RUNX1 Inhibition Using Lipid Nanoparticle-Mediated Silencing RNA Delivery as an Effective Treatment for Acute Leukemias.

Transcription factor RUNX1 plays key roles in the establishment and maintenance of the hematopoietic system. Although RUNX1 has been considered a beneficial tumor suppressor, several recent reports have described the tumor-promoting role of RUNX1 in a variety of hematopoietic neoplasms. In this study, we assessed the effect of RUNX1 depletion in multiple human leukemia cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, and confirmed that RUNX1 is in fact required for sustaining their leukemic proliferation. To achieve efficient RUNX1 inhibition in leukemia cells, we then examined the effect of lipid nanoparticle (LNP)-mediated delivery of RUNX1-targeting small interfering (si)RNA using two tumor-tropic LNPs. The LNPs containing RUNX1-targeting siRNA were efficiently incorporated into myeloid and T-cell leukemia cell lines and patient-derived primary human acute myeloid leukemia (AML) cells, downregulated RUNX1 expression, induced cell cycle arrest and apoptosis, and exhibited the growth-inhibitory effect in them. In contrast, the LNPs were not efficiently incorporated into normal cord blood CD34+ cells, indicating their minimum cytotoxicity. Thus, our study highlights RUNX1 as a potential therapeutic target to inhibit leukemogenesis, and provides the LNP-based siRNA delivery as a promising approach to deplete RUNX1 specifically in leukemia cells.

Immunohistochemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the developmental dentate gyrus and hippocampal fimbria in fetal mice.

Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice.

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain.

Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).

Tyrosine hydroxylase assay: a bioassay for aryl hydrocarbon receptor-active compounds based on tyrosine hydroxylase promoter activation.

Tyrosine hydroxylase (TH) assay is a novel bioassay for screening aryl hydrocarbon receptor (AhR)-active compounds with diverse toxicities. The TH assay employs reporter genes to evaluate TH promoter activity, which is linked to dopamine synthesis and regulated by the AhR-aryl hydrocarbon receptor responsive element-III (AHRE-III)-mediated pathway. The toxic equivalency factors for higher chlorinated dioxin congeners in the TH assay (TH-TEF) exhibit the same tendency as those for the WHO-TEF, indicating that the activity of the TH assay is consistent with that of existing methods. Furthermore, other chemicals, such as lower chlorinated dioxin congeners that were minimally investigated in the past could be detected preferentially by this assay.

A subclone of HuH-7 with enhanced intracellular hepatitis C virus production and evasion of virus related-cell cycle arrest.

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.

Specific interactions between aryl hydrocarbon receptor and dioxin congeners: ab initio fragment molecular orbital calculations.

Aryl hydrocarbon receptor (AhR) is a transcription factor and its function is activated by the binding of halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,4-trichlorodibenzo-p-dioxin (TrCDD). TCDD is highly toxic to rat, whereas its congener TrCDD shows only a weak effect on gene expression. In order to elucidate the reason of this remarkable difference in the effect of TCDD and TrCDD, we here obtained stable structures of the complexes with rat AhR (rAhR) and TCDD/TrCDD and investigated their electronic properties by using the ab initio fragment molecular orbital (FMO) method. The results indicate that TCDD binds more strongly to rAhR than TrCDD, which is consistent with the experimentally observed toxicity of TCDD and TrCDD. Furthermore, ab initio FMO calculations elucidate that His324 and Gln381 of rAhR are important for binding TCDD, while His324 and Ser334 are important for TrCDD binding.

Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study.

BACKGROUND: Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR). This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. METHODS: N2a-Rbeta cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA). RESULTS: Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III) was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE), though AHRE-III was not identical to XRE, the conventional AhR-binding motif. CONCLUSION: Our results suggest TCDD directly regulate the dopamine system by TH gene transactivation via an AhR-AHRE-III-mediated pathway. The AhR- mediated pathway could have a particular AhR-mediated genomic control pathway transmitting the effects of TCDD action to target cells in the development of dopaminergic disabilities.

Over-expression of AhR (aryl hydrocarbon receptor) induces neural differentiation of Neuro2a cells: neurotoxicology study.

BACKGROUND: Dioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s) of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR). AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE). Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron. METHODS: The influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC) was stably transfected with AhR cDNA and the established cell line was named N2a-Ralpha. The activation of exogenous AhR in N2a-Ralpha cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Ralpha based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH). RESULTS: N2a-Ralpha cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Ralpha cells exhibited two significant functional features. Morphologically, N2a-Ralpha cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Ralpha cells expressed tyrosine hydroxylase (TH) mRNA as a functional marker of catecholaminergic neurotransmitter production. Thus, exogenous AhR induced catecholaminergic differentiation in N2a-Ralpha cells. CONCLUSION: The excessive activation of AhR resulted in neural differentiation of Neuro2a cells. This result revealed that dioxins may affect the nervous system through the AhR-signaling pathway. Activated AhR may disrupt the strictly regulated brain formation with irregular differentiation occurring rather than cell death.