Gengo Kashiwazaki

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

Genetic regulation of the RUNX transcription factor family has antitumor effects

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent–conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

Cell permeability of Py–Im-polyamide-fluorescein conjugates: Influence of molecular size and Py/Im content

In order to investigate the influence of molecular size and pyrrole (Py)/imidazole (Im) content on the cell permeability of Py-Im-polyamide-fluorescein conjugates we systematically designed the Py-polyamides and Im-polyamides. Flow cytometric analysis revealed that Py-polyamides, even those with large molecular size, P-15 and P-18, showed good cellular uptake, but Im-polyamides showed very poor uptake. Fluorescence microscopy revealed that conjugate P-6 exhibited nuclear localization, while P-18 showed less nuclear stain but intracellular localization, suggesting that increased molecular size is one of the determinants in reducing nuclear access. Furthermore, results for hairpin polyamide conjugates H-1, H-2, and H-3 containing different Py/Im content indicated that cellular uptake increases as the Im residue is reduced. It appears that Py-Im-polyamide has general properties regardless of whether they have a linear or a hairpin structure.

Formation and Direct Repair of UV‐induced Dimeric DNA Pyrimidine Lesions

Direct repair of UV‐induced DNA lesions represents an elegant method for many organisms to deal with these highly mutagenic and cytotoxic compounds. Although the participating proteins are structurally well investigated, the exact repair mechanism of the photolyase enzymes remains a vivid subject of current research. In this review, we summarize and highlight the recent contributions to this exciting field.

Synthesis and Biological Properties of Highly Sequence-Specific-Alkylating N-Methylpyrrole–N-Methylimidazole Polyamide Conjugates

Four new alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1-4) with seven-base-pair (bp) recognition ability were synthesized. Evaluation of their DNA-alkylating activity clearly showed accurate alkylation at match site(s). The cytotoxicities of conjugates 1-4 were determined against six human cancer cell lines, and the effect of these conjugates on the expression levels of the whole human genome in A549 cells were also investigated. A few genes among the top 20 genes were commonly downregulated by each conjugate, which reflects their sequence specificity. Conversely, many of the top 10 genes were commonly upregulated, which may have been caused by alkylation damage to DNA. Moreover, the antitumor activities of the PI polyamide conjugates 2 and 3 were investigated using nude mice transplanted with DU145 or A549. The intravenous administration of each liposomal conjugate in water yielded tumor-suppressing effects specifically toward DU145 cells and not A549 cells, which was pertinent to cytotoxicity.

DNA ligand designed to antagonize EBNA1 represses Epstein–Barr virus‐induced immortalization

Epstein–Barr virus (EBV) transforms human B lymphocytes into immortalized cells in vitro and is associated with various malignancies in vivo. EBNA1, which is expressed in the majority of EBV‐infected cells, recognizes specific DNA sequences at the cis‐acting latent origin of plasmid replication (oriP) element of the EBV genome. EBNA1 plays a critical role in the viral episome maintenance and transactivates viral transforming genes in latently infected cells. Therefore, DNA‐targeting agents that can disrupt the EBNA1–oriP interaction will offer novel functional inhibitors of EBNA1. Pyrrole–imidazole polyamides, sequence‐specific DNA ligands, can be designed to interfere with the binding of various transcriptional factors. Here, we synthesized pyrrole–imidazole polyamides targeting EBNA1‐bound DNA sequences and developed an inhibitor for the EBNA1–oriP interaction. A pyrrole‐imidazole polyamide, designated as DSE‐3, bound adjacent to the EBNA1 recognition sequences located in the dyad symmetry element of oriP, and selectively inhibited EBNA1–oriP binding both in vitro and in vivo. DSE‐3 also inhibited the proliferation of established lymphoblastoid cell lines by eradicating EBV episomes from the cells. In addition, DSE‐3 repressed the expression of viral transforming genes after infecting human peripheral blood mononuclear cells with EBV and, as a consequence, inhibited EBV‐mediated B‐cell immortalization. These results suggest that EBNA1 functions will be an attractive pharmacological target for EBV‐associated diseases. (Cancer Sci 2011; 102: 2221–2230)

Homo- and heterobismetal complexes of 5-Hydroxy-10,15,25,30-tetrakis(pentafluorophenyl)-substituted [26]hexaphyrin(1.1.1.1.1.1).

Metalation of meso-free 5,10,20,25-tetrakis(pentafluorophenyl)-substituted [26]hexaphyrin(1.1.1.1.1.1) (4) with Cu(II) and Zn(II) ions afforded homodinuclear complexes 6 and 7, respectively. A demetalation-remetalation protocol provided monozinc(II) complex 9 selectively, from which copper(II)-zinc(II) heterodinuclear complex 10 was prepared effectively.

Comparative analysis of DNA alkylation by conjugates between pyrrole-imidazole hairpin polyamides and chlorambucil or seco-CBI

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide-chlorambucil conjugates 1-4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py-Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1-4 alkylated DNA at the sequences recognized by the Py-Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide-seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py-Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide-seco-CBI conjugate 5 was similar to that of polyamide-chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1-4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents.

Construction and characterization of Cy3- or Cy5-conjugated hairpin pyrrole–imidazole polyamides binding to DNA in the nucleosome

Sequence-specific DNA-binding modules, N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamides have been recently conjugated with fluorophores, and some of these conjugates could be used for the detection of specific DNA sequences. In this study, we synthesized two Py-Im polyamides 1 and 2, which interact with the 145-bp nucleosome positioning sequence 601. We conjugated the cyanine dye Cy3 or Cy5 with 1 or 2. In the absence of target DNA, the fluorescent conjugate of a Py-Im polyamide had lower fluorescence intensity compared with Cy3 or Cy5 alone. In the presence of either the target DNA or the nucleosome, the fluorescence intensity of the conjugates increased. Furthermore, we observed a Förster resonance energy transfer between the Cy3-Py-Im polyamide and the Cy5-Py-Im polyamide on the nucleosome. These results open up the possibilities that fluorescent conjugates of Py-Im polyamides can be used for characterization of the dynamic interactions within protein-DNA complexes.

Evaluation of PI polyamide conjugates with eight-base pair recognition and improvement of the aqueous solubility by PEGylation.

To investigate the effect of elongating base-pair (bp) recognition sequences, we synthesized N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates with eight-bp recognition (3-5). The DNA alkylating activities of conjugates 3-5 were evaluated by high-resolution denaturing polyacrylamide gel electrophoresis with a 208-bp DNA fragment. Conjugates 3-5 showed high alkylating activities at nanomolar concentrations. We then addressed the following issue about PI conjugates. Generally, PI polyamide conjugates hardly dissolve in aqueous solution. To improve the aqueous solubility, by the introduction of hydrophilic groups, we synthesized PI polyamide conjugates that were modified with a seco-CBI moiety (6-11). Conjugates 9-11 that were modified by methoxypolyethylene glycol (PEG) 750 acquired moderate solubility and stability in aqueous solution. In addition, conjugates 10 and 11 had high cytotoxicity against A549 and DU145.