Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB t... more Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB transgenic mouse carrying one copy of BamHI fragment of HBV with a deletion of Bg/II-Bg/II fragment, no RNA transcription was observed. Transgenic mice that had integrated either one or three tandem copies of HBV DNA produced RNA in various tissues except liver. However, neither initiation nor termination of transcription occurred at the correct place. Hepatitis B surface antigen and hepatitis B e antigen were not detectable in these transgenic mice. HBV DNA was methylated at all CCGG sequences, which might have resulted in aberrant RNA production. In two out of five transgenic mice HBV DNA was integrated into the Y chromosome. Although the frequency is unusually high, the mechanism is not known yet.
Most cancer recurrences are incurable and life threatening. Clinical evidence has revealed that d... more Most cancer recurrences are incurable and life threatening. Clinical evidence has revealed that disseminated tumor cells (DTCs) are present in the bone marrow (BM) in 20–40% of patients with various solid tumors at the early stage, indicating that DTCs persist somewhere in the body and may regrow a few years after a curative resection of the primary tumors. It is critical to clarify the mechanisms by which DTCs survive in the body, regrow, and metastasize. Paracrine interactions between cancer cells and the tumor microenvironment have been shown to control tumor growth and progression. Recently, it has been suggested that the BM niche acts as a supporter for DTCs, keeping these cells in a dormant state in the BM. Indeed, accumulating evidence has shown that DTCs share common properties and signaling pathways with normal stem cells within the BM niche. In this chapter, we discuss clinical evidence of DTCs and data for tumor dormancy within the BM, as well as the escape of DTC from do...
Conclusion: Our study firstly performed a systematic estimation of the incidence of SPM in NSCLC,... more Conclusion: Our study firstly performed a systematic estimation of the incidence of SPM in NSCLC, which implied the necessity of a risk predicting model. We developed the first competing risk nomogram to predict the risk of SPM, which performed well in the evaluation and might be helpful for individualized SPM screening.
The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function w... more The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G-200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.
Effect of human fibroblast interferon (IFN-m on normal human monocyte activation induced by the m... more Effect of human fibroblast interferon (IFN-m on normal human monocyte activation induced by the monocyte activating factor found in sarcoidosis sera was studied. Spreading was used as one indicator of monocyte activation. IFN-,8 was shown to inhibit spreading of normal human monocytes induced by the activating factor. The inhibitory activity of IFN-,8 against monocyte spreading was adsorbed with monoclonal antibody to human IFN-,8-Sepharose beads. Increases in phagocytosis and glucose consumption of normal human monocytes induced by the activating factor was also inhibited by IFN-,8. Recombinant IFN-,8 also showed similar inhibitions.
Publisher Summary Antisense technology has been greatly facilitated by the development of cancer ... more Publisher Summary Antisense technology has been greatly facilitated by the development of cancer therapy utilizing antisense oligodeoxynucleotides (AS-ODNs). However, prior to extensive use of natural ODNs in vivo , a number of problems must be addressed, such as rapid degradation in the presence of serum, poor membrane transport, nonspecific inhibition of other genes, and cytotoxicity at high doses. Several attempts have been made to overcome these limitations, including a chemical modification of the backbone of ODNs, which resulted in an enhanced resistance to nucleases and longer survival times in vivo. An especially critical area lies in the development of a novel delivery system for an efficient transfer of ODNs into tissue or organs; this can be accomplished in a number of ways. Of the procedures examined to date, microinjection, electroporation, and chemical reagents—such as cationic liposomes and poly( l -lysine) and receptor-mediated endocytosis—are believed to be the most effective methods. This chapter presents procedures for cationic liposome-mediated AS-ODN transfer into an in vitro organ culture system for mouse limb development.
Predictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in ... more Predictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in the selection of treatment options, including liver transplantation (LT), for HCC. The purpose of this study was to identify specific microRNAs (miRs) in exosomes from the serum of patients with recurrent HCC and to validate these molecules as novel biomarkers for HCC recurrence. We employed microarray-based expression profiling of miRs derived from exosomes in the serum of HCC patients to identify a biomarker that distinguishes between patients with and without HCC recurrence after LT. This was followed by the validation in a separate cohort of 59 HCC patients who underwent living related LT. The functions and potential gene targets of the recurrence-specific miRs were analysed using a database, clinical samples and HCC cell lines. We found that miR-718 showed significantly different expression in the serum exosomes of HCC cases with recurrence after LT compared with those without recur...
We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other gro... more We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked ...
Megakaryocyte (MK) development is dependent on the complex interaction of MK progenitors, various... more Megakaryocyte (MK) development is dependent on the complex interaction of MK progenitors, various cytokines and stromal elements. We previously reported that an injection of replication-deficient adenovirus containing HST-1/FGF-4 cDNA (Adex1HST-1) into mice caused a twofold increase in peripheral platelet count for 30 days without any other hematological or histological abnormality. In the present study using Adex1HST-1-infected human megakaryocytic Dami cells, we demonstrated for the first time that HST-1/FGF-4 promoted MK maturation, inducing increases in DNA ploidy, cytoplasmic and membrane maturation, and platelet-like particle release. Moreover, HST-1/FGF-4 acted on megakaryocytic cells to induce secretion of IL-6 and TNF-alpha, and increased adhesion of megakaryocytic cells to human endothelial cells primarily via VLA-4 and LFA-1 molecules; both mechanisms have been shown to lead to MK maturation. We also showed that HST-1/FGF-4 stimulates the proliferation of MK progenitors n...
Primary human hepatocellular carcinomas (HCC) were surveyed for oncogenes by transformation assay... more Primary human hepatocellular carcinomas (HCC) were surveyed for oncogenes by transformation assays using NIH3T3 cells and the calcium phosphate coprecipitation method. High-molecular-weight DNA obtained from the HCC tissues was employed for this purpose. One new transforming DNA, named lca (for liver cancer) was obtained, and its properties were studied in detail. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage. Restriction enzyme analyses showed that the minimum size of the lca DNA is about 10.5 kilobase pairs and that its nucleotide sequence is different from that of any known human gene or retroviral oncogene. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. It showed identical restriction enzyme cleavage profiles with its counterpart DNA obtained from normal tissue, indicating that there is no extensive DNA rearrangement associ...
Transforming activity was detected in a hepatocellular carcinoma (HCC) carrying four integrated h... more Transforming activity was detected in a hepatocellular carcinoma (HCC) carrying four integrated hepatitis B virus (HBV) DNA. This transforming gene was identified as hst-1, that lies in chromosome 11, band q13.3. One of the integrated HBV DNAs was found to lie close to the hst-1, and the hst-1 and the integrated HBV DNA were found to be co-amplified. The region of the amplification was limited. A model has been proposed that correlates the viral integration, amplification and activation of an oncogene.
How to cite this article: Kurioka, D. et al. NEK9-dependent proliferation of cancer cells lacking... more How to cite this article: Kurioka, D. et al. NEK9-dependent proliferation of cancer cells lacking functional p53. Sci. Rep. 4, 6111;
One of the most challenging issues in liver tissue engineering is the incorporation of a function... more One of the most challenging issues in liver tissue engineering is the incorporation of a functional bile duct (BD) network in the hepatic tissue. In this study, we investigated the feasibility of a three-dimensional (3D) coculture of adult rat primary biliary epithelial cells (ABECs) and rat fetal liver cells (FLCs) to form functional BD structures. 3D hetero-spheroids containing various ratios of the two cell types were successfully obtained in low-adherence, round-bottom, 96-well plates. Histological analyses and functional analysis using fluorescein diacetate demonstrated that the ABECs critically contributed to the reconstruction of continuous ductular networks. Metabolized fluorescein was transported toward cystic structures in a time- and ABEC ratio-dependent manner. In particular, the duct-like structures containing fluorescein accumulations interlinked the large cystic accumulations. Furthermore, transplantation experiments demonstrated that ABECs, but not FLCs, enabled the establishment of BD networks in vivo. This study provided important insights into the development of transplantable liver tissues with bile excretion functionality.
