Takashi Tsuruo

Publications (171)776.98 Total impact

• Article: The polar neutral and basic taxoids isolated from needles and twigs of Taxus cuspidata and their biological activity

  Liyan Wang, Liming Bai, Daisuke Tokunaga, Yusuke Watanabe, Toshiaki Hasegawa, Jun-ichi Sakai, Wanxia Tang, Yuhua Bai, Katsutoshi Hirose, Takao Yamori, Akihiro Tomida, Takashi Tsuruo, Masayoshi Ando
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  ABSTRACT: Twelve basic taxoids and 22 neutral taxoids were isolated from basic and polar neutral fractions of the extracts of needles and twigs of Taxus cuspidata. Among them, taxine NA-13, 3,11-cyclotaxinine NN-1, taxinine NN-6, 11(15→1)abeo-taxinine NN-1, taxine NA-8, and taxine NA-4 were isolated first from natural sources by us. The cytotoxic activity of isolated compounds was evaluated against three human cell lines: normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). 7-Epitaxol, 7-epicephalomannine, taxinine NN-6, taxine NA-2, taxuspine H, and taxagifine were active toward VA-13 cells and 7-epitaxol, 7-epicephalomannine, taxinine NN-1, 9,10-deacetyltaxinine, and taxagifine were active toward HepG2 cells. The multidrug-resistant (MDR) cancer reversal activity of isolated compounds was evaluated on the basis of the amount of vincristine (VCR) accumulated in MDR human ovarian cancer 2780 AD. Taxine NA-8, taxine NA-2, 5-cinnamoyl-10-acetyltaxicin II, and taxinine NN-1 indicated stronger MDR cancer reversal activity than verapamil. The result of primary screening based on 39 human cancer cell lines suggests that taxinine NN-1 belongs to a new mechanistic class and is a new anticancer agents. 7-Epicephalomannine was found to be an effective anticancer agent with tubulin as its molecular target, which is the same as paclitaxel.
  Journal of Wood Science 04/2012; 54(5):390-401. · 0.96 Impact Factor
• Article: TRF1 mediates mitotic abnormalities induced by Aurora-A overexpression.

  Tomokazu Ohishi, Toru Hirota, Takashi Tsuruo, Hiroyuki Seimiya
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  ABSTRACT: Aurora-A, a conserved serine-threonine kinase, plays essential roles in mitosis. Aberrant upregulation of Aurora-A perturbs proper mitotic progression and results in a generation of multinucleated cells with centrosome amplification. The molecular mechanisms for these mitotic defects remain elusive. Here, we show that the overexpressed Aurora-A-induced mitotic defects depend on the telomeric protein TRF1. Live and fixed cell analyses revealed that Aurora-A overexpression in HeLa cells compromises chromosome biorientation, which leads to cytokinetic failure and tetraploidization with increased centrosome numbers. TRF1 depletion by small interfering RNAs or by tankyrase-1 overexpression suppresses Aurora-A-induced occurrence of unaligned chromosomes in metaphase, thus preventing the subsequent abnormalities. We found that Aurora-A binds and phosphorylates TRF1. When TRF1 knockdown cells are complemented with wild-type TRF1, Aurora-A-induced mitotic defects recur. By contrast, a TRF1 mutant that is not phosphorylatable by Aurora-A does not restore such Aurora-A-induced phenotype. We propose that TRF1 phosphorylation by excessive Aurora-A may provoke abnormal mitosis and chromosomal instability.
  Cancer Research 02/2010; 70(5):2041-52. · 7.86 Impact Factor
• Article: Mitochondria regulate the unfolded protein response leading to cancer cell survival under glucose deprivation conditions.

  Naomi Haga, Sakae Saito, Yoshinori Tsukumo, Junko Sakurai, Aki Furuno, Takashi Tsuruo, Akihiro Tomida
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  ABSTRACT: Cancer cells consume large amounts of glucose because of their specific metabolic pathway. However, cancer cells exist in tumor tissue where glucose is insufficient. To survive, cancer cells likely have the mechanism to elude their glucose addiction. Here we show that functional mitochondria are essential if cancer cells are to avoid glucose addiction. Cancer cells with dysfunctional mitochondria, such as mitochondrial DNA-deficient rho(0) cells and electron transport chain blocker-treated cells, were highly sensitive to glucose deprivation. Our data demonstrated that this sensitization was associated with failure of the unfolded protein response (UPR), an adaptive response mediated by the endoplasmic reticulum (ER). This study suggests a link between mitochondria and the ER during the UPR under glucose deprivation conditions and that mitochondria govern cell fate, not only through ATP production and apoptosis regulation, but also through modulating the UPR for cell survival.
  Cancer Science 02/2010; 101(5):1125-32. · 3.33 Impact Factor
• Article: Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L.

