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Tomoko Takahashi,
Tamae Kawabe,
Yuichi Okazaki,
Chie Itoh,
Keisuke Noda,
Manami Tajima,
Misako Satoh,
Makoto Goto,
Youji Mitsui,
Hidetoshi Tahara,
Toshinori Ide, Yasuhiro Furuichi,
Masanobu Sugimoto
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Tomoko Takahashi,
Tamae Kawabe,
Yuichi Okazaki,
Chie Itoh,
Keisuke Noda,
Manami Tajima,
Misako Satoh,
Makoto Goto,
Youji Mitsui,
Hidetoshi Tahara,
Toshinori Ide, Yasuhiro Furuichi,
Masanobu Sugimoto
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Tomoko Takahashi,
Tamae Kawabe,
Yuichi Okazaki,
Chie Itoh,
Keisuke Noda,
Manami Tajima,
Misako Satoh,
Makoto Goto,
Youji Mitsui,
Hidetoshi Tahara,
Toshinori Ide, Yasuhiro Furuichi,
Masanobu Sugimoto
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Tomoko Takahashi,
Tamae Kawabe,
Yuichi Okazaki,
Chie Itoh,
Keisuke Noda,
Manami Tajima,
Misako Satoh,
Makoto Goto,
Youji Mitsui,
Hidetoshi Tahara,
Toshinori Ide, Yasuhiro Furuichi,
Masanobu Sugimoto
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Tomoko Takahashi,
Tamae Kawabe,
Yuichi Okazaki,
Chie Itoh,
Keisuke Noda,
Manami Tajima,
Misako Satoh,
Makoto Goto,
Youji Mitsui,
Hidetoshi Tahara,
Toshinori Ide, Yasuhiro Furuichi,
Masanobu Sugimoto
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ABSTRACT: Minor inflammation-driven aging (inflammaging) has been proposed to explain human aging mechanism. To study the inflammatory condition associated with normal human aging, highly sensitive CRP (hsCRP) was examined in the sera collected from 217 healthy Japanese individuals aged between 1 and 100years and 41 mutation-proven Japanese Werner syndrome (WS) patients. The serum hsCRP was assayed by ELISA. The serum hsCRP level increased significantly (p<0.001) with normal aging from both sexes. The serum hsCRP was significantly elevated in WS (mean±SE: 11.0±1.6μg/ml) compared with age-matched normal population (1.3±0.3μg/ml, p<0.001) and normal elderly population ages between 71 and 100years (4.2±0.7μg/ml, p<0.001). Both normal aging and WS were associated with minor inflammation that can be evaluated by serum hsCRP. WS may be a good candidate to study inflammaging.
Experimental gerontology 08/2012; · 3.34 Impact Factor
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Siddharth De,
Jyoti Kumari,
Richa Mudgal,
Priyanka Modi,
Shruti Gupta,
Kazunobu Futami,
Hideyuki Goto,
Noralane M Lindor, Yasuhiro Furuichi,
Debasisa Mohanty,
Sagar Sengupta
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ABSTRACT: Mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome (RTS). A subset of RTS patients is predisposed to cancer and is sensitive to DNA damaging agents. The enhanced sensitivity of cells from RTS patients correlates with the accumulation of transcriptionally active nuclear p53. We found that in untreated normal human cells these two nuclear proteins, p53 and RECQL4, instead colocalize in the mitochondrial nucleoids. RECQL4 accumulates in mitochondria in all phases of the cell cycle except S phase and physically interacts with p53 only in the absence of DNA damage. p53-RECQL4 binding leads to the masking of the nuclear localization signal of p53. The N-terminal 84 amino acids of RECQL4 contain a mitochondrial localization signal, which causes the localization of RECQL4-p53 complex to the mitochondria. RECQL4-p53 interaction is disrupted after stress, allowing p53 translocation to the nucleus. In untreated normal cells RECQL4 optimizes de novo replication of mtDNA, which is consequently decreased in fibroblasts from RTS patients. Wild-type RECQL4-complemented RTS cells show relocalization of both RECQL4 and p53 to the mitochondria, loss of p53 activation, restoration of de novo mtDNA replication and resistance to different types of DNA damage. In cells expressing Δ84 RECQL4, which cannot translocate to mitochondria, all the above functions are compromised. The recruitment of p53 to the sites of de novo mtDNA replication is also regulated by RECQL4. Thus these findings elucidate the mechanism by which p53 is regulated by RECQL4 in unstressed normal cells and also delineates the mitochondrial functions of the helicase.
