Levels Of Luciferase Activity Research Articles

The Biflavonoid Isoginkgetin Is a General Inhibitor of Pre-mRNA Splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected withTrypanosoma cruzi

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-kappaB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.

HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

Tip60 Is Regulated by Circadian Transcription Factor Clock and Is Involved in Cisplatin Resistance

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

A Receptor-targeted Nanocomplex Vector System Optimized for Respiratory Gene Transfer

A Receptor-targeted Nanocomplex Vector System Optimized for Respiratory Gene Transfer

Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells

To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial. We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content. Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels. Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.

A Chimeric Gene, FIP1L1-RARα, Is Isolated from t(4;17)-Positive Acute Promyelocytic Leukemia; a Report of Cloning and Functional Analysis.

Acute promyelocytic leukemia (APL) comprises 5 to 10% of acute myelogenous leukemia, the leukemia cells of which is characterized by the differentiation block at the stage of promyelocyte. In APL cells, a reciprocal translocation in chromosome 17 is usually observed, and consequently, a retinoic acid receptor alpha (RARα) gene is rearranged. The rearranged RARα gene is fused to another gene, and as a result, the product of newly composed fusion gene provokes the onset of APL. So far, there are five RARα partner genes reported; PML gene in 15q22, PLZF gene in 11q23, NPM gene in 5q35, NuMA gene in 11q13 Stat5b in 17q21. We recently experienced APL case, in which a chromosomal translocation, t(4;17) was observed. FISH analysis of leukemia cells indicated that RARα gene was translocated. To identify the RARα-partner gene, we performed 5′-rapid amplification of cDNA end (RACE) and identified FIP1L1 gene as RARα-partner gene. It is known that FIP1L1 gene is fused to PGDFRα gene in the hypereosinophilic syndrome. DNA sequence revealed that exon 15 of FIP1L1 gene was fused to exon 3 of RARα gene. In previous reports, five X-RARα have the common character;1.X-RARα manifests homodimerization.2.X-RARα suppresses retinoic acid-induced transcription.We examined that FIP1L1-RARα has these two characters. Transient transfection analysis showed that FIP1L1-RARα has the ability to form homodimer. And luciferase assay suggested that FIP1L1-RARα suppressed the retinoic acid-induced transcriptional activity at the physiological concentration of all-trans retinoic acid (ATRA). And the level of luciferase activity suppressed by FIP1L1-RARα was similar to that suppressed by PML-RARα. And the therapeutic concentration of ATRA activated the significant level of luciferase activity. Thus, in vitro analysis suggested that ATRA treatment could be effective to FIP1L1-RARα-positive APL patients. Consistent with the results of in vitro analysis, ATRA treatment was clinically effective, and the patient achieved complete remission after five weeks of ATRA treatment. Collectively, we suggest that FIP1L1-RAR? is the sixth pathogenic gene of APL.

Expression of shRNA using intron splicing

RNA is considered a highly promising candidate for several applications such as gene knock-down, gene repair and gene therapy, where double-stranded RNA and RNA with catalytic activity are key players. A group I intron, a ribozyme catalyzing its own splicing reactions in the absence of any proteins, has generated interest for its potential utility in gene repair using trans-splicing. On the other hand, the induction of small interfering RNA, via double-stranded RNA cleavage in short hairpin RNA (shRNA) by the RNase* *enzyme DICER is a convenient and powerful mechanism for gene silencing. We constructed shRNA expression vectors directed against Firefly luciferase, in which the loop region of the shRNA was interrupted by an intron. The decreased levels of luciferase activity were measured in cultured cells as an index of the ribozyme splicing activity.

Skin Sensitizers Induce Antioxidant Response Element Dependent Genes: Application to the In Vitro Testing of the Sensitization Potential of Chemicals

Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients and in vitro tests are needed to replace the current animal tests. Protein reactivity is the common feature of skin sensitizers and it is a crucial question whether a cellular in vitro assay can detect protein reactivity of diverse test chemicals. The signaling pathway involving the repressor protein Keap1 and the transcription factor nuclear factor-erythroid 2-related factor 2, which binds to the antioxidant response element (ARE) in the promoter of many phase II detoxification genes, is a potential cellular marker because Keap1 had been shown to be covalently modified by electrophiles which leads to activation of ARE-dependent genes. To evaluate whether this regulatory pathway can be used to develop a predictive cellular in vitro test for sensitization, 96 different chemicals of known skin sensitization potential were added to Hepa1C1C7 cells and the induction of the ARE-regulated quinone reductase (QR) activity was determined. In parallel, 102 chemicals were tested on the reporter cell line AREc32, which contains an eightfold repeat of the ARE sequence upstream of a luciferase gene. Among the strong/extreme skin sensitizers 14 out of 15 and 30 out of 34 moderate sensitizers induced the ARE-dependent luciferase activity and in many cases this response was paralleled by an induction of QR activity in Hepa1C1C7 cells. Sixty percent of the weak sensitizers also induced luciferase activity, and the overall accuracy of the assay was 83 percent. Only four of 30 tested nonsensitizers induced low levels of luciferase activity, indicating a high specificity of the assay. Thus, measurement of the induction of this signaling pathway provides an interesting in vitro test to screen for the skin sensitization potential of novel chemicals.