  Ryohei Katayama, Toshiyasu Ishioka, Shinji Takada, Ritsuko Takada, Naoya Fujita, Takashi Tsuruo, Mikihiko Naito
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  ABSTRACT: Cellular FLIP (cFLIP) inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) enhances Wnt signaling via inhibition of beta-catenin ubiquitylation. In this report, we present evidence that cFLIP-L translocates into the nucleus, which could have a role in modulation of Wnt signaling. cFLIP-L has a functional bipartite nuclear localization signal (NLS) at the C-terminus. Wild-type cFLIP-L (wt-FLIP-L) localizes in both the nucleus and cytoplasm, whereas NLS-mutated cFLIP-L localizes predominantly in the cytoplasm. cFLIP-L also has a nuclear export signal (NES) near the NLS, and leptomycin B, an inhibitor of CRM1-dependent nuclear export, increases the nuclear accumulation of cFLIP-L, suggesting that it shuttles between the nucleus and cytoplasm. Expression of mutant cFLIP-L proteins with a deletion or mutations in the NLS and NES confers resistance to Fas-mediated apoptosis, as does wt-FLIP-L, but they do not enhance Wnt signaling, which suggests an important role of the C-terminus of cFLIP-L in Wnt-signaling modulation. When wt-FLIP-L is expressed in the cytoplasm by conjugation with exogenous NES (NES-FLIP-L), Wnt signaling is not enhanced, whereas the NES-FLIP-L increases cytoplasmic beta-catenin as efficiently as wt-FLIP-L. cFLIP-L physically interacts with the reporter plasmid for Wnt signaling, but not with the control plasmid. These results suggest a role for nuclear cFLIP-L in the modulation of Wnt signaling.
  Journal of Cell Science 01/2010; 123(Pt 1):23-8. · 6.11 Impact Factor
• Article: Reply to Honore: Masking of Signal Sequences in CREC Proteins by cDNA Subcloning in Epitope Vectors.

  Akihiro Tomida, Yoshinori Tsukumo, Satomi Tsukahara, Sakae Saito, Takashi Tsuruo
  Journal of Biological Chemistry 12/2009; 284(49):le14. · 4.77 Impact Factor
• Article: A Novel Endoplasmic Reticulum Export Signal

  Yoshinori Tsukumo, Satomi Tsukahara, Sakae Saito, Takashi Tsuruo, Akihiro Tomida
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  ABSTRACT: NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro+2) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1–35 peptide region, including both signal peptide (amino acids 1–26) and Pro+2, was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro+2 resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro+2 was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro+2 has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca2+-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro+2 can function as a novel ER export signal of some Golgi proteins.
  Journal of Biological Chemistry 10/2009; 284(40):27500-27510. · 4.77 Impact Factor
• Article: A novel endoplasmic reticulum export signal: proline at the +2-position from the signal peptide cleavage site.

  Yoshinori Tsukumo, Satomi Tsukahara, Sakae Saito, Takashi Tsuruo, Akihiro Tomida
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  ABSTRACT: NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro(+2)) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1-35 peptide region, including both signal peptide (amino acids 1-26) and Pro(+2), was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro(+2) resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro(+2) was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro(+2) has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca(2+)-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro(+2) can function as a novel ER export signal of some Golgi proteins.
  Journal of Biological Chemistry 09/2009; 284(40):27500-10. · 4.77 Impact Factor
• Article: Dofequidar fumarate sensitizes cancer stem-like side population cells to chemotherapeutic drugs by inhibiting ABCG2/BCRP-mediated drug export.