Journal of Cell Science 02/2012; 125(Pt 10):2509-22. · 6.11 Impact Factor
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ABSTRACT: Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in the WRN DNA helicase. It is characterized by the graying and loss of hair, juvenile cataracts, sclerosis and ulceration of skin, insulin-resistant diabetes mellitus, dyslipidemia, abdominal adiposity, osteoporosis, atherosclerosis, and malignant neoplasm. Patients are usually diagnosed in their 30s or 40s, but the early pathophysiology of the syndrome is still not fully understood. Here we report a 29-year-old female patient who displayed cataracts, hair graying, and tendinous calcinosis. Her parents were first cousins. Interestingly, the patient lacked the metabolic signs typical for WS, including glucose intolerance, dyslipidemia, and visceral fat accumulation. A hyperinsulinemic response at 30 min was observed in an oral glucose tolerance test. Mutational analysis for the WRN gene revealed a homozygous nucleotide substitution 3190C>T in exon 24, resulting in a protein product with replacement of an arginine residue at position 573 by termination codon (Arg987Ter). The mutated WRN protein was unable to translocate into the nucleus in an in vitro cell assay. A WS patient with an Arg987Ter mutation has been previously reported in Switzerland, the present case is the first to be identified in Asia. This case demonstrates the early clinical features of WS and suggests that metabolic abnormality, including insulin resistance, is not an essential component of WS at disease onset. Moreover, a follow-up study of such case would be useful to understand how the various clinical symptoms in WS develop and progress over the years.
Geriatrics & Gerontology International 01/2012; 12(1):140-6.
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ABSTRACT: The repeated replication of cells shortens telomeres, culminating in their instability, after which most cells cease to replicate and die. However, a small fraction of the cells become immortalized by maintaining telomeres with activated telomerase activity. It has been proposed that WRN helicase encoded by the WRN gene, the causative gene of Werner syndrome (WS), is required for immortalization by the telomeric crisis pathway (TCP) in a system that uses lymphoblastoid cell lines transformed by the Epstein-Barr virus. Taken together, these characteristics indicate that WRN helicase is also required for the immortalization of epithelial cells by TCP and consequent carcinogenesis, suggesting that the tumorigenesis of epithelial cells by TCP is suppressed in WS lacking the WRN helicase function. Notably, in WS the pathway of alternative lengthening of telomeres without activation of telomerase activity has been suggested to be involved in immortalization and tumorigenesis. This factor is consistent with the abundance of non-epithelial cancers in WS in that the ratio of epithelial to non-epithelial cancers is approximately 1:1 in WS patients compared to 10:1 in the general population. A hypothetical scheme showing the role of WRN helicase in immortalization by means of the supposed 'breakage-fusion-bridge cycle' of chromosomes at telomeric crisis is described.
Oncology letters 07/2011; 2(4):609-611. · 0.11 Impact Factor
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Akihito Arai,
Tokuhiro Chano,
Kazunobu Futami, Yasuhiro Furuichi,
Kaichiro Ikebuchi,
Takuma Inui,
Hitosuke Tameno,
Yasuko Ochi,
Taketoshi Shimada,
Yasuo Hisa,
Hidetoshi Okabe
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ABSTRACT: RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyper-recombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers.
