Biomedical subjects

Ken-ichi Shinohara

Publications and source records attributed to Ken-ichi Shinohara.

11 recordsLinked to original sources

PubMed2006

Sequence-specific alkylation of double-strand human telomere repeat sequence by pyrrole-imidazole polyamides with indole linkers.

We designed and synthesized pyrrole (Py)-imidazole (Im) hairpin polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates 1 and 2, which target both strands of the double-stranded region of the human telomere repeat sequences, 5'-d(TTAGGG)(n)-3'/5'-d(CCCTAA)(n)-3'. High-resolution denaturing polyacrylamide gel electrophoresis demonstrated that conjugates 1 and 2 alkylated DNA at the 3' A of 5'-ACCCTA-3' and 5'-AGGGTTA-3', respectively. Cytotoxicities of conjugates 1 and 2 were evaluated using 39 human cancer cell lines; averages of log IC(50) values for conjugates 1 and 2 were -6.96 (110 nM) and -7.24 (57.5 nM), respectively. Conjugates 1 and 2 have potential as antitumor drugs capable of targeting telomere repeat sequence.

Alkylation↗

PubMed2006

Single molecular anatomy of solvophobic effects in host-guest interactions based on surface tension using atomic force microscopy.

The effectiveness of the mechanism of surface tension to analyze the host-guest interaction was demonstrated by measuring the unbinding force of the beta-Cyclodextrin (CyD)--adamantane molecules in a liquid environment at the single-molecule level. The contribution of the solvophobic effect was quantitatively distinguished from the intrinsic van der Waals force, and the effective length that produces the detected force through employing the notion of surface tension was determined to be , which is consistent with the perimeter of the adamantane molecule, . Specific strong interaction of water molecules was also confirmed on the nanoscale.

Adamantane↗

PubMed2006

Alkylation of template strand of coding region causes effective gene silencing.

We recently developed a new type of pyrrole (Py)-imidazole (Im) polyamide-tetrahydrocyclopropabenzindolone (CBI) conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing activities of polyamides A, B and C, which selectively alkylate specific sequences in the promoter region, non-coding strand and coding strand, respectively, of the green fluorescent protein (GFP) gene. GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells were treated with 100 nM of the polyamides for 24 h. Fluorescence microscopy indicated that a significant reduction of GFP fluorescence was only observed in the cells that were treated with polyamide C. In clear contrast, polyamides A and B did not show such activity. Moreover, real-time PCR demonstrated selective reduction of the expression of GFP mRNA following treatment with polyamide C. These results suggest that alkylating Py-Im polyamides that target the coding strand represent a novel approach for sequence-specific gene silencing.

Alkylation↗

PubMed2006

The biological impact of sequence-specific DNA alkylation by pyrrole-imidazole polyamides.

We have developed a series of novel DNA alkylating polyamides possessing indole linkers. Investigations using high-resolution gel electrophoresis revealed that the indole linked Py-Im polyamide alkylated at A of a targeted nine base pair matching sequence. Evaluation in human cancer cell lines revealed that the indole linked Py-Im polyamides have strong cytotoxicities. Furthermore, we showed that alkylation of the template strand of the coding region by these polyamides causes effective gene silencing.

Alkylation↗

PubMed2006

Synthesis and evaluation of sequence-specific DNA alkylating agents: effect of alkylation subunits.

We have demonstrated that hairpin pyrrole (Py)- imidazole (Im) polyamide-CBI conjugates selectively alkylate predetermined sequences. In this study, we investigated the effect of alkylation subunits, for example conjugates 1-4 with three types of DNA alkylating units, and Py-Im polyamides with indole linker. Conjugate 3 and 4 selectively alkylated the predetermined sequences as described previously, while conjugates 1 and 2 alkylate at mismatched sites.

Antineoplastic Agents, Alkylating↗

PubMed2006

Molecular design of DNA alkylating pyrrole-imidazole polyamides with longer recognition sequence.

The sequence-specificity, and DNA alkylating activity of the conjugate 1, which consists of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with indole linker, were investigated in the absence or presence of partner Py-Im polyamide 2. High-resolution denaturing polyacrylamide gel electrophoresis showed that the specificity of DNA alkylation by 1 modulated in the presence of partner 2. We found that sequence-specific DNA alkylation by 1 and 2 with 10 base pair (bp) match recognition sequence through heterodimer formation. This result indicates one possibility of DNA alkylation with longer recognition sequence by different two molecules.