  Ryohei Katayama, Sumie Koike, Shigeo Sato, Yoshikazu Sugimoto, Takashi Tsuruo, Naoya Fujita
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  ABSTRACT: The ATP-binding cassette (ABC) transporters (ABC-T) actively efflux structurally and mechanistically unrelated anticancer drugs from cells. As a consequence, they can confer multidrug resistance (MDR) to cancer cells. ABC-T are also reported to be phenotypic markers and functional regulators of cancer stem/initiating cells (CSC) and believed to be associated with tumor initiation, progression, and relapse. Dofequidar fumarate, an orally active quinoline compound, has been reported to overcome MDR by inhibiting ABCB1/P-gp, ABCC1/MDR-associated protein 1, or both. Phase III clinical trials suggested that dofequidar had efficacy in patients who had not received prior therapy. Here we show that dofequidar inhibits the efflux of chemotherapeutic drugs and increases the sensitivity to anticancer drugs in CSC-like side population (SP) cells isolated from various cancer cell lines. Dofequidar treatment greatly reduced the cell number in the SP fraction. Estimation of ABC-T expression revealed that ABCG2/breast cancer resistance protein (BCRP) mRNA level, but not the ABCB1/P-gp or ABCC1/MDR-associated protein 1 mRNA level, in all the tested SP cells was higher than that in non-SP cells. The in vitro vesicle transporter assay clarified that dofequidar had the ability to suppress ABCG2/BCRP function. Dofequidar treatment sensitized SP cells to anticancer agents in vitro. We compared the antitumor efficacy of irinotecan (CPT-11) alone with that of CPT-11 plus dofequidar in xenografted SP cells. Although xenografted SP tumors showed resistance to CPT-11, treatment with CPT-11 plus dofequidar greatly reduced the SP-derived tumor growth in vivo. Our results suggest the possibility of selective eradication of CSC by inhibiting ABCG2/BCRP.
  Cancer Science 08/2009; 100(11):2060-8. · 3.33 Impact Factor
• Article: Biodistribution and ultrastructural localization of single-walled carbon nanohorns determined in vivo with embedded Gd2O3 labels.

  Jin Miyawaki, Sachiko Matsumura, Ryota Yuge, Tatsuya Murakami, Shigeo Sato, Akihiro Tomida, Takashi Tsuruo, Toshinari Ichihashi, Takako Fujinami, Hiroshi Irie, Kunihiro Tsuchida, Sumio Iijima, Kiyotaka Shiba, Masako Yudasaka
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  ABSTRACT: Single-walled carbon nanohorns (SWNHs) are single-graphene tubules that have shown high potential for drug delivery systems. In drug delivery, it is essential to quantitatively determine biodistribution and ultrastructural localization. However, to date, these determinations have not been successfully achieved. In this report, we describe for the first time a method that can achieve these determinations. We embedded Gd(2)O(3) nanoparticles within SWNH aggregates (Gd(2)O(3)@SWNHag) to facilitate detection and quantification. Gd(2)O(3)@SWNHag was intravenously injected into mice, and the quantities of Gd in the internal organs were measured by inductively coupled plasma atomic emission spectroscopy: 70-80% of the total injected material accumulated in liver. The high electron scattering ability of Gd allows detection with energy dispersive X-ray spectroscopy and facilitates the ultrastructural localization of individual Gd(2)O(3)@SWNHag with transmission electron microscopy. In the liver, we found that the Gd(2)O(3)@SWNHag was localized in Kupffer cells but were not observed in hepatocytes. In the Kupffer cells, most of the Gd(2)O(3)@SWNHag was detected inside phagosomes, but some were in another cytoplasmic compartment that was most likely the phagolysosome.
  ACS Nano 07/2009; 3(6):1399-406. · 10.77 Impact Factor
• Article: Intestinal epithelial cancer cell anoikis resistance: EGFR-mediated sustained activation of Src overrides Fak-dependent signaling to MEK/Erk and/or PI3-K/Akt-1.

  Marie-Josée Demers, Sonya Thibodeau, Dominique Noël, Naoya Fujita, Takashi Tsuruo, Rémy Gauthier, Mélina Arguin, Pierre H Vachon
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  ABSTRACT: Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3-K/Akt-1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down-activation, cancer cells nevertheless exhibit sustained Fak-Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3-K/Akt-1 down-activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3-K/Akt-1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak-mediated signaling to MEK/Erk and PI3-K/Akt-1 in T84 cells, as in undifferentiated IECs, whereas PI3-K/Akt-1 is Src-independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak-Src interactions and down-activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3-K/Akt-1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR-mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak-Src interactions and MEK/Erk activation, thus not only overriding Fak-mediated signaling to MEK/Erk and/or PI3-K/Akt-1, but also the requirement of Fak and/or PI3-K/Akt-1 for survival.
  Journal of Cellular Biochemistry 06/2009; 107(4):639-54. · 2.87 Impact Factor
• Article: Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide.

