Yoshinori Okamoto

PRDM14 promotes active DNA demethylation through the Ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

Combined Activation of Methyl Paraben by Light Irradiation and Esterase Metabolism toward Oxidative DNA Damage

Methyl paraben (MP) is often used as a preservative in foods, drugs, and cosmetics because of its high reliability in safety based on the rapid excretion and nonaccumulation following administration. Light irradiation sometimes produces unexpected activity from chemicals such as MP; furthermore, there is ample opportunity for MP to be exposed to sunlight. Here, we investigated whether MP shows DNA damage after sunlight irradiation. Two major photoproducts, p-hydroxybenzoic acid (PHBA) and 3-hydroxy methyl paraben (MP-3OH), were detected after sunlight irradiation to an aqueous MP solution. Both photoproducts were inactive in the in vitro DNA damage assay that measures oxidized guanine formed in calf thymus DNA in the presence of divalent copper ion, a known mediator of oxidative DNA damage. Simulated MP metabolism using dermal tissues after light irradiation produced these two photoproducts, which reacted with a microsomal fraction (S9) of the skin. A metabolite from MP-3OH, not PHBA, caused distinct DNA damage in the in vitro assay. This active metabolite was identified as protocatechuic acid, a hydrolyzed MP-3OH product. In addition, NADH, a cellular reductant, enhanced DNA damage by approximately five times. These results suggest that reactive oxygen species generated by the redox cycle via metal ion and catechol autoxidation are participating in oxidative DNA damage. This study reveals that MP might cause skin damage involving carcinogenesis through the combined activation of sunlight irradiation and skin esterases.

Anti-Breast Cancer Potential of SS5020, a Novel Benzopyran Antiestrogen

Treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. The carcinogenic effect is thought to involve initiation and/or promotion resulting from DNA damage induced by TAM as well as its estrogenic action. To minimize this serious side‐effect while increasing the anti‐breast cancer potential, a new benzopyran antiestrogen, 2E‐3‐{4‐[(7‐hydroxy‐2‐oxo‐3‐phenyl‐2H‐chromen‐4‐yl)‐methyl]‐phenyl}‐acrylic acid (SS5020), was synthesized. Unlike TAM, SS5020 exhibits no genotoxic activity to damage DNA. Furthermore, SS5020 does not present significant uterotrophic potential in rats; in contrast, the structurally related compounds, TAM, toremifene, raloxifene (RAL) and SP500263 all have uterotrophic activity. At the human equivalent molar dose of TAM (0.33 or 1.0 mg/kg), SS5020 had much stronger antitumor potential than those same antiestrogens against 7,12‐dimethylbenz(a)anthracene‐induced mammary carcinoma in rats. The growth of human MCF‐7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS5020. SS5020, lacking genotoxic and estrogenic actions, could be a safer and stronger antiestrogen alternative to TAM and RAL for breast cancer therapy and prevention.

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry

Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.

Oxidative DNA damage in Xpc-knockout and its wild mice treated with equine estrogen

Long-term hormone replacement therapy with equine estrogens is associated with a higher risk of breast, ovarian, and endometrial cancers. Reactive oxygen species generated through redox cycling of equine estrogen metabolites may damage cellular DNA. Such oxidative stress may be linked to the development of cancers in reproductive organs. Xeroderma pigmentosa complementation group C-knockout ( Xpc-KO) and wild-type mice were treated with equilenin (EN), and the formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) was determined as a marker of typical oxidative DNA damage, using liquid chromatography electrospray tandem mass spectrometry. The level of hepatic 8-oxodG in wild-type mice treated with EN (5 or 50 mg/kg/day) was significantly increased by approximately 220% after 1 week, as compared with mice treated with vehicle. In the uterus also, the level of 8-oxodG was significantly increased by more than 150% after 2 weeks. Similar results were observed with Xpc-KO mice, indicating that Xpc does not significantly contribute to the repair of oxidative damage. Oxidative DNA damage generated by equine estrogens may be involved in equine estrogen carcinogenesis.

