Fatty acid 2-hydroxylase (FA2H) as a stimulatory molecule responsible for breast cancer cell migration.

The functional role of fatty acid 2-hydroxylase (FA2H) is controversial in the field of cancer biology due to the dual role of FA2H, particularly related to its interaction with triple-negative breast cancer (TNBC). A previous biochemical- and clinical-focused study suggested that FA2H could dampen TNBC aggressiveness. However, another epidemiological study demonstrated that FA2H expression is associated with shorter disease-free survival in TNBC cases. We reported that FA2H is a peroxisome proliferator-activated receptor α (PPARα)-regulated gene in human breast cancer MDA-MB-231 cells, in vitro experimental models for TNBC analysis. PPARα activation by its ligand reportedly results in an aggressive MDA-MB-231 cell phenotype, as well as estrogen receptor α (ERα)-positive MCF-7 cells. The results of this study show that i) MDA-MB-231 cells express very low levels of FA2H compared to the MCF-7 cells, reflecting a low basal-level PPARα-driven transcriptional activity compared to the MCF-7 cells, and ii) the increased FA2H expression stimulates the MDA-MB-231 and MCF-7 breast cancer cell migration without affecting proliferation. Taken together, our findings indicate that FA2H might be a breast cancer cell migration stimulator, independently of the ERα expression status.

Products of Oxidative Guanine Damage Form Base Pairs with Guanine.

Among the natural bases, guanine is the most oxidizable base. The damage caused by oxidation of guanine, commonly referred to as oxidative guanine damage, results in the formation of several products, including 2,5-diamino-4H-imidazol-4-one (Iz), 2,2,4-triamino-5(2H)-oxazolone (Oz), guanidinoformimine (Gf), guanidinohydantoin/iminoallantoin (Gh/Ia), spiroiminodihydantoin (Sp), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), urea (Ua), 5-guanidino-4-nitroimidazole (NI), spirodi(iminohydantoin) (5-Si and 8-Si), triazine, the M+7 product, other products by peroxynitrite, alkylated guanines, and 8,5'-cyclo-2'-deoxyguanosine (cG). Herein, we summarize the present knowledge about base pairs containing the products of oxidative guanine damage and guanine. Of these products, Iz is involved in G-C transversions. Oz, Gh/Ia, and Sp form preferably Oz:G, Gh/Ia:G, and Sp:G base pairs in some cases. An involvement of Gf, 2Ih, Ua, 5-Si, 8-Si, triazine, the M+7 product, and 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA) in G-C transversions requires further experiments. In addition, we describe base pairs that target the RNA-dependent RNA polymerase (RdRp) of RNA viruses and describe implications for the 2019 novel coronavirus (SARS-CoV-2): When products of oxidative guanine damage are adapted for the ribonucleoside analogs, mimics of oxidative guanine damages, which can form base pairs, may become antiviral agents for SARS-CoV-2.

Analysis of nucleotide insertion opposite 2,2,4-triamino-5(2H)-oxazolone by eukaryotic B- and Y-family DNA polymerases.

Mutations induced by oxidative DNA damage can cause diseases such as cancer. In particular, G:C-T:A and G:C-C:G transversions are caused by oxidized guanine and have been observed in the p53 and K-ras genes. We focused on an oxidized form of guanine, 2,2,4-triamino-5(2H)-oxazolone (Oz), as a cause of G:C-C:G transversions based on our earlier elucidation that DNA polymerases (Pols) α, ß, γ, ε, η, I, and IV incorporate dGTP opposite Oz. The nucleotide insertion and extension of Pols δ, ζ, ι, κ, and REV1, belonging to the B- and Y-families of DNA polymerases, were analyzed for the first time. Pol δ incorporated dGTP, in common with other replicative DNA polymerases. Pol ζ incorporated dGTP and dATP, and the efficiency of elongation up to full-length beyond Oz was almost the same as that beyond G. Although nucleotide incorporation by Pols ι or κ was also error-prone, they did not extend the primer. On the other hand, the polymerase REV1 predominantly incorporated dCTP opposite Oz more efficiently than opposite 8-oxo-7,8-dihydroguanine, guanidinohydantoin, or tetrahydrofuran. Here, we demonstrate that Pol ζ can efficiently replicate DNA containing Oz and that REV1 can prevent G:C-C:G transversions caused by Oz.