of biologically active siRNA in its final form. The advantage of such a system is the avoidance o... more of biologically active siRNA in its final form. The advantage of such a system is the avoidance of the need to translocate the nuclear membrane in non-dividing cells. Cationic oligopeptides of varying sequence (CK 6 C, CK 8 C, CK 10 C, CH 3 K 3 H 3 C, CH 6 K 3 H 6 C) were synthesised and subjected to oxidative polycondensation to produce reducible polycations (RPCs). Complexation of RPCs with siRNA form 100nm particles stable in the weakly reducing extracellular environment but designed to disintegrate in the intracellular reducing environment. Complexation completely retarded the mobility of siRNA on an agarose gel, with the amount of RPC required dependent on the lysine content. Treatment with 8mM glutathione restored siRNA mobility, indicating cleavage of disulphide bonds within the RPC, thus simulating the reducing conditions within the cytoplasm. Indeed a punctate pattern of glutathione localisation was demonstrated within liver cells using two-photon laser scanning microscopy. A GFP-expressing stable liver cell line was generated to test the biological activity of siRNA delivered by various RPCs. siRNA delivered using pH-responsive histidine containing RPCs exhibited the greatest level of GFP repression consistent with improved endosomal escape and decomplexation of particles within the cytoplasm. The intracellular fate of complexes is being assessed using Cy3 labelled siRNA. Furthermore RPCs terminated in hepatocyte-binding oligopeptides will be employed to improve cell uptake via receptormediated endocytosis. Ultimately it is hoped this vector system will provide a platform for delivery of siRNA targeting hepatitis B and C proteins for treatment of liver disease.
An auxiliary liver represents a promising alternative for liver transplantation. The use of a lar... more An auxiliary liver represents a promising alternative for liver transplantation. The use of a large amount of mature hepatocytes, however, despite their high function, is limited in a clinical setting. Here, we propose a novel transplantation system that dramatically improved a diseased animal by incorporating fetal liver cells (FLCs) as a cell source, the mesentery as a transplantation site and a hyaluronic acid (HA) sponge as a cell scaffold. We transplanted wild-type Long Evans Agouti rat FLCs embedded in HA sponges onto the mesentery of Long Evans Cinnamon (LEC) rats, an animal model for Wilson's disease. The FLC-loaded HA sponges successfully grafted and consequently prevented jaundice. Accordingly, the treated animals showed a significant reduction in blood copper concentration, which consequently led to significant decreases in serum total bilirubin and direct bilirubin, and to a significant increase in albumin productivity. Furthermore, haematoxylin and eosin staining of the host livers demonstrated that fibrosis at the periportal area was moderated in the treated animals. In conclusion, we transplanted FLC-loaded HA sponges onto the mesenteric blood vessels, leading to thick, liver-like tissue possessing blood vessels, and the liver tissue engineered thus exhibited a remarkable therapeutic effect on the copper metabolism deficiency of LEC rats.
