concentrations. Based on DNA microarray analysis, AM6-36 was found to up-regulate the expression of CDKN1A, a target gene of RXR, by ∼40-fold. In accordance with this response, the protein level of CDKN1A (p21WAF1/CIP1) was increased in the presence of AM6-36. Induction of p21 by AM6-36 was abrogated following transient transfection of RXR siRNA, demonstrating the effect of AM6-36 on the expression of p21 is closely related to modulation of RXR transcriptional activity. These data suggest AM6-36 is a promising lead compound for the treatment or prevention of breast cancer. A synthetic procedure has been devised for large-scale production, moderate absorption and slow metabolism has been demonstrated with rats (p.o. administration), and more advanced anti-tumor evaluation is underway. (Supported by program project P01 CA48112 awarded by the National Cancer Institute.) Citation Information: Cancer Prev Res 2010;3(1 Suppl):B91.

Aldose Reductase Regulates Hepatic Peroxisome Proliferator-activated Receptor α Phosphorylation and Activity to Impact Lipid Homeostasis

Aldose reductase (AR) is implicated in the development of a number of diabetic complications, but the underlying mechanisms remain to be fully elucidated. We performed this study to determine whether and how AR might influence hepatic peroxisome proliferator-activated receptor alpha (PPARalpha) activity and lipid metabolism. Our results in mouse hepatocyte AML12 cells show that AR overexpression caused strong suppression of PPARalpha/delta activity (74%, p < 0.001) together with significant down-regulation of mRNA expression for acetyl-CoA oxidase and carnitine palmitoyltransferase-1. These suppressive effects were attenuated by the selective AR inhibitor zopolrestat. Furthermore, AR overexpression greatly increased the levels of phosphorylated PPARalpha and ERK1/2. Moreover, AR-induced suppression of PPARalpha activity was attenuated by treatment with an inhibitor for ERK1/2 but not that for phosphoinositide 3-kinase, p38, or JNK. Importantly, similar effects were observed for cells exposed to 25 mm glucose. In streptozotocin-diabetic mice, AR inhibitor treatment or genetic deficiency of AR resulted in significant dephosphorylation of both PPARalpha and ERK1/2. With the dephosphorylation of PPARalpha, hepatic acetyl-CoA oxidase and apolipoprotein C-III mRNA expression was greatly affected and that was associated with substantial reductions in blood triglyceride and nonesterified fatty acid levels. These data indicate that AR plays an important role in the regulation of hepatic PPARalpha phosphorylation and activity and lipid homeostasis. A significant portion of the AR-induced modulation is achieved through ERK1/2 signaling.

Transfection of Primary Cultures of Rat Hepatocytes.

Five different transfection reagents-calcium phosphate, TransFast™ Transfection Reagent, Superfect™ Transfection Reagent, Effectene™ Transfection Reagent, and Tfx™-20-were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids. The efficiency of transfection of each reagent was monitored using the dual luciferase reporter gene assay system. Superfect Transfection Reagent, Effectene Transfection Reagent and Tfx-20 were the most effective for the transfection of primary hepatocytes and gave comparable transfection efficiencies. Calcium phosphate was found to be the least effective transfection reagent and gave the most variable transfection results. Tfx-20 gave the least variable transfection results when different hepatocyte preparations were compared.

Characterization of the 5' regulatory region of the mouse angiopoietin-like protein 4.

The 5' regulatory region of the mouse angiopoietin-like protein 4 (mANGPTL4), a remarkably versatile secreted protein responsible for hyperlipidaemia and angiogenesis, was cloned and functionally characterized. Three potential transcriptional start sites were determined by 5'-RACE and found to be at -129, -126 and -118, relative to the translation initiation codon. The activities of the putative promoters were confirmed using a firefly luciferase reporter gene assay system, following transient transfection into COS-1 cells. The PPAR alpha-regulated region and the minimal region required for basal activity of the mANGPTL4 promoter were determined by generating a series of deletion constructs, and were found to be encoded by a sequence between -2761 to -383 and -50 to -30, relative to the transcription start site. Putative recognition sequences for the transcription factor AP2 were identified in the minimal promoter sequences. These results are the first molecular characterization of the regulatory region of this important gene.

Analysis of the coupling of G12/13 to G protein-coupled receptors using a luciferase reporter assay.

G protein-coupled receptors (GPCRs) transduce signals from cell surface to intracellular effectors through various G proteins. In this chapter, a luciferase reporter gene assay system is described for the study of the coupling of GPCRs to G alpha 12/13. This assay system, in which ligand-stimulated production of the reporter luciferase under the control of a modified serum-responsive element (SRE) in a cell line derived from mice lacking G alpha q/11 that are transfected with a GPCR of interest, allows for easy, fast, and valid determination of the coupling of the GPCR to G alpha 12/13.