Alkylating Agents↗

PubMed2006

Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (R<0). These results suggest that differences in the sequence specificity of DNA alkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

Adenine↗

PubMed2005

Sequence-specific gene silencing by alkylating Py-Im polyamide.

We have demonstrated that hairpin pyrrole (Py)-imidazole (Im) polyamide-CPI conjugates selectively induced luciferase gene silencing by sequence-specific alkylation of the coding region. Recently, we developed a new type of Py-Im polyamide CBI conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing ability of polyamides A, B and C, which potentially target specific sequences in the promoter region, non-coding strand, and coding strand of the green fluorescent protein (GFP) gene, respectively. The GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells treated with 100 nM of the polyamides for 24 h. Using direct observation of cell by fluorescence microscopy, a significant GFP-gene silencing effect was only seen with treatment with polyamide C. Polyamides A and B did not show such activity.

Alkylating Agents↗

PubMed2003

Human neutrophils isolated from peripheral blood contain Ku protein but not DNA-dependent protein kinase.

Ku protein, a heterodimer of 70kDa (Ku70) and 86kDa (Ku86) polypeptides, is involved in non-homologous DNA end-joining (NHEJ) of DNA double-strand break repair and V(D)J recombination in combination with the catalytic component of DNA-dependent protein kinase (p470). Although Ku protein is known to be ubiquitously present in eukaryotic cells, it was previously reported to be absent in mature neutrophils. Using a mixture of protease inhibitors in the isolation procedure of neutrophils from human peripheral blood, we were able to detect Ku in the neutrophils by immunoblot and flow-cytometric analyses. Transcripts of Ku70 and Ku86 genes were also detected by the reverse transcriptase-polymerase chain reaction (RT-PCR), and Ku protein was shown to be localized in the nucleus of neutrophils as a heterodimer. Like poly(ADP-ribose) polymerase-1, neither mRNA nor protein of p470 was detected in the neutrophils. These results suggest that Ku is involved independently of p470 in DNA metabolism and signal transduction.

Antigens, Nuclear↗

PubMed2003

Involvement of DNA-dependent protein kinase in down-regulation of cell cycle progression.

The catalytic polypeptide of DNA-dependent protein kinase (p470) is encoded by the gene responsible for murine severe combined immunodeficiency (SCID) devoid of DNA double-strand break repair and V(D)J recombination. Here, we have characterized the role of p470 in cell proliferation using SCID mice and the cell lines. In accord with DNA histogram patterns, SCID cell lines (SD/SD-eA and SC3VA2) expressing extremely low level of DNA-PK activity grew faster than a normal mouse cell line (CB/CB-eB) and SC3VA2 complemented with human p470 gene (RD13B2). In regenerating liver after partial hepatectomy, de novo DNA synthesis determined by [(3)H]thymidine incorporation started at 30h in C.B-17/Icr-SCID (SCID) mice and at around 36h in C.B-17/Icr (C.B-17) mice. Compared with normal cells, SCID cells contained slightly higher levels of transcripts of cyclin A, cyclin E, B-Myb and dihydrofolate reductase, which are regulated by E2F-1. E2F-1 playing a key role in G1- to S-phase progression was phosphorylated in vitro by DNA-PK. Importantly, the E2F-1 promoter transcriptional activity in SCID cell lines (SD/SD-eA and SC3VA2) was 4-5-fold higher than that in CB/CB-eB and RD13B2. These results suggest that p470 is involved in down-regulation of cell cycle progression through E2F-1-responsible genes.

Animals↗

PubMedDate not supplied

Efficient DNA alkylation by a pyrrole-imidazole CBI conjugate with an indole linker: sequence-specific alkylation with nine-base-pair recognition.

Conjugates 7, 8, and 10 of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) with a 5-amino-1H-indole-2-carbonyl linker were synthesized by Fmoc solid-phase synthesis and a subsequent liquid-phase coupling procedure. The DNA alkylating abilities of conjugates 7, 8, 6b, and 10 were examined using Texas Red-labeled PCR fragments and high-resolution denaturing gel electrophoresis. CBI conjugates 7 and 8 exhibited highly efficient sequence-specific DNA alkylation comparable with previous CBI conjugates with a vinyl linker. In particular, conjugate 10, with a 10-ringed hairpin Py-Im polyamide, alkylated at the adenine of 5'-ACAAATCCA-3'. Introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides.

Alkylation↗