Oral DNA immunization in the second trimester fetal lamb and secondary immune responses in the neonate

We previously demonstrated that oral DNA vaccination of the third trimester fetus may be an effective strategy to prevent vertical disease transmission despite an immature immune system and the possibility of developing tolerance. The present investigation examined oral DNA vaccine delivery and immunogenicity in the second trimester fetal lamb (50–100 days of gestation (dg)). Histological examination revealed nucleated cells in the superficial layers of the oral mucosa. Therefore, luciferase-encoding DNA plasmid was injected into the oral cavity of 65–70 dg fetuses to monitor plasmid expression. Luciferase activity was detected in the oral mucosa of all fetuses but the level of luciferase activity varied among individual fetuses. Luciferase activity was also detected within the tonsils and lymph nodes draining the oral cavity. Oral DNA immunization between 55 and 83 dg with a truncated glycoprotein D (tgD)-encoding plasmid induced germinal centres in the draining lymph nodes and detectable tgD-specific serum antibody titers and/or IFNγ-secreting cell responses in 16 of 24 (67%) fetuses. The tgD-specific antibody and IFNγ responses persisted until birth in some fetuses but the magnitude of antibody titers in second trimester fetuses was low relative to antibody titers induced following oral DNA immunization in the third trimester. Lambs immunized during the second trimester of gestation responded to neonatal DNA immunization and anamnestic responses were detected in some lambs immunized as early as 67–72 dg. These observations confirmed that oral DNA immunization of the early second trimester fetus induced antigen-specific immune responses with no evidence of tolerance induction.

Construction of Modular Tandem Expression Vectors for the Green Alga Chlamydomonas Reinhardtii Using the Cre/lox-System

The successful expression of foreign genes mainly depends on both a reliable method for transformation and a suitable promoter sequence. We created a series of modular plasmids that facilitate the rapid construction of large tandem vectors for transgene expression under the control of different promoter sequences in Chlamydomonas reinhardtii. Tandem vectors carrying expression cassettes for Renilla luciferase and a metabolic selection marker (ARG7) were manufactured by fusing two plasmids in vitro using Cre/lox site-specific recombination. Supercoiled and linear plasmids were used to transform an arginine auxotrophic Chlamydomonas strain, and rates of co-expression as well as levels of luciferase activity were monitored for frequently used promoters (HSP70A, LHCB1, PSAD, and the chimeric HSP70A/RBCS2). Linearized tandem vectors generally increased the co-expression frequency (up to 77%) compared with standard cotransformation protocols. Most transformants showed a single and complete integration event confirming the close linkage of active selectable marker and reporter gene within the nuclear genome. The analysis of luciferase activity showed expression levels within three orders of magnitude for the promoters used, with the artificial HSP70A/RRBCS2 being the most active. For 69% of all luminescent transformants carrying the HSP70A promoter luciferase expression was enhanced by heatshock, indicating physiological promoter function in a transgenic context.

Design of PCR‐amplified DNA fragments for in vivo gene delivery: Size‐dependency on stability and transgene expression

PCR-amplified DNA fragments can be more efficient and safer vectors than conventional plasmid DNA because of their smaller size and fewer numbers of immunostimulatory cytosine-phosphate-guanine (CpG) motifs. In the present study, the expression unit of plasmid DNA encoding farnesylated enhanced green fluorescent protein (EGFPF; pEGFP-F) or firefly luciferase (pLuc) was amplified by polymerase chain reaction (PCR) to obtain DNA fragments (EGFPF-mini, Luc-mini). EGFPF-mini was as effective as pEGFP-F on the basis of the number of EGFPF-expressing cells after intravenous injection into mice by the hydrodynamics-based procedure. Then, the effects of the length of DNA fragments on transgene expression were examined using luciferase-expressing DNA preparations. Luc-mini preparations showed high levels of luciferase activity in cultured cells as well as in mouse liver, even although the levels did not exceed that of pLuc. An elongation of the DNA fragment on either side of the minimal expression unit was effective in increasing the transgene expression and the stability against nucleases. PCR-amplified DNA fragments showed a sustained luciferase activity in mouse liver compared with pLuc, indicating that they are effective in achieving a prolonged expression. Their stabilization against nucleases will further increase the potential of such short, structure-controlled and synthetic DNA fragments for in vivo gene delivery.

