Shinobu Ueda

High susceptibility of human c-Ha-ras proto-oncogene transgenic rats to carcinogenesis: A cancer-prone animal model

Transgenic animals carrying human c‐Ha‐ras proto‐oncogene, v‐Ha‐ras transgenic mice, pim‐1 transgenic mice and several knockout mice deficient of tumor suppressor genes, such as p53, have been shown to exhibit increased carcinogen susceptibility. As a result, studies into practical application and medium‐term screening of environmental carcinogens are under way. Given the advantages of rat models characterized by larger organ size, abundant information regarding preneoplasias and virus‐free constitution, we have concentrated on the generation of transgenic rats bearing copies of the human c‐Ha‐ras proto‐oncogene and shown the Hras128 strain to be extremely sensitive to the induction of mammary carcinomas, and to a lesser extent, lesions in the urinary bladder, esophagus and skin. In most, if not all, the mammary cancers mutations of the transgene but not the endogenous H‐ras gene are present, appearing to occur early in the process of tumorigenesis, which involves proliferation of cells in TEB and intraductal hyperplasia before carcinomas arise. Preliminary findings suggest that this is independent of endogenous ovarian hormones, although inhibited by soy isoflavones and promoted by atrazine and nonylphenols. Although further studies of the mechanisms are clearly necessary, the model appears to have great potential for screening purposes, not only for modifiers active in the breast, but also other organs where tumors characterized by ras gene mutations develop. (Cancer Sci 2005; 96: 309–316)

Systemically Injected Exosomes Targeted to EGFR Deliver Antitumor MicroRNA to Breast Cancer Cells

Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2-/- mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.

miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer

MicroRNAs (miRNAs) belong to a class of endogenously expressed non‐coding small RNAs that function primarily as gene regulators. Growing evidence suggests that miRNAs play a significant role in tumor development, making them potential biomarkers for cancer diagnosis and prognosis. The miR‐17–92 cluster has emerged as an important locus, being highly overexpressed in several cancers in association with cancer development and progression. The miR‐17–92 miRNA cluster generates a single polycistronic primary transcript that yields six mature miRNAs: miR‐17, miR‐18a, miR‐19a, miR‐20a, miR‐19b, and miR‐92a. In colon cancer development, the pathophysiologic roles of these transcripts and their targets are largely unknown. In the present study, we performed copy number analyses of the six miRNAs transcribed from the miR‐17–92 cluster in colon tumor tissues. We determined that miR‐92a was transcribed at higher levels than the other five miRNAs in both adenomas and carcinoma. In addition, miR‐92a directly targeted the anti‐apoptotic molecule BCL‐2‐interacting mediator of cell death (BIM) in colon cancer tissues. An anti‐miR‐92a antagomir induced apoptosis of colon cancer‐derived cell lines. These data indicate that miR‐92a plays a pivotal role in the development of colorectal carcinoma. (Cancer Sci 2011; 102: 2264–2271)

Establishment of Rat Embryonic Stem Cells and Making of Chimera Rats

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases.

Rapid induction of skin and mammary tumors in human c‐Ha‐ras proto‐oncogene transgenic rats by treatment with 7,12‐dimethylbenz[a]anthracene followed by 12‐O‐tetradecanoylphorbol 13‐acetate

We have established a transgenic rat line carrying 3 copies of the human c‐Ha‐ras proto‐oncogene with its own promoter region (Jcl/SD‐TgN(HrasGen)128Ncc) (Hras128 rat), expression being detectable in almost all organs. We have already demonstrated that the rat is highly sensitive to mammary, esophagus and bladder carcinogenesis. In the present study, male and female transgenic and wild‐type littermates were topically treated with 2.5 mg of 7,12‐dimethylbenz[a]anthracene (DMBA) dissolved in 1.0 ml of acetone on the back skin at 50 days after birth. Starting 1 week thereafter, they were again topically treated with 100 nmol of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) dissolved in 0.5 ml of acetone 3 times weekly for the following 31 weeks. In males treated with DMBA and/or TPA, skin tumors, including both squamous cell papillomas (SCP) and carcinomas (SCC), were preferentially induced at the DMBA‐TPA painting sites: DMBA‐TPA, 15/15 (100%); DMBA, 6/8 (75%); TPA, 1/6 (16.7%). They were also, unexpectedly, induced on remote scrotal skin: DMBA‐TPA, 13/15 (86.7%); DMBA, 5/8 (62.5%); TPA, 0/6 (0%). Lesions were thus more frequent in the DMBA‐TPA group than with DMBA or TPA alone. In females, adenomas and adenocarcinomas of the mammary glands were preferentially induced: DMBA‐TPA, 12/14 (85.7%); DMBA, 6/8 (75%); TPA, 3/6 (50%), with only a few small skin papillomas at painting sites. Incidences and numbers of the mammary and skin tumors were much greater in Hras128 rats than in their wild‐type counterparts. PCR‐RFLP analysis of the transgene indicated that the percentage of the cell populations harboring a mutation in codons 12 and/or 61 ranged from 2% to 60% in individual tumors; skin tumors showed more mutations in codon 61 in the DMBA‐treated groups. In contrast, no mutations were detected in the endogenous rat c‐Ha‐ras gene. These results indicate that the Hras128 rat is highly susceptible to DMBA‐TPA skin and mammary carcinogenesis, thus providing a unique painting model for skin as well as mammary gland carcinogenesis, that would be suitable for investigating the role of transgene mutations.