  Tetsuo Mashima, Shigeo Sato, Sachiko Okabe, Satoshi Miyata, Masaaki Matsuura, Yoshikazu Sugimoto, Takashi Tsuruo, Hiroyuki Seimiya
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  ABSTRACT: Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl-CoA synthetase (ACS), which converts long-chain fatty acids to acyl-CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c-resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax-induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well-known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non-toxic dose enhanced the antitumor efficacy of a low-dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide.
  Cancer Science 06/2009; 100(8):1556-62. · 3.33 Impact Factor
• Article: Chemical genomics identifies the unfolded protein response as a target for selective cancer cell killing during glucose deprivation.

  Sakae Saito, Aki Furuno, Junko Sakurai, Asami Sakamoto, Hae-Ryong Park, Kazuo Shin-Ya, Takashi Tsuruo, Akihiro Tomida
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  ABSTRACT: Glucose deprivation, a cell condition that occurs in solid tumors, activates the unfolded protein response (UPR). A key feature of the UPR is the transcription program activation, which allows the cell to survive under stress conditions. Here, we show that the UPR transcription program is disrupted by the antidiabetic biguanides metformin, buformin, and phenformin depending on cellular glucose availability. These drugs inhibit production of the UPR transcription activators XBP1 and ATF4 and induce massive cell death during glucose deprivation as did the antitumor macrocyclic compound versipelostatin. Gene expression profiling shows remarkable similarity in the modes of action of biguanides and versipelostatin determined by the broad range of glucose deprivation-inducible genes. Importantly, during glucose deprivation, most of the biguanide suppression genes overlap with the genes induced by tunicamycin, a chemical UPR inducer. Gene expression profiling also identifies drug-driven signatures as a tool for discovering pharmacologic UPR modulators. Our findings show that disrupting the UPR during glucose deprivation could be an attractive approach for selective cancer cell killing and could provide a chemical genomic basis for developing UPR-targeting drugs against solid tumors.
  Cancer Research 06/2009; 69(10):4225-34. · 7.86 Impact Factor
• Article: PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

  Hiroshi Tanaka, Yutaka Hoshikawa, Tomoko Oh-hara, Sumie Koike, Mikihiko Naito, Tetsuo Noda, Hiroyuki Arai, Takashi Tsuruo, Naoya Fujita
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  ABSTRACT: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments
  Molecular Cancer Research 05/2009; 7(4):557-69. · 4.29 Impact Factor
• Article: Preventing the unfolded protein response via aberrant activation of 4E-binding protein 1 by versipelostatin.

  Junichi Matsuo, Yoshinori Tsukumo, Junko Sakurai, Satomi Tsukahara, Hae-Ryong Park, Kazuo Shin-ya, Toshiki Watanabe, Takashi Tsuruo, Akihiro Tomida
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  ABSTRACT: We recently isolated a macrocyclic compound, versipelostatin (VST), that exerts in vivo antitumor activity. VST shows unique, selective cytotoxicity to glucose-deprived tumor cells by preventing the unfolded protein response (UPR). Here we show that eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of eukaryotic initiation factor 4E-mediated protein translation, plays a role in the UPR-inhibitory action of VST. Indeed, 4E-BP1 is aberrantly activated by VST. This activation occurs specifically during glucose deprivation and results in profound translation repression and prevents induction of the typical UPR markers glucose-regulated protein (GRP) 78 and activating transcription factor (ATF) 4. Our overexpression and knockdown experiments showed that 4E-BP1 can regulate GRP78 and ATF4 expression. These mechanisms appear to be specific for VST. By contrast, rapamycin, which activates 4E-BP1 regardless of cellular glucose availability, has only marginal effects on the expression of GRP78 and ATF4. Our present findings demonstrate that aberrant 4E-BP1 activation can contribute to UPR preventing by VST, possibly through a mechanism that does not operate in rapamycin-treated cells.
  Cancer Science 01/2009; 100(2):327-33. · 3.33 Impact Factor
• Chapter: Therapeutic Targets and Drugs III: Tankyrase 1, Telomere-Binding Proteins, and Inhibitors

