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ABSTRACT: H-ferritin (HF) is a core subunit of the iron storage protein ferritin, and plays a central role in the regulation of cellular iron homeostasis. Recent studies revealed that ferritin and HF are involved in a wide variety of iron-independent functions, including regulating biological processes during physiological and pathological conditions, and can be overexpressed in some human diseases. To investigate the in vivo function of HF, we generated transgenic (tg) mice overexpressing the human HF gene (hHF-tg). We established two independent hHF-tg mouse lines. Although both lines of hHF-tg mice were viable, they showed reduced body size compared to wild-type (WT) mice at 4-12 weeks of age. Serum iron concentration and blood parameters of hHF-tg mice such as hemoglobin and red blood cell counts were comparable to those of WT mice. At 3-5 weeks of age, hHF-tg mice exhibited temporary loss of coat hair on the trunk, but not on the head or face. Histological analyses revealed that although initial hair development was normal, hHF-tg mice had epidermal hyperplasia with hyperkeratosis, dilated hair follicles, bended hair shafts and keratinous debris during the hairless period. In conclusion, we showed that hHF-tg mice exhibited mild growth retardation and temporary hairless phenotype. Our findings highlight the physiological roles of HF and demonstrate that hHF-tg mice are useful for understanding the in vivo functions of HF.
Transgenic Research 10/2012; · 2.75 Impact Factor
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ABSTRACT: H-ferritin (HF) is a core subunit of the iron storage protein ferritin and is related to the pathogenesis of malignant diseases. HF overexpression is present in human hematologic malignancies, suggesting that HF overexpression may contribute to the development of hematologic cancers. However, in vivo evidence that HF is directly linked to hematologic tumorigenesis has not yet been shown. In this study, we show that transgenic (tg) mice overexpressing the human HF gene (hHF-tg) developed aggressive radiation-induced thymic lymphoma/leukemia (TL) compared with wild-type (WT) mice, providing evidence that HF overexpression promotes leukemia/lymphomagenesis. Fractionated X-irradiation of hHF-tg mice caused a higher incidence and earlier onset of TL compared with WT mice. Immunological and pathological features of TLs were similar in both groups. However, proliferative activity of hHF-tg lymphoma cells was higher than that of WT lymphoma cells, and microarray analyses revealed that some leukemia/lymphoma-related genes were differentially expressed in hHF-tg TLs compared with WT TLs. To investigate whether cell damage induced by irradiation is related to leukemia/lymphomagenesis, we evaluated apoptotic levels in the thymus and bone marrow (BM) of hHF-tg and WT groups after fractionated X-irradiation. Apoptosis was augmented in the hHF-tg BM, but not in the thymus, compared with the WT BM, suggesting a possible linkage between increased BM apoptosis by HF overexpression and accelerated radiation-induced TL development. Our findings indicate that HF overexpression is closely related to the development of leukemia/lymphoma, which could have implications for the prevention of malignant hematologic diseases.
Carcinogenesis 07/2012; · 5.70 Impact Factor
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ABSTRACT: Malignant mesothelioma (MM) is a fatal malignancy with a rapidly increasing incidence in industrialized countries because of the widespread use of asbestos in the past centuries. Early diagnosis of MM is critical for a better prognosis, but this is often difficult because of the lack of disease-specific diagnostic imaging. Here, we report that manganese-enhanced magnetic resonance imaging (MEMRI) represents a promising approach for a more selective mesothelioma imaging by monitoring a high-level expression of manganese-superoxide dismutase (Mn-SOD), which is observed in many MM. We found that most human MM cells overexpressed Mn-SOD protein compared with human mesothelial cells and that NCI-H226 human MM cells highly expressed Mn-SOD and augmented Mn accumulation when loaded with manganese chloride (MnCl(2)). The cells showed marked T(1)-signal enhancement on in vitro MRI after incubation with MnCl(2) because of the T(1) shortening effect of Mn(2+). H226 subcutaneous tumor was preferentially enhanced compared with a lung adenocarcinoma cell tumor and another human MM cell tumor in MnCl(2)-enhanced T(1)-weighted MR image (T(1)WI), correlating with their respective Mn-SOD expression levels. Moreover, in a more clinically relevant setting, H226 xenografted pleural tumor was markedly enhanced and readily detected by MEMRI using manganese dipyridoxyl diphosphate (MnDPDP), a clinically used contrast agent, as well as MnCl(2). Therefore, we propose that MEMRI can be a potentially powerful method for noninvasive detection of MM, with high spatial resolution and marked signal enhancement, by targeting Mn-SOD.