Possible Application of Human c-Ha-ras Proto-Oncogene Transgenic Rats in a Medium-Term Bioassay Model for Carcinogens

With the aim of developing a medium-term assay for screening of environmental carcinogens, we exposed mammary carcinogen sensitive human c-Ha-ras proto-oncogene transgenic (Hras128) rats to various carcinogens, including compounds that do not normally induce mammary tumors. Seven-week-old Hras128 rats and wild-type littermates received administrations of 3-methylcholanthrene (3-MC), benzo[a]pyrene (B[a]P), anthracene, pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), dimethylarsinic acid (DMA), diethylnitrosamine (DEN) or azoxymethane (AOM) and were sacrificed at week 12 (females) (at week 10 for the 3-MC group) or week 20 (males). Female Hras128 rats receiving NNK, DEN, or DMA showed a significant increase in mammary tumor incidence and/or multiplicity compared to the respective values with olive oil or deionized distilled water (DDW) vehicles. In male Hras128 rats, a significant increase in mammary tumors was also observed in groups administered 3-MC, B[a]P, anthracene, IQ, and NNK. Mutations of transgenes were observed in codons 12 and/or 61 in the induced tumors by PCR-RFLP except in the DEN group in female and in the MeIQx group in male Hras128 rats. Thus various carcinogens, not necessarily limited to those normally targeting the breast, were found to induce mammary carcinomas in Hras128 rats, especially in females, pointing to potential use for medium-term screening.

Terminal Endbuds and Acini as the Respective Major Targets for Chemical and Sporadic Carcinogenesis in the Mammary Glands of Human c-Ha-ras Protooncogene Transgenic Rats

A rat strain carrying the human c-Ha-ras protooncogene, established by our laboratory, is highly susceptible to mammary chemical carcinogens. The transgenic rats exhibit increased number of terminal endbuds (TEBs) at the tips of developing ducts in the mammary gland compared to non-transgenic littermates. Confocal microscopy revealed the level of active mitogen-activated protein kinase to be elevated in these TEBs, and a close correlation between their numbers and tumorigenic response initiated by 7,12-dimethylbenz[a]anthracene was confirmed. Single injections of N-methyl-N-nitrosourea into the transgenic rats caused mutations in codon 12 of human c-Ha-ras transgene in TEBs before tumor development, supporting the conclusion that these structures are the major targets of chemical carcinogens. In contrast, with spontaneous development of lesions, alveolar hyperplasia with elevated expression levels of rat and human c-Ha-ras protooncogenes is the first morphological alteration which becomes apparent. Some but not all hyperplastic alveolar nodules were found to harbor mutations in the transgene. The results indicate that elevated expression of c-Ha-ras protooncogene is sufficient in itself to cause a highly proliferative phenotype of mammary alveoli. Our data suggest that TEBs and acini are the major targets for chemical and sporadic carcinogenesis, respectively, in the mammary glands of human c-Ha-ras protooncogene transgenic rats.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

Summary The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

Adult-Specific Systemic Over-Expression Reveals Novel In Vivo Effects of the Soluble Forms of ActRIIA, ActRIIB and BMPRII

Bone morphogenetic proteins (BMPs)/growth differentiation factors (GDFs), which belong to the TGF-beta superfamily, are pleiotropic factors that play a role in regulating the embryonic development and postnatal homeostasis of various organs and tissues by controlling cellular differentiation, proliferation and apoptosis. Conventional transgenic and knockout (KO) mouse approaches have provided only limited information regarding the in vivo functions of BMP signaling in adult animals due to the effects on prenatal development and the difficulty in manipulating multiligand signals simultaneously. We recently produced transgenic chimeric mice(Tg chimeras) in which the soluble IgG1-Fc fusion protein of three BMP type II receptors (ActRIIA, ActRIIB, BMPRII) was highly circulated (281-709 μg/ml), specifically in adult mouse blood. Since each BMP receptor can bind to multiple BMP ligands, these Tg chimeras should be useful to investigate the effects of trapping multiple BMP ligands. Remarkably, some phenotypes were unexpected based on previous studies, such as KO mouse analyses, presumably representing the effects of the multiple ligand trapping. These phenotypes included increased red blood cells (RBCs) and decreased viability in adults. In a further study, we focused on the phenotype of increased RBCs and found that extramedullary hematopoiesis in the spleen, not in the bone marrow, was increased using histological and flow cytometric analyses. Although it remains to be elucidated whether the transgene products affect the tissues directly or indirectly, our data provide novel and important insight into the biological functions of the soluble IgG1-Fc fusion protein of three BMP type II receptors in adults, and our approach should have broad applications to research on other ligand receptor families and studies involving mouse models.