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Background: Pancreatic ductal adenocarcinoma (PDA) is associated with very poor prognoses. Therefore, new therapies and preclinical models are urgently needed. In the present study, we sought to develop more realistic experimental models for use in PDA research. Methods: We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), which we integrated to create 13 matched “trios” – i.e., patient-derived tumor models of PDA. We then compared and contrasted histological and molecular alterations between these three model systems. Results: Orthotopic implantation (OI) of the PDCLs resulted in tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDXs and OI-PDXs with passaged tumors revealed that the histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. When gene expression levels in the PDCLs, ectopic tumors, and OI tumors were compared, the distant metastasis-promoting gene CXCR4 was specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation. Conclusion: These patient-derived tumor models provide useful tools for monitoring responses to antineoplastic agents and for studying PDA biology.

Supplemental digital content is available in the text., Objectives Peritoneal dissemination (PD) is an important cause of morbidity and mortality among patients with pancreatic ductal adenocarcinoma (PDAC). We sought to develop and characterized a novel PD mouse model by using a previously established PDAC cell line TCC-Pan2. Methods TCC-Pan2 cell line was characterized for growth rate, tumor markers, histology, and somatic mutations. TCC-Pan2 cells were implanted orthotopically to produce PD. TCC-Pan2 cells from these metastatic foci were expanded in vitro and then implanted orthotopically in mice. This PD model was used for comparing the antitumor effect of paclitaxel and NK105. Results Orthotopically implanted TCC-Pan2 cells caused tumor formation and PD with high frequency in mice. A potent metastatic subline—Pan2M—was obtained. NK105 exerted a stronger antitumor effect than paclitaxel against Pan2M cells harboring a luciferase gene (Pan2MmLuc). Notably, the survival rate on day 80 in the Pan2MmLuc mouse model was 100% for the NK105 group and 0% for the paclitaxel group. Conclusion TCC-Pan2 cell line and Pan2MmLuc PD model can serve as useful tools for monitoring the responses to antineoplastic agents and for studying PDAC biology.

SiRNAs are strong gene-silencing agents that function in a target sequence-specific manner. Although siRNAs might one day be used in therapy for intractable diseases such as cancers, a number of problems with siRNAs must first be overcome. In this study, we developed 16 different types of lipid-conjugated siRNAs (lipid-siRNAs) that could effectively inhibit the expression of target genes. We determined the hybridization properties, cellular uptake efficacies, and RNAi potencies of the resulting lipid-siRNAs. The lipid-siRNAs exhibited a mild interaction with Lipofectamine RNAiMAX (LFRNAi) as a transfection reagent, and a high membrane permeability was observed in all lipid-siRNAs-LFRNAi complexes; the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed an especially good cellular uptake efficacy. The in vitro RNAi effect of lipid-siRNAs targeted to a β-catenin gene exhibited a strong RNAi potency compared with those of unmodified siRNAs. In particular, the conjugate siRNAs composed of 16-18 carbon chains as fatty acids showed excellent RNAi potencies with prolonged effectivities. Interestingly, the RNAi potencies of conjugate siRNAs containing 18 carbon chains with a trans-form (elaidic acid and trans-vaccenic acid) were inferior to those of the carbon chains with a cis-form (oleic acid and cis-vaccenic acid). These lipid-siRNAs can solve the many problems hindering the clinical application of siRNAs.

Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (EGSH/GSSG = − 333 mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (EGSH/GSSG > − 256 mV) and relatively high H2O2 production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that H2O2 production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time., Highlights • Palmitoylated versions of redox sensors were constructed. • The sensors successfully visualized local redox environment near membranes. • The cytosol is oxidative near endocytosed vesicles containing EGFR., Graphical abstract fx1