Fully Codon-Optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock

We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.

An improved and secreted luciferase reporter for schistosomes

Schistosomes are multicellular parasites of humans exhibiting interesting biological adaptations to their parasitic lifestyle. Concerted and in depth analyses of these adaptations and their cell and molecular biology requires further development of molecular genetic tools in schistosomes. In the current study, we demonstrate that a Gaussia luciferase reporter leads to significantly higher levels of luciferase activity in schistosomes compared to other tested luciferases. In addition, Gaussia luciferase can be secreted into culture media enabling non-invasive analysis of reporter activity. The secretion of Gaussia luciferase should allow a variety of new experimental paradigms for schistosome studies. Comparison of biolistic and electroporation transfection methods using luciferase RNA reporters and the luciferase acitivty produced indicates that electroporation of sporocysts and schistosomula is the most efficient transfection method for the four stages analyzed. These data should facilitate additional studies in schistosomes and provide a framework for further development of DNA transfection and gene expression analysis.

Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication

Similar to all other viruses, human immunodeficiency virus type 1 (HIV-1) depends heavily on cellular factors for its successful replication. In this study we have investigated the interaction of HIV-1 integrase (IN) with several host nuclear import factors using co-immunoprecipitation assays. Our results indicate that IN interacts specifically with host importin 7 (Imp7) in vivo, but does not interact with importin 8 (Imp8) or importin alpha (Rch1). In contrast, another HIV-1 karyophilic protein MAp17, which is capable of binding Rch1, fails to interact with Imp7, suggesting that IN and Map17 may interact with different cellular pathways during HIV-1 replication. Genetic analysis revealed that the C-terminal domain of IN is the region responsible for interaction between IN with Imp7, and an IN mutant (K240A,K244A/R263A,K264A) disrupted the Imp7 binding ability of the protein, indicating that both regions ((235)WKGPAKLLWKG and (262)RRKAK) within the C-terminal domain of IN are required for efficient IN/Imp7 interaction. Using a vesicular stomatitis virus G glycoprotein pseudotyped HIV single-cycle replication system, we showed that the IN/Imp7 interaction-deficient mutant was unable to mediate viral replication and displayed impairment at both viral reverse transcription and nuclear import steps. Moreover, transient knockdown of Imp7 in both HIV-1 producing and target cells resulted in a 2.5-3.5-fold inhibition of HIV infection. Altogether, our results indicate that HIV-1 IN specifically interacts with Imp7, and this viral/cellular protein interaction contributes to efficient HIV-1 infection.

A Sequence Motif in the Simian Virus 40 (SV40) Early Core Promoter Affects Alternative Splicing of Transcribed mRNA

To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.

Dysregulation of Cardiogenesis, Cardiac Conduction, and Cell Cycle in Mice Lacking miRNA-1-2

MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

Protein kinase Cα is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the Aγ globin‐promoter in cellular models of hemoglobin switching

PKCalpha was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

Establishment of a Human Hepatoma Cell Line HepG2-A10 for a Reporter Gene Assay of Arylhydrocarbon Receptor Activators

To establish a human hepatic cell line for a convenient reporter gene assay of arylhydrocarbon receptor (AhR) activators, the chimera plasmid containing xenobiotic responsible element (XRE), minimal SV40 promoter, and luciferase reporter gene and the expression vector pRC/CMV containing a neomycin-resistant gene were co-transfected into a human hepatoma cell line, HepG2. Then, antibiotic (G418)-resistant HepG2 cells were selected and cloned. A cell clone, HepG2-A10, showed the highest responsibility to 3-methylcholanthrene (MC)-mediated induction of luciferase among the clones obtained. Expression levels of luciferase activity in HepG2-A10 cells were increased in a dose- and time-dependent manner by treatment with either MC, an AhR-ligand type activator, or omeprazole (OME), a non-AhR ligand type activator. In addition, expression levels of cytochrome P4501A subfamily genes (CYP1A1 and CYP1A2) were also increased in a dose- and time-dependent manner by treatment with MC. The present findings demonstrate that a newly established human HepG2-A10 is a useful cell line for a convenient reporter gene assay of AhR activators.