  Hiroyuki Seimiya, Takashi Tsuruo
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  ABSTRACT: Telomere maintenance by telomerase enables cancer cells to proliferate indefinitely. Telomerase inhibitors resume the end replication problem and gradually shorten telomeres in telomerase-positive cancer cells. Critically shortened telomeres elicit a DNA damage response and induce senescence, apoptosis, or both. Accordingly, telomerase inhibition is one of the rational strategies for cancer therapy. Meanwhile, there are increasing numbers of telomere-binding proteins that maintain telomere integrity. Among them, tankyrase 1, a telomeric poly(ADP-ribose) polymerase, is one of the most druggable targets, whose enzymatic inhibition enhances the anticancer impact of telomerase inhibitors. Telomere capping is accomplished by sufficient lengths of double- and single-stranded telomeric DNA and their association with shelterin, which consists of TRF1, TRF2, TIN2, Rap1, TPP1, and POT1. Disruption of shelterin function leads to prompt telomere decapping, followed by DNA damage response and growth inhibition. In this chapter, we review telomere length regulation by tankyrase 1 and telomere protection by TRF2 and POT1, as potential target events for telomere-directed molecular cancer therapeutics. KeywordsTankyrase 1–TRF1–Poly(ADP-ribosyl)ation–3′-Overhang–Shelterin–TRF2–POT1
  12/2008: pages 281-291;
• Article: Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

  Tae Nakasaki, Toshiyuki Tanaka, Shinichi Okudaira, Michi Hirosawa, Eiji Umemoto, Kazuhiro Otani, Soojung Jin, Zhongbin Bai, Haruko Hayasaka, Yoshinori Fukui, Katsuyuki Aozasa, Naoya Fujita, Takashi Tsuruo, Keiichi Ozono, Junken Aoki, Masayuki Miyasaka
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  ABSTRACT: Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.
  American Journal Of Pathology 10/2008; 173(5):1566-76. · 4.89 Impact Factor
• Article: IL12RB2 and ABCA1 genes are associated with susceptibility to radiation dermatitis.

  Minoru Isomura, Natsuo Oya, Seiji Tachiiri, Yuko Kaneyasu, Yasumasa Nishimura, Tetsuo Akimoto, Masato Hareyama, Tadasi Sugita, Norio Mitsuhashi, Takashi Yamashita, Masahiko Aoki, Heitetsu Sai, Yutaka Hirokawa, Koh-Ichi Sakata, Kumiko Karasawa, Akihiro Tomida, Takashi Tsuruo, Yoshio Miki, Tetsuo Noda, Masahiro Hiraoka
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  ABSTRACT: Severe acute radiation dermatitis is observed in approximately 5% to 10% of patients who receive whole-breast radiotherapy. Several factors, including treatment-related and patient-oriented factors, are involved in susceptibility to severe dermatitis. Genetic factors are also thought to be related to a patient's susceptibility to severe dermatitis. To elucidate genetic polymorphisms associated with a susceptibility to radiation-induced dermatitis, a large-scale single-nucleotide polymorphism (SNP) analysis using DNA samples from 156 patients with breast cancer was conducted. Patients were selected from more than 3,000 female patients with early breast cancer who received radiotherapy after undergoing breast-conserving surgery. The dermatitis group was defined as patients who developed dermatitis at a National Cancer Institute Common Toxicity Criteria grade of > or =2. For the SNP analysis, DNA samples from each patient were subjected to the genotyping of 3,144 SNPs covering 494 genes. SNPs that mapped to two genes, ABCA1 and IL12RB2, were associated with radiation-induced dermatitis. In the ABCA1 gene, one of these SNPs was a nonsynonymous coding SNP causing R219K (P = 0.0065). As for the IL12RB2 gene, the strongest association was observed at SNP-K (rs3790568; P = 0.0013). Using polymorphisms of both genes, the probability of severe dermatitis was estimated for each combination of genotypes. These analyses showed that individuals carrying a combination of genotypes accounting for 14.7% of the Japanese population have the highest probability of developing radiation-induced dermatitis. Our results shed light on the mechanisms responsible for radiation-induced dermatitis. These results may also contribute to the individualization of radiotherapy.
  Clinical Cancer Research 10/2008; 14(20):6683-9. · 7.74 Impact Factor
• Article: Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt pathway in epidermal growth factor signaling.