Calculation of the HOMO localization of Tetrahymena and Oxytricha telomeric quadruplex DNA.

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3'-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5'-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K(+) and d(TAGGGT)4T +2K(+) localized at the 5'-guanine, and that the HOMO shifted from the 5'-guanine to the 3'-guanine by the addition of a 5'-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K(+) and d(TAGGGT)4T +2K(+) matched the experimental results and suggest that d(TGGGGT)4 contains a 5'-capping cation in solution.

Transcriptional regulation of Tal2 gene by all-trans retinoic acid (atRA) in P19 cells.

TAL2 is a transcription factor required in the normal development of mouse brain. In a previous study, we demonstrated that the expression of Tal2 gene is induced by the complex of all-trans retinoic acid (atRA) and retinoic acid receptor α (RARα) in mouse embryonal carcinoma P19 cells. atRA is also known to be important in inducing P19 cells to differentiate into the neural lineage. Therefore, we believe that the function of TAL2 in neural differentiation may be clarified by utilizing P19 cells. As the atRA-RARα complex induced the expression of Tal2, we focused on the regulatory region that is involved in its transcription. The atRA-RARα complex occupies a characteristic retinoic acid response element (RARE) located in the promoter of target genes. Therefore, we searched for RARE on the mouse Tal2 and found that a RARE-like element was located in the intron. We also found that a TATA-box-like element was located in the 5'-region of Tal2. Involvement between transcriptional activity and the TATA-box-like element was confirmed in the luciferase assay, and TATA-box binding protein was bound to this element upstream of Tal2 in P19 cells. atRA signaling activated the transcription through the RARE-like element, and RARα was bound to this element on Tal2 in P19 cells. In addition, the interaction between these elements on Tal2 was shown in the chromatin immunoprecipitation assay. These results suggest that the transcription of Tal2 is coordinately mediated by two distal regulatory elements.

Synthesis and structure-activity relationship study of triazine-based inhibitors of the DNA binding of NF-κB.

Nuclear transcription factor nuclear factor-kappa B (NF-κB) has diverse pathophysiological functions, and NF-κB inhibitors are considered to be candidates for multiple therapeutic applications. We previously reported a novel triazine-based NF-κB inhibitor, 2-anilino-4,6-dichloro-1,3,5-triazine (NI241), that directly inhibits DNA binding of NF-κB. Here, we report synthesis of a series of triazine derivatives and evaluation of their structure-activity relationships for NF-κB inhibition. We found that 2-amino-4,6-dichloro-1,3,5-triazine substructure is essential for the inhibitory activity of the lead compound NI241, and modification of NI241 by introduction of an m-methoxy substituent on the phenyl ring afforded the more potent derivative 28. The structure-activity relationships identified in this study suggested a possible mechanism of irreversible NF-κB inhibition by NI241, and should be helpful in the design of other NF-κB inhibitors.

Formation of a flavin-linked peptide.

In a previous study, we showed that formylmethylflavin (FMF) can bind to cysteine. In this study, FMF was reacted with native peptides (CG and CKLVFF) containing an N-terminal cysteine. The formation of flavin-CG and flavin-CKLVFF was confirmed using HPLC and ESI-MS. Storage of flavin-CKLVFF in DMSO at -30 °C for 7 days resulted in no detectable deposition. In contrast, flavin-CKLVFF formed deposits when stored in water at -30 °C for 1 day, but no deposit was observed in the aqueous solution of flavin-CKLVFF after 7 days storage in the presence of 0.1% Triton X-100.

Calculating distortions of short DNA duplexes with base pairing between an oxidatively damaged guanine and a guanine.