Anti-breast cancer potential of SS1020, a novel antiestrogen lacking estrogenic and genotoxic actions

Long‐term treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. Several antiestrogens developed in last decades have been discontinued from clinical testing because of their undesirable effects on the uterus. To avoid such serious side‐effect while increasing the drugs anti‐breast cancer potential, new triphenylethylene antiestrogens, 2E‐3‐{4‐[(E)‐4‐chloro‐1‐(4‐hydroxyphenyl)‐2‐phenylbut‐1‐enyl]‐phenyl} acrylic acid (SS1020) and 2E‐3‐{4‐[(Z)‐4‐chloro‐1,2‐diphenylbut‐1‐enyl]phenyl}acrylic acid (SS1010), were designed as safer alternatives. Unlike TAM, SS1020 does not present significant uterotrophic potential in rats; in contrast, SS1010, a compound removing a 4‐OH moiety from SS1020, represented weak uterotrophic activity. The structurally related compounds 4‐hydroxytamoxifen, toremifene, ospemifene, raloxifene (RAL) and GW5638 all have uterotrophic activity. In addition, SS1020 and SS1010 exhibit no genotoxic activity to damage hepatic DNA in rats. Therefore, SS1020 was selected as a safer antiestrogen candidate and used for evaluating the antitumor potential in animals. At the human equivalent doses of TAM, SS1020 had antitumor potential much higher than that of TAM, RAL and GW5638 against 7,12‐dimethylbenz(a)anthracene‐induced mammary carcinoma in rats. The growth of human MCF‐7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS1020. SS1020, lacking estrogenic and genotoxic actions and having strong antitumor potency superior to that of TAM and RAL, could be a safer alternative for breast cancer therapy and prevention.

Different Mechanisms Between Copper and Iron in Catecholamines-Mediated Oxidative DNA Damage and Disruption of Gene Expression In Vitro

Catechols produce reactive oxygen species (ROS) and induce oxidative DNA damage through reduction–oxidation reactions with metals such as copper. Here, we examined oxidative DNA damage by neurotransmitter catecholamines in the presence of copper or iron and evaluated the effects of this damage on gene expression in vitro. Dopamine induced strand breaks and base oxidation in calf thymus DNA in the presence of Cu(II) or Fe(III)-NTA (nitrilotriacetic acid). The extent of this damage was greater for Cu(II) than for Fe(III)-NTA. For the DNA damage induced by dopamine, the responsible reactive species were hydrogen peroxide and Cu(I) for Cu(II) and hydroxyl radicals and Fe(II) for Fe(III)-NTA. Cu(II) induced DNA conformational changes, but Fe(III)-NTA did not in the presence of dopamine. These differences indicate different modes of action between Cu and Fe-NTA with regard to the induction of DNA damage. Expression of the lacZ gene coded on plasmid DNA was inhibited depending on the extent of the oxidative damage and strand breaks. Endogenous catecholamines (dopamine, adrenaline, and noradrenaline) were more potent than catechols (no aminoalkyl side chains) or 3,4-dihydroxybenzylamine (aminomethyl side chain). These results suggest that the metal-mediated DNA damage induced by dopamine disrupts gene expression, and leukoaminochromes (further oxidation products of O-quinones having aminoethyl side chain) are involved in the DNA damage. These findings indicate a possibility that metal (especially iron and copper)-mediated oxidation of catecholamines plays an important role in the pathogenesis of neurodegenerative disorders including Parkinson’s disease.

Modulation of oxidative DNA damage and DNA-crosslink formation induced by cis-diammine-tetrachloro-platinum(IV) in the presence of endogenous reductants.