  Akito Nakamura, Mikihiko Naito, Takashi Tsuruo, Naoya Fujita
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  ABSTRACT: The phosphoinositide 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathway regulates various cellular functions, especially cell survival and cell cycle progression. In contrast to other survival pathways, there have been few reports of scaffold proteins that regulate signaling cascade specificity in this pathway. Here we identify a 5' repressor element under dual-repression binding protein 1 (Freud-1)/Akt kinase-interacting protein 1 (Aki1) as a novel scaffold for the PDK1/Akt pathway. Freud-1/Aki1 (also known as CC2D1A) expression induced formation of a PDK1/Akt complex and regulated Akt activation in a concentration-dependent biphasic manner. Freud-1/Aki1 also associated with epidermal growth factor (EGF) receptor in response to EGF stimulation and was required for Akt activation induced by EGF, but not by insulin-like growth factor 1. Freud-1/Aki1 gene silencing decreased Akt kinase activity, resulting in induction of apoptosis and increased sensitivity toward chemotherapeutic agents. Our results suggest that Freud-1/Aki1 is a novel receptor-selective scaffold protein for the PDK1/Akt pathway and present a new activation mechanism of Akt.
  Molecular and cellular biology 08/2008; 28(19):5996-6009. · 6.06 Impact Factor
• Article: Pim kinases promote cell cycle progression by phosphorylating and down-regulating p27Kip1 at the transcriptional and posttranscriptional levels.

  Daisuke Morishita, Ryohei Katayama, Kazuhisa Sekimizu, Takashi Tsuruo, Naoya Fujita
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  ABSTRACT: The serine/threonine kinase Pim is known to promote cell cycle progression and to inhibit apoptosis leading to tumorigenesis. However, the precise mechanisms remain unclear. We show, herein, that all the Pim family members (Pim1, Pim2, and Pim3) bind to and directly phosphorylate the cyclin-dependent kinase inhibitor p27(Kip1) at threonine-157 and threonine-198 residues in cells and in vitro. The Pim-mediated phosphorylation induced p27(Kip1) binding to 14-3-3 protein, resulting in its nuclear export and proteasome-dependent degradation. Ectopic expression of Pim kinases overcome the G(1) arrest mediated by wild-type p27(Kip1) but not by phosphorylation-resistant T157A-p27(Kip1) or T198A-p27(Kip1). In addition to the posttranslational regulations, p27(Kip1) promoter assay revealed that Pim kinases also had the ability to suppress p27(Kip1) transcription. Pim-mediated phosphorylation and inactivation of forkhead transcription factors, FoxO1a and FoxO3a, was involved in the transcriptional repression of the p27(Kip1) gene. In contrast, inhibition of Pim signaling by expressing the dominant-negative form of Pim1 increased nuclear p27(Kip1) level and attenuated cell proliferation. Because the CDK inhibitor p27(Kip1) plays a crucial role in tumor suppression by inhibiting abnormal cell cycle progression, Pim kinases promote cell cycle progression and tumorigenesis by down-regulating p27(Kip1) expression at both transcriptional and posttranslational levels.
  Cancer Research 08/2008; 68(13):5076-85. · 7.86 Impact Factor
• Article: Tetraspanin family member CD9 inhibits Aggrus/podoplanin-induced platelet aggregation and suppresses pulmonary metastasis.

  Youya Nakazawa, Shigeo Sato, Mikihiko Naito, Yukinari Kato, Kazuhiko Mishima, Hiroyuki Arai, Takashi Tsuruo, Naoya Fujita
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  ABSTRACT: CD9 has been reported to play a role in tumor metastasis suppression. However, it is not fully understood how CD9 affects the hematogenous spread of tumor cells. To clarify a new mechanism (or mechanisms), we generated HT1080 cells that had been transfected with a CD9-expressing plasmid. Ectopic expression of CD9 in HT1080 cells actually reduced their metastatic ability. CD9 expression reduced lung retention and platelet aggregation activity of the transfectants. Because HT1080 cells express the metastasis-promoting, platelet aggregation-inducing factor Aggrus/podoplanin on their surface, we examined the relationship between CD9 and Aggrus. We discovered that CD9 formed a complex with Aggrus via transmembrane domains 1 and 2 (TM1 and TM2) of CD9. Investigation of the interaction revealed that each CD9 and Aggrus interacted homophilically, and that they colocalized in low-density membrane fractions. Deleting TM1 and TM2 attenuated the ability of CD9 to interact homophilically or to localize in low-density membrane fractions. The expression of CD9-wild-type (WT), but not CD9 lacking TM1 and TM2, attenuated the platelet aggregation and metastasis induced by forced expression of Aggrus in CHO cells. Therefore, CD9 may act as a metastasis suppressor, at least in part, by neutralizing Aggrus-mediated platelet aggregation.
  Blood 07/2008; 112(5):1730-9. · 9.90 Impact Factor