Cancer Research 05/2011; 71(13):4598-607. · 7.86 Impact Factor
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ABSTRACT: RecQL1 in the human RecQ DNA helicase family participates in DNA repair and recombination pathways in cell cycle replication. Immunohistochemical analysis of human hepatocellular carcinoma (HCC) tissues showed that RecQL1 expression is strongly correlated with histological grade and MIB-1 indices of HCC, and that the expression was greater in simple HCCs inducing extranodular growth or portal vein invasion than in HCCs not inducing extranodular growth or portal vein invasion. These histological data reveal the potential of RecQL1 as a biological marker predicting the malignancy and progression of liver cancer. High expression profiles were also produced by various HCC cells, including HCC cell lines established by us. When RecQL1 expression was silenced by siRNA in vitro, most HCC cells died of mitotic catastrophe. In a mouse orthotopic xenograft model of liver cancer with transplanted human HCC, RecQL1-siRNA mixed with cationic liposomes exhibited a strong anticancer effect that prevented the growth of the cancer. RecQL1-siRNA inhibited the growth of human HCC in the mouse liver, confirming that RecQL1 is an excellent molecular agent against liver cancer and suggests that RecQL1-siRNA formulated with liver-prone liposomes has excellent potential as a therapeutic drug against liver cancers.
International Journal of Molecular Medicine 04/2010; 25(4):537-45. · 1.98 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA) has excellent pharmacological features and is expected to be used for therapeutic drug development. To this end, however, new RNA technology needs to be established so that extremely small amounts (less than 1 pmol) of siRNA can be detected in organs of experimental animals and in human blood to facilitate pharmacokinetics studies. An important feature is that this new technology is not dependent on radioisotopes and can detect siRNA molecules identical to those used for drug development in preclinical tests with experimental animals or in clinical tests with humans. We report a convenient method that can detect small amounts of siRNA. The method uses high-power confocal microscopic analysis of fluorescence polarization in DNA probes that are bound to one of the strands of siRNA and directly quantitates the copy number of siRNA molecule after extraction from specimens. A pharmacokinetic study to examine the blood retention time of siRNA/cationic liposomes in mice showed that this straightforward method is consistent with the other reverse transcriptase polymerase chain reaction amplification-based method. We believe that the entire process is simple and applicable for a high-throughput analysis, which provides excellent technical support for fundamental research on RNA interference and development of siRNA drugs.
Nucleic Acids Research 04/2009; 37(7):e56. · 8.03 Impact Factor
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ABSTRACT: Accurate estimation of small interfering RNA (siRNA) concentration in cells and blood is increasingly important for pharmacokinetic studies required to develop siRNA drugs. We report a method that detects siRNA having 3'-terminal deoxynucleotide overhangs, such as 3'-dTdT, present in most chemically synthesized siRNAs. Short overhangs were elongated to oligo-dG by incubation with terminal deoxynucleotidyl transferase and dGTP and were used as priming sites for reverse transcription of siRNA to complementary DNA (cDNA). The resultant cDNA was used as a template for quantitation by polymerase chain reaction. This method was reliable for determining the pharmacokinetics of siRNA in blood of injected mice.
Analytical Biochemistry 01/2009; 385(2):386-8. · 3.00 Impact Factor
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Keiichi Izumikawa,
Mitsuaki Yanagida,
Toshiya Hayano,
Hiroyuki Tachikawa,
Wataru Komatsu,
Akira Shimamoto,
Kazunobu Futami, Yasuhiro Furuichi,
Takashi Shinkawa,
Yoshio Yamauchi,
Toshiaki Isobe,
Nobuhiro Takahashi
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ABSTRACT: Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.
Biochemical Journal 09/2008; 413(3):505-16. · 4.90 Impact Factor
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ABSTRACT: Small interfering RNAs (siRNAs) are expected to have a medical application in human therapy as drugs with a high specificity for their molecular target mRNAs. RecQL1 DNA helicase in the human RecQ helicase family participates in DNA repair and recombination pathways in the cell cycle of replication. Silencing the RecQL1 expression by RecQL1-siRNA induces mitotic death in vitro specifically in growing cancer cells. By contrast, the same RecQL1 silencing does not affect the growth of normal cells, emphasizing that RecQL1 helicase is an ideal molecular target for cancer therapy. In this study, we show that local and systemic administration of RecQL1-siRNA mixed with polyethyleneimine polymer or cationic liposomes prevented cancer cell proliferation in vivo in mouse models of cancer without noticeable adverse effects. The results indicate that RecQL1-siRNA in a complex with a cationic polymer is a very promising anticancer drug candidate, and that in particular, RecQL1-siRNA formulated with a cationic liposome has an enormous potential to be used by intravenous injection for therapy specific for liver cancers, including metastasized cancers from the colon and pancreas.