Cancer-specific antigens are promising targets for the specific delivery of certain drugs or gene... more Cancer-specific antigens are promising targets for the specific delivery of certain drugs or genes to cancer cells in cancer therapy. Carcinoembryonic antigen (CEA) is one of the cancer-associated antigens predominantly detected in the gastrointestinal cancer of the colon and stomach. Targeting strategies for CEA-producing cancer cells have been thoroughly developed mainly by the production of monoclonal antibodies to CEA and further single-chain variable fragment (scFv) antibodies. Here, we have generated Moloney murine leukemia virus-derived retroviral vectors co-displaying an anti-CEA scFv-envelope chimeric protein and an unmodified envelope protein to deliver a gene for hepres simplex virus thymidine kinase (HSV-tk) or Escherichia coli /3-galactosidase. The harvested viruses successfully incorporated the chimeric envelope protein as well as the unmodified envelope into the viral particles, and specifically bound to and infected human CEAproducing cancer cells via recognition of CEA, depending on the CEA-producing phenotype of the target cells. These results may have significant implications for the use of scFv directed against tumor-specific antigens for targeting specific antigen-producing cancer cells, a potential step toward in vivo cancer therapy. OVERVIEW SUMMARY antibodies should then be selected; and appropriate scFv insertion sites in the envelope protein, an appropriate linker Targeting to tumor-specific antigens is a powerful approach size between the scFv and the envelope, and an approprito obtain safe and efficient therapeutic effects in cancer gene ate expression ratio of the chimeric envelope to the untherapy. The present study showed that a murine leukemia modified envelope should be determined. virus-derived virus co-displaying an anti-CEA scFv-envelope protein and an unmodified envelope could infect human CEA-producing cancer cells and deliver a suicide gene INTRODUCTION to human CEA-producing cancer cells specifically through the recognition of CEA, depending on the CEA-producing r • iargeting to human tumor-specihc antigens has a great phenotype of the target cells. This strategy may possibly M. potential in cancer therapy for selective delivery of theralead to a specific killing of the antigen-producing cancer peutic agents or genes to cancer cells. Carcinoembryonic anticells by specific suicide gene delivery, and may also be ap-gen (CEA) is one of tiie most extensively characterized tumorplied to other tumor-specific cell-surface antigens or recep-associated antigens detected on the surface of cancer of tiie tors expressed solely on cancer cells. However, further im-colon, stomach, pancreas, lung, and breast as well as fetal inprovement is required to achieve more efTicient and specific testine (Gold and Freedman, 1965a,b). CEA is a glycosylphosretroviral gene transfer. For this purpose, the following is-phatidylinositol (GPI)-anchored glycoprotein (Hefta ef al, sues should be addressed: Appropriate target antigens or 1988) belonging to the immunoglobulin superfamily (Oikawa receptors on target cells should be selected to facilitate more et al, 1987; Zknmermann et al, 1987; Sato et al, 1988). The efficient viral infection of the target cells; high-affinity scFv normal counterparts of CEA such as NCA-2 (Burtin etal, 1973)
Liver transplants represent the only way to treat patients suffering from terminal liver failure,... more Liver transplants represent the only way to treat patients suffering from terminal liver failure, but they are associated with numerous problems, including a chronic shortage of donors, high cost, rejection, and side effects for the donor. It is anticipated that regenerative medicine will provide an alternative to liver transplants for such patients. Regenerative medicine refers to the academic field of eliciting the inherent capacity of organisms for self-regeneration to the greatest possible extent in order to develop new methods of treatment for intractable disorders. From this perspective, much is expected from the use of human embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells), and the vigorous development of technology to induce the differentiation of such stem cells into cells possessing hepatic functions is underway. Clinical applications of these human stem cells, however, have yet to reach even the earliest stages of implementation. Facing off against these versatile ES cells are stem cells derived from somatic cells present within organisms, which are attracting attention owing to their superiority in terms of ethics and safety, with many research institutes now in the process of elucidating the details of stem cell separation and identification as well as their plasticity and pluripotency. Bone marrow cells are the best-known somatic-cell-derived stem cells, but the use of mesenchymal stem cells (MSCs) found in adipose tissue has also recently attracted attention. This paper will review the differentiation ability and mechanisms of these various stem cell types to hepatocytes and their application to liver regeneration and the future outlook.
To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we an... more To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we analyzed genomic imprinting in Dicer1-hypomorphic mice, in which Dicer1 expression was significantly reduced. We did not observe any abnormality in the allelic expression of imprinted genes in these mice or their offspring, suggesting that reduced expression of Dicer1 did not significantly affect the maintenance and reprogramming of imprinting.
Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by ... more Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg +/and Parg-/-) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg-/embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg +/+ ES cells. Parg-/-ES cells exhibited the same growth rate as Parg +/+ ES cells in culture. Sensitivity of Parg-/-ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and-irradiation was examined by clonogenic survival assay. Parg-/-ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and-irradiation compared with wildtype (Parg +/+) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo-and radiation therapies.
Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB t... more Three lines of transgenic mice carrying hepatitis B virus (HBV) DNA were produced. In a pSHB1BB transgenic mouse carrying one copy of BamHI fragment of HBV with a deletion of Bg/II-Bg/II fragment, no RNA transcription was observed. Transgenic mice that had integrated either one or three tandem copies of HBV DNA produced RNA in various tissues except liver. However, neither initiation nor termination of transcription occurred at the correct place. Hepatitis B surface antigen and hepatitis B e antigen were not detectable in these transgenic mice. HBV DNA was methylated at all CCGG sequences, which might have resulted in aberrant RNA production. In two out of five transgenic mice HBV DNA was integrated into the Y chromosome. Although the frequency is unusually high, the mechanism is not known yet.
Most cancer recurrences are incurable and life threatening. Clinical evidence has revealed that d... more Most cancer recurrences are incurable and life threatening. Clinical evidence has revealed that disseminated tumor cells (DTCs) are present in the bone marrow (BM) in 20–40% of patients with various solid tumors at the early stage, indicating that DTCs persist somewhere in the body and may regrow a few years after a curative resection of the primary tumors. It is critical to clarify the mechanisms by which DTCs survive in the body, regrow, and metastasize. Paracrine interactions between cancer cells and the tumor microenvironment have been shown to control tumor growth and progression. Recently, it has been suggested that the BM niche acts as a supporter for DTCs, keeping these cells in a dormant state in the BM. Indeed, accumulating evidence has shown that DTCs share common properties and signaling pathways with normal stem cells within the BM niche. In this chapter, we discuss clinical evidence of DTCs and data for tumor dormancy within the BM, as well as the escape of DTC from do...
Conclusion: Our study firstly performed a systematic estimation of the incidence of SPM in NSCLC,... more Conclusion: Our study firstly performed a systematic estimation of the incidence of SPM in NSCLC, which implied the necessity of a risk predicting model. We developed the first competing risk nomogram to predict the risk of SPM, which performed well in the evaluation and might be helpful for individualized SPM screening.
The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function w... more The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G-200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.
Effect of human fibroblast interferon (IFN-m on normal human monocyte activation induced by the m... more Effect of human fibroblast interferon (IFN-m on normal human monocyte activation induced by the monocyte activating factor found in sarcoidosis sera was studied. Spreading was used as one indicator of monocyte activation. IFN-,8 was shown to inhibit spreading of normal human monocytes induced by the activating factor. The inhibitory activity of IFN-,8 against monocyte spreading was adsorbed with monoclonal antibody to human IFN-,8-Sepharose beads. Increases in phagocytosis and glucose consumption of normal human monocytes induced by the activating factor was also inhibited by IFN-,8. Recombinant IFN-,8 also showed similar inhibitions.
Publisher Summary Antisense technology has been greatly facilitated by the development of cancer ... more Publisher Summary Antisense technology has been greatly facilitated by the development of cancer therapy utilizing antisense oligodeoxynucleotides (AS-ODNs). However, prior to extensive use of natural ODNs in vivo , a number of problems must be addressed, such as rapid degradation in the presence of serum, poor membrane transport, nonspecific inhibition of other genes, and cytotoxicity at high doses. Several attempts have been made to overcome these limitations, including a chemical modification of the backbone of ODNs, which resulted in an enhanced resistance to nucleases and longer survival times in vivo. An especially critical area lies in the development of a novel delivery system for an efficient transfer of ODNs into tissue or organs; this can be accomplished in a number of ways. Of the procedures examined to date, microinjection, electroporation, and chemical reagents—such as cationic liposomes and poly( l -lysine) and receptor-mediated endocytosis—are believed to be the most effective methods. This chapter presents procedures for cationic liposome-mediated AS-ODN transfer into an in vitro organ culture system for mouse limb development.
Predictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in ... more Predictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in the selection of treatment options, including liver transplantation (LT), for HCC. The purpose of this study was to identify specific microRNAs (miRs) in exosomes from the serum of patients with recurrent HCC and to validate these molecules as novel biomarkers for HCC recurrence. We employed microarray-based expression profiling of miRs derived from exosomes in the serum of HCC patients to identify a biomarker that distinguishes between patients with and without HCC recurrence after LT. This was followed by the validation in a separate cohort of 59 HCC patients who underwent living related LT. The functions and potential gene targets of the recurrence-specific miRs were analysed using a database, clinical samples and HCC cell lines. We found that miR-718 showed significantly different expression in the serum exosomes of HCC cases with recurrence after LT compared with those without recur...
We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other gro... more We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked ...
Megakaryocyte (MK) development is dependent on the complex interaction of MK progenitors, various... more Megakaryocyte (MK) development is dependent on the complex interaction of MK progenitors, various cytokines and stromal elements. We previously reported that an injection of replication-deficient adenovirus containing HST-1/FGF-4 cDNA (Adex1HST-1) into mice caused a twofold increase in peripheral platelet count for 30 days without any other hematological or histological abnormality. In the present study using Adex1HST-1-infected human megakaryocytic Dami cells, we demonstrated for the first time that HST-1/FGF-4 promoted MK maturation, inducing increases in DNA ploidy, cytoplasmic and membrane maturation, and platelet-like particle release. Moreover, HST-1/FGF-4 acted on megakaryocytic cells to induce secretion of IL-6 and TNF-alpha, and increased adhesion of megakaryocytic cells to human endothelial cells primarily via VLA-4 and LFA-1 molecules; both mechanisms have been shown to lead to MK maturation. We also showed that HST-1/FGF-4 stimulates the proliferation of MK progenitors n...
Primary human hepatocellular carcinomas (HCC) were surveyed for oncogenes by transformation assay... more Primary human hepatocellular carcinomas (HCC) were surveyed for oncogenes by transformation assays using NIH3T3 cells and the calcium phosphate coprecipitation method. High-molecular-weight DNA obtained from the HCC tissues was employed for this purpose. One new transforming DNA, named lca (for liver cancer) was obtained, and its properties were studied in detail. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage. Restriction enzyme analyses showed that the minimum size of the lca DNA is about 10.5 kilobase pairs and that its nucleotide sequence is different from that of any known human gene or retroviral oncogene. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. It showed identical restriction enzyme cleavage profiles with its counterpart DNA obtained from normal tissue, indicating that there is no extensive DNA rearrangement associ...
Transforming activity was detected in a hepatocellular carcinoma (HCC) carrying four integrated h... more Transforming activity was detected in a hepatocellular carcinoma (HCC) carrying four integrated hepatitis B virus (HBV) DNA. This transforming gene was identified as hst-1, that lies in chromosome 11, band q13.3. One of the integrated HBV DNAs was found to lie close to the hst-1, and the hst-1 and the integrated HBV DNA were found to be co-amplified. The region of the amplification was limited. A model has been proposed that correlates the viral integration, amplification and activation of an oncogene.
How to cite this article: Kurioka, D. et al. NEK9-dependent proliferation of cancer cells lacking... more How to cite this article: Kurioka, D. et al. NEK9-dependent proliferation of cancer cells lacking functional p53. Sci. Rep. 4, 6111;
One of the most challenging issues in liver tissue engineering is the incorporation of a function... more One of the most challenging issues in liver tissue engineering is the incorporation of a functional bile duct (BD) network in the hepatic tissue. In this study, we investigated the feasibility of a three-dimensional (3D) coculture of adult rat primary biliary epithelial cells (ABECs) and rat fetal liver cells (FLCs) to form functional BD structures. 3D hetero-spheroids containing various ratios of the two cell types were successfully obtained in low-adherence, round-bottom, 96-well plates. Histological analyses and functional analysis using fluorescein diacetate demonstrated that the ABECs critically contributed to the reconstruction of continuous ductular networks. Metabolized fluorescein was transported toward cystic structures in a time- and ABEC ratio-dependent manner. In particular, the duct-like structures containing fluorescein accumulations interlinked the large cystic accumulations. Furthermore, transplantation experiments demonstrated that ABECs, but not FLCs, enabled the establishment of BD networks in vivo. This study provided important insights into the development of transplantable liver tissues with bile excretion functionality.