Cachexia, a negative prognostic factor, worsens a patient's quality of life. We established 2 novel cachexia models with the human stomach cancer cell line MKN-45, which was subcloned to produce potent cachexia-inducing cells by repeating the xenografts in immune-deficient mice. After subsequent xenografts, we isolated potent cachexia-inducing cells (MKN45cl85 and 85As2mLuc). Xenografts of MKN45cl85 cells in mice led to substantial weight loss and reduced adipose tissue and musculature volumes, whereas xenografts of 85As2mLuc cells resulted in highly metastatic and cachectic mice. Surgical removal of tumor tissues helped the mice regain body-weight in both mouse models. In vitro studies using these cells showed that isoflavones reduced their proliferation, implying that the isoflavones possess antiproliferative effects of these cancer cell lines. Isoflavone treatment on the models induced tumor cytostasis, attenuation of cachexia, and prolonged survival whereas discontinuation of the treatment resulted in progressive tumor growth and weight loss. The inhibitory effects of tumor growth and weight loss by isoflavones were graded as soy isoflavone aglycone AglyMax > daidzein > genistein. These results demonstrated that the 2 novel cachectic mouse models appear useful for analyzing the mechanism of cancer cachexia and monitoring the efficacy of anticachectic agents.

Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

Short interfering RNAs (siRNAs), used in RNA interference (RNAi) technology, are powerful tools for target-gene silencing in a sequence-specific manner. In this study, Dicer-substrate 27-nucleotide (nt) double-stranded RNAs (dsRNAs), which are known to have a highly potent RNAi effect, were conjugated with palmitic acid at the 5'-end of the sense strand to enhance intracellular delivery and RNAi efficacy. The palmitic acid-conjugated 27-nt dsRNAs (C16-ds27RNAs) were prepared by our simple synthesis strategy in good yield. The C16-ds27RNAs were cleaved by a Dicer enzyme, leading to the release of 21-nt siRNAs. The high level of stability in serum using C16-ds27RNAs was also confirmed. The C16-ds27RNAs showed enhanced RNAi potency targeted to both an exogenous luciferase and an endogenous vascular endothelial growth factor (VEGF) gene in the presence or absence of a transfection reagent, such as Lipofectamine 2000. In addition, the C16-ds27RNAs had a more potent gene-silencing activity than the other lipid-conjugated 21-nt siRNAs and 27-nt dsRNAs. The C16-ds27RNAs also exhibited significant membrane permeability. These results suggested that the C16-ds27RNAs will be useful for next-generation RNAi molecules that can address the problems of RNAi technology.

Effects of artekeiskeanol A, a newly isolated coumarin derivative from Artemisa keiskeana Miq. (Compositae), the extract of which is used for treatment of rheumatoid arthritis as a folk medicine, on the antigen-induced activation of RBL-2H3 cells were examined. RBL-2H3 cells were sensitized with dinitrophenol (DNP)-specific IgE, and then stimulated with the antigen DNP-conjugated human serum albumin (DNP-HSA). Artekeiskeanol A at 10 to 100 microM inhibited the antigen-induced degranulation in a concentration-dependent manner, the IC(50) value being 38.0 + or - 0.2 microM. Degranulation induced by thapsigargin or A23187 also was inhibited by artekeiskeanol A at 10 to 100 microM. The antigen-induced increase in the levels of mRNA for tumor necrosis factor (TNF)-alpha and interleukin (IL)-13 and phosphorylations of Akt, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p44/42 MAPK were also suppressed by artekeiskeanol A. Our findings suggested that the effectiveness of the extract of A. keiskeana might partly be due to the inhibition of mast cell activation by artekeiskeanol A.

To improve our understanding of ionizing radiation effects on immune cells, we investigated steps leading to radiation-induced cell death in MOLT-4, a thymus-derived human leukemia cell. After exposure of MOLT-4 cells to 4 Gy of X-rays, irradiated cells sequentially showed increase in intracellular reactive oxygen species (ROS), decrease in mitochondrial membrane potential, and eventually apoptotic cell death. In the presence of the caspase inhibitor z-VAD-fmk, irradiated cells exhibited necrotic characteristics such as mitochondrial swelling instead of apoptosis. ROS generation was not detected during this necrotic cell death process. These results indicate that radiation-induced apoptosis in MOLT-4 cells requires elevation of intracellular ROS as well as activation of a series of caspases, whereas the cryptic necrosis program—which is independent of intracellular ROS generation and caspase activation—is activated when the apoptosis pathway is blocked.