Cancer Science 07/2008; 99(6):1227-36. · 3.33 Impact Factor
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ABSTRACT: Werner syndrome (WS) is an autosomal recessive genetic disorder causing premature aging, and WRN has been identified as the causative gene of WS. The product of the WRN gene (WRN) acts as a DNA helicase with exonuclease activity, and data have accumulated showing that the WRN gene strongly participates in carcinogenesis: (1) the normal WRN gene likely participates in the immortalization of B-lymphoblastoid cell lines through telomeric crisis caused by telomere shortening, (2) a much higher incidence of rare cancers occurs in WS patients than in other kinds of patients, and (3) levels of WRN expressed in virus-transformed cells and cancer cells are usually markedly up-regulated and are inversely correlated with the sensitivity of these cells against various genotoxins, including camptothecin. In this paper, we review the events that show a close correlation of the WRN gene and WRN with carcinogenesis and their underlying molecular mechanisms.
Cancer Science 06/2008; 99(5):843-8. · 3.33 Impact Factor
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ABSTRACT: The inducible transcription factor NF-kappaB regulates divergent signaling pathways including inflammatory response and cancer development. Selective inhibitors for NF-kappaB signaling are potentially useful for treatment of inflammation and cancer. NF-kappaB is canonically activated by preferential disposal of its inhibitory protein; IkappaB, which suppresses the nuclear translocation of NF-kappaB. IkappaBalpha (a major member of IkappaB family proteins) is phosphorylated with an IkappaB kinase (IKK) and subsequently polyubiquitylated by SCF(betaTrCP1) ubiquitin-ligase in the presence of E1 and E2 prior to proteasomal degradation. Here, we describe a novel inhibitor termed GS143, which suppressed IkappaBalpha ubiquitylation, but not IkappaBalpha phosphorylation, MDM2-directed p53 ubiquitylation, and proteasome activity in vitro. GS143 markedly suppressed the destruction of IkappaBalpha stimulated by TNFalpha and a set of downstream responses coupled to NF-kappaB signaling but not those of p53 and beta-catenin in vivo. Our results indicate that GS143 serves as an effective inhibitor of multiple pathways served by NF-kappaB signaling.
Biochemical and Biophysical Research Communications 05/2008; 368(4):1007-13. · 2.48 Impact Factor
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ABSTRACT: RecQL1 DNA helicase of the human RecQ helicase family participates in DNA repair and recombination pathways during cell-cycle replication. When we examined the effect of RecQL1 suppression on cell growth, we found that RecQL1 silencing by small interference RNA efficiently prevented proliferation of a wide range of cancer cells by inducing mitotic catastrophe and mitotic cell death. In contrast, such mitotic cell death was not seen in the growing normal fibroblasts used as controls, even if RecQL1 expression was fully downregulated. Our results support the hypothesis that endogenous DNA damage that occurs during DNA replication and remains unrepaired in cancer cells due to RecQL1 silencing induces cancer cell-specific mitotic catastrophe through a less-strict checkpoint in cancer cells than in normal cells. We speculate that normal cells are exempt from such mitotic cell death, despite slow growth, because cell-cycle progression is controlled strictly by a strong checkpoint system that detects DNA damage and arrests progression of the cell cycle until DNA damage is repaired completely. These results suggest that RecQL1 helicase is an excellent molecular target for cancer chemotherapy.
Cancer Science 02/2008; 99(1):71-80. · 3.33 Impact Factor