Platinum(IV) [Pt(IV)] complex, satraplatin, is currently in clinical trials for the treatment of various cancers. As a key step of the anti-cancer effect exertion, satraplatin is supposed to be reduced by endogenous reductants to platinum(II) [Pt(II)] complex. In this study, we investigated the interaction of DNA, Pt(IV), and the endogenous reductants such as ascorbic acid (AsA) and glutathione (GSH). As a model Pt(IV) compound, cis-diammine-tetrachloro-Pt(IV) [cis-Pt(IV)], which is a prodrug of cisplatin [cis-diammine-dichloro-Pt(II), cis-Pt(II)], was incubated with calf thymus DNA in the presence of AsA or GSH. In the presence of AsA, cis-Pt(IV) induced oxidative DNA damage. Hydroxyl radical scavengers suppressed the AsA-associated oxidative damage, thereby suggesting that hydroxyl radicals are involved in the DNA oxidation. cis-Pt(II)-like CD spectral change and crosslink formation in calf thymus DNA were also observed during this DNA oxidation, suggesting cis-Pt(IV) reduction by AsA and DNA conformational change induced by the newly formed cis-Pt(II) binding to DNA. GSH did not induce oxidative DNA damage likely due to its own hydroxyl radical scavenging ability. Further, GSH suppressed the Pt(II)-mediated DNA conformational change and crosslink formation, suggesting that GSH sequesters the cis-Pt(II) away from DNA by GSH-cis-Pt(II) complex formation.

Thiol-mediated multiple mechanisms centered on selenodiglutathione determine selenium cytotoxicity against MCF-7 cancer cells

Selenium (Se) is an essential antioxidative micronutrient but can exert cancer-selective cytotoxicity if the nutritional levels are too high. Selenodiglutathione (GSSeSG) is a primary Se metabolite conjugated with two glutathione (GSH) moieties. GSSeSG has been suggested to be an important molecule for cytotoxicity. Here, we propose the underlying mechanisms for the potent cytotoxicity of GSSeSG: cellular intake; reductive metabolism; production of reactive oxygen species; oxidative damage to DNA; apoptosis induction. GSSeSG rather than selenite decreased cell viability and induced apoptosis accompanied by increases in intracellular Se contents. Therefore, GSSeSG-specific cytotoxicity may be ascribed to its preferable incorporation. Base oxidation and strand fragmentation in genomic DNA preceded cell death, suggesting that oxidative stress (including DNA damage) is crucial for GSSeSG cytotoxicity. Strand breaks of purified DNA were caused by the coexistence of GSSeSG and thiols (GSH, cysteine, homocysteine), but not the oxidized form or non-thiol reductants. This implies the important role of intracellular thiols in the mechanism of Se toxicity. GSH-assisted DNA strand breaks were inhibited by specific scavengers for hydrogen peroxide or hydroxyl radicals. The GSSeSG metabolite selenide induced some DNA strand breaks without GSH, whereas elemental Se did so only with GSH. These observations suggest involvement of Fenton-type reaction in the absence of transition metals and reactivation of inert elemental Se. Overall, our results suggest that chemical interactions between Se and the sulfur of thiols are crucial for the toxicity mechanisms of Se.

Increased antitumor potential of the raloxifene prodrug, raloxifene diphosphate.

Raloxifene (RAL) significantly reduced the incidence of breast cancer in women at high risk of developing the disease. Unlike tamoxifen (TAM), an increased incidence of endometrial cancer was not observed in women treated with RAL. However, RAL, having two hydroxyl moieties, can be conjugated rapidly through phase II metabolism and excreted, making it difficult to achieve adequate bioavailability by oral administration in humans. As a result, higher doses must be administered to obtain an efficacy equivalent to that achieved with TAM. To improve oral bioavailability and antitumor potential, RAL diphosphate was prepared as a prodrug. RAL diphosphate showed several orders of magnitude lower binding potential to both ERα and ERβ and weak antiproliferative potency on cultured human MCF‐7 and ZR‐75‐1 breast cancer cells, as compared to RAL. However, RAL diphosphate has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL against both 7,12‐dimethylbenz(a)anthracene‐induced mammary carcinoma in rats and human MCF‐7 breast cancer implanted in athymic nude mice. The RAL prodrug may provide greater clinical benefit for breast cancer therapy and prevention.