Background: Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRα without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRα is decreased by apicidin and n-butyrate. Methods: EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRα and phosphorylated eIF-2α were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRα protein. Results: When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRα was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2α for up to 8 days. Conclusions: The decrease in the level of FIP1L1-PDGFRα protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA.

Short interfering RNAs are used in RNA interference technology and are powerful tools for target gene silencing in a sequence-specific manner. In this study, we synthesized Dicer-substrate siRNAs consisting of 27-nt double-stranded RNAs conjugated with palmitic acid at the 5'-end of the sense strand and investigated their RNA interference efficacies in vitro and in vivo. The palmitic acid-conjugated 27-nt DsiRNAs (C16-Dsi27RNAs) were prepared by our simple synthesis strategy and achieved a good yield. C16-Dsi27RNAs showed enhanced in vitro RNA interference potency compared with not only non-modified Dsi27RNAs but also cholesterol-conjugated Dsi27RNAs against both an exogenous enhanced green fluorescent protein and the endogenous vascular endothelial growth factor gene in a human scirrhous-type gastric cancer cell line that stably expressed the enhanced green fluorescent protein gene (GCIY-eGFP). Additionally, C16-Dsi27RNAs had potent gene silencing activity against both enhanced green fluorescent protein and vascular endothelial growth factor as target genes in a subcutaneous tumor mouse model generated from GCIY-eGFP cells administered by intratumoral injection. These results suggest that the C16-Dsi27RNAs will be useful next-generation RNA interference molecules that can overcome the problems associated with RNA interference technology.

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.

Ataxia-telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradiated and X-irradiated EBV (Epstein-Barr virus)-transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM + / + ), AT heterozygotes (ATM + / - ), or AT homozygotes (ATM - / - ), respectively. In ATM + / + LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5-fold within 1 h of exposure to 10 Gy of X-rays, while the ATM protein levels had increased by 1.5- to 2.0-fold within 2 to 3 h of irradiation. The wild-type mRNA and protein levels both returned to their basal values fairly quickly after this time. The results obtained with the ATM + / - LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X-irradiation in any ATM + / - LCL. Twelve of the mutations in the ATM - / - LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM - / - LCLs produced mutant ATM proteins that were identical in molecular weight to the wild-type ATM protein. When cells from three of these five clones were exposed to X-rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up-regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way.

Ionizing radiation is a well known risk factor of thyroid cancer development, but the mechanism of radiation induced carcinogenesis is not clear. The RET/PTC oncogene, an activated form of the RET proto-oncogene, is frequently observed in papillary thyroid carcinoma (PTC); RET/PTC1, -2 and -3 are known to be the three major forms. High frequencies of RET/PTC rearrangements have been observed in radiation-associated PTC, such as those appearing post-Chernobyl or post-radiotherapy, but the rearrangement types differ between these two populations. We investigated whether a specific type of RET/PTC rearrangement was induced by X-rays in vivo and in vitro. In human normal thyroid tissues transplanted in scid mice, the RET/PTC1 rearrangement was predominantly detected throughout the observation period (up to 60 days) after X-ray exposure of 50 Gy. On the other hand, RET/PTC3 was detected only 7 days after X-irradiation, and no transcript of RET/PTC2 was detected. These results are supported by the results of an in vitro study. The RET/PTC1 rearrangement was preferentially induced in a dose-dependent manner by X-rays within a high dose range (10, 50 and 100 Gy) in four cell lines. On the other hand, RET/PTC3 was induced at a much lower frequency, and no induction of RET/PTC2 was observed. These results suggest that the preferential induction of the RET/PTC1 rearrangement may play an important role in the early steps of thyroid carcinogenesis induced by acute X-irradiation.