of biologically active siRNA in its final form. The advantage of such a system is the avoidance o... more of biologically active siRNA in its final form. The advantage of such a system is the avoidance of the need to translocate the nuclear membrane in non-dividing cells. Cationic oligopeptides of varying sequence (CK 6 C, CK 8 C, CK 10 C, CH 3 K 3 H 3 C, CH 6 K 3 H 6 C) were synthesised and subjected to oxidative polycondensation to produce reducible polycations (RPCs). Complexation of RPCs with siRNA form 100nm particles stable in the weakly reducing extracellular environment but designed to disintegrate in the intracellular reducing environment. Complexation completely retarded the mobility of siRNA on an agarose gel, with the amount of RPC required dependent on the lysine content. Treatment with 8mM glutathione restored siRNA mobility, indicating cleavage of disulphide bonds within the RPC, thus simulating the reducing conditions within the cytoplasm. Indeed a punctate pattern of glutathione localisation was demonstrated within liver cells using two-photon laser scanning microscopy. A GFP-expressing stable liver cell line was generated to test the biological activity of siRNA delivered by various RPCs. siRNA delivered using pH-responsive histidine containing RPCs exhibited the greatest level of GFP repression consistent with improved endosomal escape and decomplexation of particles within the cytoplasm. The intracellular fate of complexes is being assessed using Cy3 labelled siRNA. Furthermore RPCs terminated in hepatocyte-binding oligopeptides will be employed to improve cell uptake via receptormediated endocytosis. Ultimately it is hoped this vector system will provide a platform for delivery of siRNA targeting hepatitis B and C proteins for treatment of liver disease.
An auxiliary liver represents a promising alternative for liver transplantation. The use of a lar... more An auxiliary liver represents a promising alternative for liver transplantation. The use of a large amount of mature hepatocytes, however, despite their high function, is limited in a clinical setting. Here, we propose a novel transplantation system that dramatically improved a diseased animal by incorporating fetal liver cells (FLCs) as a cell source, the mesentery as a transplantation site and a hyaluronic acid (HA) sponge as a cell scaffold. We transplanted wild-type Long Evans Agouti rat FLCs embedded in HA sponges onto the mesentery of Long Evans Cinnamon (LEC) rats, an animal model for Wilson's disease. The FLC-loaded HA sponges successfully grafted and consequently prevented jaundice. Accordingly, the treated animals showed a significant reduction in blood copper concentration, which consequently led to significant decreases in serum total bilirubin and direct bilirubin, and to a significant increase in albumin productivity. Furthermore, haematoxylin and eosin staining of the host livers demonstrated that fibrosis at the periportal area was moderated in the treated animals. In conclusion, we transplanted FLC-loaded HA sponges onto the mesenteric blood vessels, leading to thick, liver-like tissue possessing blood vessels, and the liver tissue engineered thus exhibited a remarkable therapeutic effect on the copper metabolism deficiency of LEC rats.
