テロメア及びテロメラーゼを標的としたがん治療法及びがん診断法の開発

テロメラーゼによるテロメアDNA伸長反応（テロメラーゼ反応）は細胞のがん化を導く。そこで本研究では、テロメラーゼ反応の阻害によるがん治療法及び、 テロメラーゼ活性測定を介したがん診断法の開発を行った。１．がん治療法の開発：テロメアDNAにより形成されるG-quadruplex構造に結合するリガンドは、テロメラーゼ反応を阻害することが知られている。しかし従来のG-quadruplexリガンドの多くは、細胞内では染色体二本鎖DNAとも非特異的に結合するため、競合的にG-quadruplexへの結合が阻害される。さらに最近、細胞内の分子クラウディング（Molecular Crowding：MC）環境による水の活量低下も、G-quadruplexリガンドの結合を阻害することが報告された。そこでこれら阻害要因に対するG-quadruplexリガンドの官能基、中心金属、π平面の大きさの影響について系統的に解析した。その結果、アニオン性官能基を有するフタロシアニンは、 二本鎖DNAとは静電的に反発するため、G-quadruplexへの特異性が高いことが見出された。さらにアニオン性フタロシアニンはG-quadruplexに結合する際に水分子を取り込まないため、水の活量低下によるG-quadruplexへの結合阻害が生じないことが示された。そのためアニオン性フタロシアニンは、大量の二本鎖DNA存在下 及びMC条件下においてもテロメラーゼ反応を効率よく阻害することが示された。以上の結果は、アニオン性フタロシアニンが細胞核内においても効率よくテロメアDNAのG-quadruplexに結合し、テロメラーゼ反応を阻害することを示唆している。２．がん診断法の開発：従来のテロメラーゼ活性測定技術は、テロメラーゼ反応とPolymerase Chain Reaction（PCR）を組み合わせた手法である。そのため生体試料中のPCR阻害分子によって擬陰性結果が生じることがあった。そこで本研究では、テロメラーゼ反応後の反応産物を磁性ビーズ上に固定化、洗浄することで、PCR阻害分子を取り除く手法を開発した。さらにその後に行うPCRの産物をサイクリングプローブ法によって高感度に検出する方法を開発した。その結果、これらの手法を組み合わせることによってPCR阻害分子である胆汁酸、ビリルビン、ヘモグロビン存在下においても、阻害の影響を受けることなくがん細胞中のテロメラーゼ活性を測定できることが示された。また、検出感度50細胞という非常に高い感度でがん細胞を検出できることが示された。甲南大学平成25年度(2013年度

Interaction between the CheY response regulator and the histidine-containing phosphotransfer (HPt) domain of the ArcB sensory kinase in Escherichia coli

AbstractBacteria have devised sophisticated His–Asp phosphorelay signaling systems for eliciting a variety of adaptive responses to their environment. The histidine-containing phosphotransfer (HPt) domain, found in many signal transduction protein, functions as a mediator of the His–Asp phosphorelay. The ArcB anaerobic sensor of E. coli contains such a HPt domain, although its function is not fully understood. In this study, we provide in vivo and in vitro evidence that the HPt domain is capable of interacting with the CheY receiver, which contains a phospho-accepting aspartate residue

A mRNA-Responsive G-Quadruplex-Based Drug Release System

G-quadruplex-based drug delivery carriers (GDDCs) were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II) phthalocyanine (CuAPC). As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR) mRNA. Structural analysis using circular dichroism (CD) spectroscopy showed that the GDDCs form a (3 + 1) type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM) at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM) at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA

A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF) cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity

Specific Binding of Anionic Porphyrin and Phthalocyanine to the G-Quadruplex with a Variety of in Vitro and in Vivo Applications

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property

A mRNA-Responsive G-Quadruplex-Based Drug Release System

G-quadruplex-based drug delivery carriers (GDDCs) were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II) phthalocyanine (CuAPC). As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR) mRNA. Structural analysis using circular dichroism (CD) spectroscopy showed that the GDDCs form a (3 + 1) type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM) at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM) at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA

Sub-micro-liter electrochemical single-nucleotide-polymorphism detector for lab-on-a-chip system

A sub-micro-liter single-nucleotide-polymorphism (SNP) detector for lab-on-a-chip applications is developed. This detector enables a fast, sensitive, and selective SNP detection directly from human blood. The detector is fabricated on a Si substrate by a standard complementary metal oxide semiconductor/micro electro mechanical systems (CMOS/MEMS) process and Polydimethylsiloxane (PDMS) molding. Stable and reproducible measurements are obtained by implementing an on-chip Ag/AgCl electrode and encapsulating the detector. The detector senses the presence of SNPs by measuring the concentration of pyrophosphoric acid generated during selective DNA amplification. A 0.5-mu L-volume detector enabled the successful performance of the typing of a SNP within the ABO gene using human blood. The measured sensitivity is 566 pA/mu M. (C) 2012 The Japan Society of Applied Physic

Highly Sensitive Telomerase Assay Insusceptible to Telomerase and Polymerase Chain Reaction Inhibitors for Cervical Cancer Screening Using Scraped Cells

A sensitive telomerase assay based on asymmetric-polymerase chain reaction (A-PCR) on magnetic beads and subsequent application of cycling probe technology, STAMC, which is insusceptible to DNase and PCR inhibitors, was for the first time applied to clinical specimens in addition to a conventional telomeric repetitive amplification protocol (TRAP). The electrophoresis results showed that an increase in scraped cervical cancer cells not only reduced TRAP products but also increased smaller products, suggesting the unreliability of TRAP for clinical samples. To achieve the required sensitivity of STAMC for clinical application, the sequence and concentration conditions were explored for the forward and reverse primers for A-PCR, which resulted in a detection limit of only two HeLa cells with 1 μM TS primer (5′-AATCC­GTCGAGC­AGAGTT-3′) and 0.04 μM ACX primer (5′-GCGCGGC­TTACCCTTA­CCCTTACC­CTAACC-3′). Under the same primer conditions, the fluorescence signal of STAMC increased as scraped cervical cancer cells increased despite showing a negligible intensity for benign tumors. Furthermore, STAMC showed no signal for a cervical cancer patient treated with irradiation therapy. These results indicate that STAMC is useful for not only cervical cancer screening but also investigating the effect of cancer treatments such as radiation therapy and drug administration