DNA is constantly being oxidized, and oxidized DNA is prone to mutation; moreover, guanine is highly sensitive to several oxidative stressors. Several oxidatively damaged forms of guanine-including 2,2,4-triamino-5(2H)-oxazolone (Oz), iminoallantoin (Ia), and spiroiminodihydantoin (Sp)-can be paired with guanine, and cause G:C-C:G transversions. Previous findings indicate that guanine is incorporated more efficiently opposite Oz than opposite Ia or Sp, and that these differences in efficiency cannot be explained by differences in the stabilities of G:Oz, G:Ia, and G:Sp base pairs calculated ab initio. Here, to explain previous experimental result, we used a 3-base-pair model DNA duplex to calculate the difference in the stability and the distortion of DNA containing a G:Oz, G:Ia, or G:Sp base pair. We found that the stability of the structure containing 5' and 3' base pairs adjacent to G:Oz was more stable than that containing the respective base pairs adjacent to G:Ia or G:Sp. Moreover, the distortion of the structure in the DNA model duplex that contained a G:Oz was smaller than that containing a G:Ia or G:Sp. Therefore, our discussion can explain the previous results involving translesion synthesis past an oxidatively damaged guanine.

A DNA oligomer containing 2,2,4-triamino-5(2H)-oxazolone is incised by human NEIL1 and NTH1.

The nucleobase derivative, 2,2,4-triamino-5(2H)-oxazolone (Oz), is an oxidation product of guanine or of 8-oxo-7,8-dihydroguanine that causes G-to-C transversions in DNA. Human NEIL1 (hNEIL1) and NTH1 (hNTH1) are homologues of two prokaryotic base excision repair enzymes, FPG/NEI and NTH, respectively. Here, we demonstrated that hNEIL1 and hNTH1 cleave Oz sites as efficiently as 5-hydroxyuracil sites. Thus, hNEIL1 and hNTH1 can repair Oz lesions. Furthermore, the nicking activities of these enzymes are largely independent of nucleobases opposite Oz; this finding indicates that removing Oz from Oz:G and Oz:A base pairs might cause an increase in the rate of point mutations in human cells.

Calculation of the stabilization energies of oxidatively damaged guanine base pairs with guanine.

DNA is constantly exposed to endogenous and exogenous oxidative stresses. Damaged DNA can cause mutations, which may increase the risk of developing cancer and other diseases. G:C-C:G transversions are caused by various oxidative stresses. 2,2,4-Triamino-5(2H)-oxazolone (Oz), guanidinohydantoin (Gh)/iminoallantoin (Ia) and spiro-imino-dihydantoin (Sp) are known products of oxidative guanine damage. These damaged bases can base pair with guanine and cause G:C-C:G transversions. In this study, the stabilization energies of these bases paired with guanine were calculated in vacuo and in water. The calculated stabilization energies of the Ia:G base pairs were similar to that of the native C:G base pair, and both bases pairs have three hydrogen bonds. By contrast, the calculated stabilization energies of Gh:G, which form two hydrogen bonds, were lower than the Ia:G base pairs, suggesting that the stabilization energy depends on the number of hydrogen bonds. In addition, the Sp:G base pairs were less stable than the Ia:G base pairs. Furthermore, calculations showed that the Oz:G base pairs were less stable than the Ia:G, Gh:G and Sp:G base pairs, even though experimental results showed that incorporation of guanine opposite Oz is more efficient than that opposite Gh/Ia and Sp.

Analysis of nucleotide insertion opposite urea and translesion synthesis across urea by DNA polymerases.

Urea (Ua) is produced in DNA as the result of oxidative damage to thymine and guanine. It was previously reported that Klenow fragment (Kf) exo- incorporated dATP opposite Ua, and that DNA polymerase ß was blocked by Ua. We report here the following nucleotide incorporations opposite Ua by various DNA polymerases: DNA polymerase α, dATP and dGTP (dATP > dGTP); DNA polymerase δ, dATP; DNA polymerase ζ, dATP; Kf exo-, dATP; Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), dGTP and dATP (dGTP > dATP); and DNA polymerase η, dCTP, dGTP, dATP, and dTTP (dCTP > dGTP > dATP > dTTP). DNA polymerases ß and ε were blocked by Ua. Elongation by DNA polymerases δ and ζ stopped after inserting dATP opposite Ua. Importantly, the elongation efficiency to full-length beyond Ua using DNA polymerase η and Dpo4 were almost the same as that of natural DNA.