Cancer-specific antigens are promising targets for the specific delivery of certain drugs or gene... more Cancer-specific antigens are promising targets for the specific delivery of certain drugs or genes to cancer cells in cancer therapy. Carcinoembryonic antigen (CEA) is one of the cancer-associated antigens predominantly detected in the gastrointestinal cancer of the colon and stomach. Targeting strategies for CEA-producing cancer cells have been thoroughly developed mainly by the production of monoclonal antibodies to CEA and further single-chain variable fragment (scFv) antibodies. Here, we have generated Moloney murine leukemia virus-derived retroviral vectors co-displaying an anti-CEA scFv-envelope chimeric protein and an unmodified envelope protein to deliver a gene for hepres simplex virus thymidine kinase (HSV-tk) or Escherichia coli /3-galactosidase. The harvested viruses successfully incorporated the chimeric envelope protein as well as the unmodified envelope into the viral particles, and specifically bound to and infected human CEAproducing cancer cells via recognition of CEA, depending on the CEA-producing phenotype of the target cells. These results may have significant implications for the use of scFv directed against tumor-specific antigens for targeting specific antigen-producing cancer cells, a potential step toward in vivo cancer therapy. OVERVIEW SUMMARY antibodies should then be selected; and appropriate scFv insertion sites in the envelope protein, an appropriate linker Targeting to tumor-specific antigens is a powerful approach size between the scFv and the envelope, and an approprito obtain safe and efficient therapeutic effects in cancer gene ate expression ratio of the chimeric envelope to the untherapy. The present study showed that a murine leukemia modified envelope should be determined. virus-derived virus co-displaying an anti-CEA scFv-envelope protein and an unmodified envelope could infect human CEA-producing cancer cells and deliver a suicide gene INTRODUCTION to human CEA-producing cancer cells specifically through the recognition of CEA, depending on the CEA-producing r • iargeting to human tumor-specihc antigens has a great phenotype of the target cells. This strategy may possibly M. potential in cancer therapy for selective delivery of theralead to a specific killing of the antigen-producing cancer peutic agents or genes to cancer cells. Carcinoembryonic anticells by specific suicide gene delivery, and may also be ap-gen (CEA) is one of tiie most extensively characterized tumorplied to other tumor-specific cell-surface antigens or recep-associated antigens detected on the surface of cancer of tiie tors expressed solely on cancer cells. However, further im-colon, stomach, pancreas, lung, and breast as well as fetal inprovement is required to achieve more efTicient and specific testine (Gold and Freedman, 1965a,b). CEA is a glycosylphosretroviral gene transfer. For this purpose, the following is-phatidylinositol (GPI)-anchored glycoprotein (Hefta ef al, sues should be addressed: Appropriate target antigens or 1988) belonging to the immunoglobulin superfamily (Oikawa receptors on target cells should be selected to facilitate more et al, 1987; Zknmermann et al, 1987; Sato et al, 1988). The efficient viral infection of the target cells; high-affinity scFv normal counterparts of CEA such as NCA-2 (Burtin etal, 1973)
Liver transplants represent the only way to treat patients suffering from terminal liver failure,... more Liver transplants represent the only way to treat patients suffering from terminal liver failure, but they are associated with numerous problems, including a chronic shortage of donors, high cost, rejection, and side effects for the donor. It is anticipated that regenerative medicine will provide an alternative to liver transplants for such patients. Regenerative medicine refers to the academic field of eliciting the inherent capacity of organisms for self-regeneration to the greatest possible extent in order to develop new methods of treatment for intractable disorders. From this perspective, much is expected from the use of human embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells), and the vigorous development of technology to induce the differentiation of such stem cells into cells possessing hepatic functions is underway. Clinical applications of these human stem cells, however, have yet to reach even the earliest stages of implementation. Facing off against these versatile ES cells are stem cells derived from somatic cells present within organisms, which are attracting attention owing to their superiority in terms of ethics and safety, with many research institutes now in the process of elucidating the details of stem cell separation and identification as well as their plasticity and pluripotency. Bone marrow cells are the best-known somatic-cell-derived stem cells, but the use of mesenchymal stem cells (MSCs) found in adipose tissue has also recently attracted attention. This paper will review the differentiation ability and mechanisms of these various stem cell types to hepatocytes and their application to liver regeneration and the future outlook.
To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we an... more To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we analyzed genomic imprinting in Dicer1-hypomorphic mice, in which Dicer1 expression was significantly reduced. We did not observe any abnormality in the allelic expression of imprinted genes in these mice or their offspring, suggesting that reduced expression of Dicer1 did not significantly affect the maintenance and reprogramming of imprinting.
Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by ... more Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg +/and Parg-/-) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg-/embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg +/+ ES cells. Parg-/-ES cells exhibited the same growth rate as Parg +/+ ES cells in culture. Sensitivity of Parg-/-ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and-irradiation was examined by clonogenic survival assay. Parg-/-ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and-irradiation compared with wildtype (Parg +/+) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo-and radiation therapies.
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Papers by T. Ochiya