International Journal of Cancer 05/2011; 128(9):2138-46. · 5.44 Impact Factor
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ABSTRACT: Aberrant expression of ferritin, a major iron-binding protein, has shown to be involved in neurodegenerative diseases. In this study, we generated transgenic (Tg) mice of human ferritin heavy chain (FTH) gene and investigated the effects of ferritin overexpression in FTH-Tg brain by (1)H-MRI and (1)H-MRS. The mice displayed no apparent neurological symptoms, and no specific morphological and T(2) alterations were found in the brain by MRI, and not even by histological studies. (1)H-MRS, however, revealed that some metabolic markers were significantly altered in FTH-Tg brains compared to wild-type control brains, such as decreases in myo-inositol and glutamine, and an increase in lactate. Our present studies suggested that despite the absence of neurological, morphological, T(2), and histological signatures, brain metabolisms were significantly affected in FTH-Tg mice. This study also highlights the usefulness of (1)H-MRS in the analysis of transgenic mouse models.
Magnetic Resonance Imaging 02/2011; 29(2):179-84. · 1.99 Impact Factor
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ABSTRACT: Manganese-enhanced magnetic resonance imaging (MEMRI), used to trace neuronal connections and visualize brain activity, has recently been suggested useful for tumor detection, but the mechanism of tumor enhancement by manganese (Mn) is poorly understood. Our recent report of preferential enhancement of human mesothelioma cells with higher levels of manganese-superoxide dismutase (Mn-SOD) expression may suggest a correlation between Mn-SOD expression and enhancement. We investigate this possibility further using engineered human ovarian cancer cells overexpressing Mn-SOD.
We subcutaneously implanted SK-OV-3 human ovarian cancer cells stably overexpressing Mn-SOD (SK-Mn-SOD) into athymic nude mice and SK-OV-3 cells with plasmid DNAs carrying neomycin-resistant genes (SK-neo) into the same mice for controls. We conducted MEMRI in the tumor-bearing mice and compared enhancement between the 2 tumors.
Subcutaneous SK-Mn-SOD tumors were preferentially enhanced in MEMRI compared to SK-neo tumors. After Mn enhancement, the T(1)-relaxation rate (R(1)=1/T(1)) increased significantly for SK-Mn-SOD but not SK-neo tumors.
In some tumors, high expression of Mn-SOD may be a biological factor responsible for enhanced signal in MEMRI.
Magnetic Resonance in Medical Sciences 01/2011; 10(3):155-8. · 0.97 Impact Factor
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ABSTRACT: Molecular imaging using magnetic resonance (MR) imaging has been actively investigated and made rapid progress in the past decade. Applied to cancer gene therapy, the technique's high spatial resolution allows evaluation of gene delivery into target tissues. Because noninvasive monitoring of the duration, location, and magnitude of transgene expression in tumor tissues or cells provides useful information for assessing therapeutic efficacy and optimizing protocols, molecular imaging is expected to become a critical step in the success of cancer gene therapy in the near future. We present a brief overview of the current status of molecular MR imaging, especially in vivo reporter gene imaging using ferritin and other reporters, discuss its application to cancer gene therapy, and present our research of MR imaging detection of electroporation-mediated cancer gene therapy using the ferritin reporter gene.