A quantitatively-modeled homozygosity mapping algorithm, qHomozygosityMapping, utilizing whole genome single nucleotide polymorphism genotyping data.

Homozygosity mapping is a powerful procedure that is capable of detecting recessive disease-causing genes in a few patients from families with a history of inbreeding. We report here a homozygosity mapping algorithm for high-density single nucleotide polymorphism arrays that is able to (i) correct genotyping errors, (ii) search for autozygous segments genome-wide through regions with runs of homozygous SNPs, (iii) check the validity of the inbreeding history, and (iv) calculate the probability of the disease-causing gene being located in the regions identified. The genotyping error correction restored an average of 94.2% of the total length of all regions with run of homozygous SNPs, and 99.9% of the total length of them that were longer than 2 cM. At the end of the analysis, we would know the probability that regions identified contain a disease-causing gene, and we would be able to determine how much effort should be devoted to scrutinizing the regions. We confirmed the power of this algorithm using 6 patients with Siiyama-type α1-antitrypsin deficiency, a rare autosomal recessive disease in Japan. Our procedure will accelerate the identification of disease-causing genes using high-density SNP array data.

The oxidation of 8-oxo-7,8-dihydroguanine by iodine.

8-Oxo-7,8-dihydroguanine was specifically oxidized by iodine with aqueous KI. Under acidic conditions, the major product was dehydro-guanidinohydantoin. Under basic conditions, two diastereoisomers of spirohydantoin were chiefly obtained. In addition, unstable diimine was detected for the first time.

Photoirradiation products of flavin derivatives, and the effects of photooxidation on guanine.

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.

A new small molecule that directly inhibits the DNA binding of NF-kappaB.

Nuclear factor-kappaB (NF-kappaB) has been considered as a good target for the treatment of many diseases. Although a lot of NF-kappaB inhibitors have already been reported, many of them have several common problems. Thus, we attempted to identify novel NF-kappaB inhibitors to be unique lead compounds for creating new pharmaceuticals. In the present study, we screened our chemical library for compounds that directly inhibit the DNA binding of NF-kappaB by using fluorescence correlation spectroscopy (FCS). Consequently, we identified a promising compound, 4,6-dichloro-N-phenyl-1,3,5-triazin-2-amine, referred to as NI241. It mediated a dose-dependent inhibition of the DNA binding of NF-kappaB p50. Its analogues also showed dose-dependent inhibition and their inhibitory effects were altered by the substituents on the N-phenyl group. Furthermore, we predicted the binding mode of NI241 with p50 in silico. In this model, NI241 forms three hydrogen bonds with Tyr60, His144, and Asp242 on p50, which are important amino acid residues for the interaction with DNA. These results suggest that NI241 with structural novelty may serve as a useful scaffold for the creation of new NF-kappaB inhibitors by rational optimization.

Cadmium down-regulates apolipoprotein E (ApoE) expression during malignant transformation of rat liver cells: direct evidence for DNA hypermethylation in the promoter region of ApoE.

There is adequate evidence for the carcinogenicity of cadmium (Cd). However, a significant unaddressed question remains as to how this metal actually causes malignant transformation (tumor initiation). Since it has been shown that Cd only has the weak direct interaction potential with DNA, the metal is recognized as an indirect genotoxicant and mutagen. Currently, Cd-mediated "epigenetic" modifications, such as changes in DNA methylation resulting in alteration in target gene expression, coupled with cancer progression, are the focus of mechanistic research. We have reported that the apolipoprotein E (ApoE) gene, a suppressor of cell invasion, is an early Cd target, and is involved in the malignant transformation of TRL 1215 rodent liver cells. Cd exposure suppresses ApoE expression which can be re-activated with 5-aza-2'-deoxycytidine, a DNA demethylating agent. In the present study, we sought direct evidence of Cd-induced DNA hypermethylation of the ApoE promoter region by performing bisulfite sequencing and real-time quantitative methylation-specific PCR. Our data clearly suggest that Cd can down-regulate the expression of ApoE via introduction of excess DNA methylation in the promoter region of ApoE during malignant transformation of TRL 1215 cells.