Magnetic Resonance in Medical Sciences 01/2010; 9(2):37-47. · 0.97 Impact Factor
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ABSTRACT: The recent success with small fish as an animal model of cancer with the aid of fluorescence technique has attracted cancer modelers' attention because it would be possible to directly visualize tumor cells in vivo in real time. Here, we report a medaka model capable of allowing the observation of various cell behaviors of transplanted tumor cells, such as cell proliferation and metastasis, which were visualized easily in vivo. We established medaka melanoma (MM) cells stably expressing GFP and transplanted them into nonirradiated and irradiated medaka. The tumor cells were grown at the injection sites in medaka, and the spatiotemporal changes were visualized under a fluorescence stereoscopic microscope at a cellular-level resolution, and even at a single-cell level. Tumor dormancy and metastasis were also observed. Interestingly, in irradiated medaka, accelerated tumor growth and metastasis of the transplanted tumor cells were directly visualized. Our medaka model provides an opportunity to visualize in vivo tumor cells "as seen in a culture dish" and would be useful for in vivo tumor cell biology.
Proceedings of the National Academy of Sciences 09/2009; 106(33):13832-7. · 9.68 Impact Factor
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ABSTRACT: Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.
ChemBioChem 12/2008; 9(16):2682-91. · 3.94 Impact Factor
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ABSTRACT: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD).
There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn.
In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.
Journal of Trace Elements in Medicine and Biology 02/2008; 22(3):248-55. · 1.68 Impact Factor
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ABSTRACT: Exposure to asbestos is a known etiological factor in malignant mesothelioma (MM). However, in vitro cell culture studies have provided paradoxical evidence that asbestos exposure to mesothelial cells causes cytotoxicity or apoptosis rather than malignant transformation. Although it has been shown that the iron associated with asbestos participates in the cell toxicity and probably MM pathogenesis via generation of reactive oxygen species (ROS), the molecular mechanisms largely remain unknown. Here, we demonstrate that ferritin heavy chain (FHC), a core subunit of iron-binding protein ferritin, works as an anti-apoptotic protein against toxic asbestos and oxidative stress in human mesothelial cells and MM cells. We found that FHC was induced in asbestos-exposed MeT-5A human mesothelial cells. The mesothelial cell line stably expressing FHC generated less amount of hydrogen peroxide (H2O2), one of the main ROS, after asbestos exposure and was more resistant to apoptosis induced by H2O2 compared with the cells transfected with the empty vector. Next, we investigated biological roles of FHC in human MM cell. We found that NCI-H2052, a human MM cell line, had a higher expression of endogenous FHC than MeT-5A and used the cell to address FHC function in MM. NCI-H2052 showed reduced H2O2 production and an apoptosis-resistant phenotype compared with MeT-5A. Suppression of the over-expressed FHC by using FHC small interfering RNA rendered the MM cells sensitive to apoptosis, suggesting the contribution of FHC to apoptosis resistance of the MM cells. Our findings highlight the potential role of FHC in the pathogenesis of asbestos-induced mesothelioma.
Carcinogenesis 10/2007; 28(9):2047-52. · 5.70 Impact Factor
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ChemBioChem 07/2006; 7(6):925-8. · 3.94 Impact Factor
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ABSTRACT: The internal guiding sequence (IGS) is normally located at the 5' end of trans-splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans-splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS-containing substrate, and the IGS-lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans-splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self-assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.
FEBS Letters 04/2006; 580(6):1592-6. · 3.54 Impact Factor
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Naoto Yoshizuka,
Ryozo Moriuchi,
Tsuyoshi Mori,
Kenji Yamada, Sumitaka Hasegawa,
Takahiro Maeda,
Takako Shimada,
Yasuaki Yamada,
Shimeru Kamihira,
Masao Tomonaga,
Shigeru Katamine
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ABSTRACT: By screening cDNA expression libraries derived from fresh leukemic cells of adult T-cell leukemia for the potential to transform murine fibroblasts, NIH3T3, we have identified a novel transforming gene, designated Tgat. Expression of Tgat in NIH3T3 resulted in the loss of contact inhibition, increase of saturation density, anchorage-independent growth in a semisolid medium, tumorigenicity in nude mice, and increased invasiveness. Sequence comparison revealed that an alternative RNA splicing of the Trio gene was involved in the generation of Tgat. The Tgat cDNA encoded a protein product consisting of the Rho-guanosine nucleotide exchange factor (GEF) domain of a multifunctional protein, TRIO, and a unique C-terminal 15-amino acid sequence, which were derived from the exons 38-46 of the Trio gene and a novel exon located downstream of its last exon (exon 58), respectively. A Tgat mutant cDNA lacking the C-terminal coding region preserved Rho-GEF activity but lost the transforming potential, indicating an indispensable role of the unique sequence. On the other hand, treatment of Tgat-transformed NIH3T3 cells with Y-27632, a pharmacological inhibitor of Rho-associated kinase, abrogated their transforming phenotypes, suggesting the coinvolvement of Rho-GEF activity. Thus, alternative RNA splicing, resulting in the fusion protein with the Rho-GEF domain and the unique 15 amino acids, is the mechanism generating the novel oncogene, Tgat.
Journal of Biological Chemistry 11/2004; 279(42):43998-4004. · 4.77 Impact Factor
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ABSTRACT: The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants.
Journal of the American Chemical Society 07/2004; 126(23):7158-9. · 9.91 Impact Factor
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ABSTRACT: Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, beta-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of beta-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.
Proceedings of the National Academy of Sciences 01/2004; 100(25):14892-6. · 9.68 Impact Factor
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Sumitaka Hasegawa,
Tomoyuki Sato,
Hiroshi Akazawa,
Hitoshi Okada,
Akiteru Maeno,
Masaki Ito,
Yoshinobu Sugitani,
Hiroyuki Shibata,
Jun-ichi Miyazaki Ji,
Motoya Katsuki,
Yasutaka Yamauchi,
Ken-ichi Yamamura Ki,
Shigeru Katamine,
Tetsuo Noda
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ABSTRACT: Apc is a gene associated with familial adenomatous polyposis coli (FAP) and its inactivation is a critical step in colorectal tumor formation. The protein product, adenomatous polyposis coli (APC), acts to down-regulate intracellular levels of beta-catenin, a key signal transducer in the Wnt signaling. Conditional targeting of Apc in the neural crest of mice caused massive apoptosis of cephalic and cardiac neural crest cells at about 11.5 days post coitum, resulting in craniofacial and cardiac anomalies at birth. Notably, the apoptotic cells localized in the regions where beta-catenin had accumulated. In contrast to its role in colorectal epithelial cells, inactivation of APC leads to dysregulation of beta-catenin/Wnt signaling with resultant apoptosis in certain tissues including neural crest cells.
Proceedings of the National Academy of Sciences 02/2002; 99(1):297-302. · 9.68 Impact Factor
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Sumitaka Hasegawa,
Tomoyuki Sato,
Hiroshi Akazawa,
Hitoshi Okada,
Akiteru Maeno,
Masaki Ito,
Yoshinobu Sugitani,
Hiroyuki Shibata,
Jun-ichi Miyazaki,
Motoya Katsuki,
Yasutaka Yamauchi,
Ken-Ichi Yamamura,
Shigeru Katamine,
Tetsuo Noda
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ABSTRACT: Apc is a gene associated with familial adenomatous polyposis coli (FAP) and its inactivation is a critical step in colorectal tumor formation. The protein product, adenomatous polyposis coli (APC), acts to down-regulate intracellular levels of β-catenin, a key signal transducer in the Wnt signaling. Conditional targeting of Apc in the neural crest of mice caused massive apoptosis of cephalic and cardiac neural crest cells at about 11.5 days post coitum, resulting in craniofacial and cardiac anomalies at birth. Notably, the apoptotic cells localized in the regions where β-catenin had accumulated. In contrast to its role in colorectal epithelial cells, inactivation of APC leads to dysregulation of β-catenin/Wnt signaling with resultant apoptosis in certain tissues including neural crest cells.