Chitin promotes antigen-specific Th2 cell-mediated murine asthma through induction of IL-33-mediated IL-1ß production by DCs.

Chitin, which is a major component of house dust mites (HDM), fungi, crustaceans, etc., can activate immune cells, suggesting that it contributes to development of allergic disorders such as asthma. Although the pathophysiological sensitization route of asthmatic patients to allergens is considered via the respiratory tract, the roles of intranasally-administered chitin in development of asthma remain unclear. After ovalbumin (OVA) challenge, development of airway inflammation was profoundly exacerbated in mice sensitized with OVA in the presence of chitin. The exacerbation was dependent on IL-33, but not IL-25, thymic stromal lymphopoietin or IL-17A. Chitin enhanced IL-33-dependent IL-1ß production by dendritic cells (DCs). Furthermore, chitin- and IL-33-stimulated DC-derived IL-1ß promoted OVA-specific Th2 cell activation, resulting in aggravation of OVA-induced airway inflammation. These findings indicate the adjuvant activity of chitin via a new mechanism and provide important clues for development of therapeutics for allergic disorders caused by HDM, fungi and crustaceans.

Generation, repair and replication of guanine oxidation products.

Guanine is the most readily oxidized of the four DNA bases, and guanine oxidation products cause G:C-T:A and G:C-C:G transversions through DNA replication. 8-Oxo-7,8-dihydroguanine (8-oxoG) causes G:C-T:A transversions but not G:C-C:G transversions, and is more readily oxidized than guanine. This review covers four major findings. (i) 2,2,4-Triamino-5(2H)-oxazolone (Oz) is produced from guanine and 8-oxoG under various oxidative conditions. Guanine is incorporated opposite Oz by DNA polymerases, except REV1. (ii) Several enzymes exhibit incision activity towards Oz. (iii) Since the redox potential of GG is lower than that of G, contiguous GG sequences are more readily oxidized by a one-electron oxidant than a single guanine, and OzOz is produced from GG in double-stranded DNA. Unlike most DNA polymerases, DNA polymerase ζ efficiently extends the primer up to full-length across OzOz. (iv) In quadruplex DNA, 3'-guanine is mainly damaged by one-electron oxidation in quadruplex DNA, and this damage depends on the highest occupied molecular orbital (HOMO). The oxidation products in quadruplex DNA are different from those in single-stranded or double-stranded DNA.

Cadmium-induced malignant transformation of rat liver cells: Potential key role and regulatory mechanism of altered apolipoprotein E expression in enhanced invasiveness.

Cadmium is a transition metal that is classified as human carcinogen by the International Agency for Research on Cancer (IARC) with multiple target sites. Many studies using various model systems provide evidence of cadmium-induced malignancy formation in vivo or malignant cell transformation in vitro. Nonetheless, further studies are needed to completely understand the mechanisms of cadmium carcinogenicity. Our prior studies have utilized a rat liver epithelial cell line (TRL 1215) as a model for cadmium-induced malignant transformation. In the present study, we focused on the molecular mechanisms of this malignant transformation, especially with regard to hyper-invasiveness stimulated by cadmium transformation. By performing a series of biochemical analyses on cadmium transformed cells, it was determined that cadmium had significantly down-regulated the expression of apolipoprotein E (ApoE). ApoE was recently established as a suppressor of cell invasion. A key factor in the suppression of ApoE by cadmium appeared to be that the metal evoked a 5-aza-2'-deoxycytidine-sensitive hypermethylation of the regulatory region of ApoE, coupled with interference of the action of liver X receptor α (LXRα), a transcriptional regulator for ApoE. Furthermore, the expression of LXRα itself was suppressed by cadmium-mediated epigenetic modification. Re-expression of ApoE clearly abrogated the cell invasion stimulated by cadmium-induced malignant transformation. Together, the current results suggest that the cadmium-mediated enhanced cell invasion is linked to down-regulation of ApoE during malignant transformation these liver cells.

Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, ß, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, ß, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, ß, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.