The mechanism and effects of epigenetic alterations in human carcinogenesis are not well understood, except that cancers often have alterations in the methylation status of their genomes. Additionally, human cancers, including aggressive T-cell leukemias and lymphomas, have a high frequency of p53 mutations, particularly missense mutations, which raises the possibility of gain-of-new-function proteins, but the new proteins' oncogenic functions are mechanistically ill-defined. To investigate the mechanisms behind the high prevalence of p53 tumor suppressor gene mutations in aggressive or relapsed T-cell leukemias, we transfected Jurkat cells null for p53 protein with a temperature-sensitive p53 mutant. We showed that this mutant p53 abrogated expression of the T-cell antigen receptor (TCR) by affecting the methylation of an at least 20-kb region of DNA, 5'to the TCR beta-chain gene enhancer region, which includes TCRbetaC1 and betaC2. Expression of the TCR is restored when the temperature is reduced to 32 degrees C, at which temperature the mutant p53 regains wild-type function. The TCR, a common site of dysfunction in T-cell malignancies, is the principal signal transduction moiety controlling both T-cell activation and activation-induced apoptosis. These results suggest a new role for mutant p53-as an epigenetic mutator, bridging p53, methylation, and transcriptional silencing-and suggest novel mechanisms in immunosuppression and cancer progression.

To evaluate the intrinsic lifespan of human memory T-cells in the absence of T-cell receptor signaling, we used radiation-induced mutant CD4+ T-cells lacking surface expression of TCR/CD3 complex as an in vivo cell marker. We analyzed the long-term kinetics of TCR/CD3 - mutant T-cells among CD4+ CD45RA+ naive and CD4+ CD45RA- memory T-cell fractions in peripheral blood of gynecological cancer patients receiving radiotherapy. Both the proportion and number of these mutant T-cells decayed exponentially with time following radiotherapy. The estimated half-life of mutant memory T-cells was 2 to 3 years and did not differ from that of mutant naive T-cells. These results indicate that the lifespan of mature CD4+ T-cells is limited regardless of their memory or naive phenotype in the absence of TCR/CD3 expression. This finding may suggest that continued T-cell receptor signaling is required for lifetime maintenance of human memory T-cells.

Ionizing radiation is a well-known risk factor of cancer development, but the mechanism of radiation induced carcinogenesis is not clear. Chromosomal rearrangements induced by radiation most likely are one of the principal genetic alterations resulting in malignant transformation. The chimeric BCR-ABL associated with chronic myelogenous leukemia (CML) and H4-RET oncogenes associated with thyroid papillary carcinoma are the result of a translocation and inversion, respectively. In vitro studies showed these genes were induced by high-doses of X-irradiation in cell lines. Studies also show that therapeutic external X-ray doses as high as 60 Gy for treatment of various childhood cancers including Hodgkin's disease significantly increase the risk of thyroid cancer. Therefore, we examined the induction and persistence of these chimeric genes in human thyroid tissues transplanted in scid mice after 50 Gy exposure as a function of time for 2 months to elucidate the early events of thyroid carcinogenesis. The H4-RET genes were detected on day 2 and throughout the 2 month period. On the other hand, BCR-ABL genes were detected on day 2 and were undetectable subsequently. These results suggest that ionizing radiation causes various oncogene activations, but cells with only specific gene alteration uniquely associated with thyroid carcinogenesis are selectively retained demonstrating one of the early events in the beginnings of radiation carcinogenesis in human thyroid tissues.

An experimental system was established to study in vivo T-cell receptor αβ (TCR) mutations in murine CD4 + T-lymphocytes. The frequency of TCR-defective mutant T-cells that have the CD3 − 4 + surface phenotype, was measured using two-color flow cytometry of splenic T-cells passed through nylon wool. The spontaneous TCR mutant frequency (MF) in BALB/c mice (2.3×10 −4 ) was significantly lower than the frequencies of C57BL/6 (4.0×10 −4 ) and C3H/He (4.2×10 −4 ) mice. The general trend of the TCR MF started to increase at 3 days after whole-body X-irradiation, reached a peak level at 2–3 weeks, and then gradually decreased with a half-life of about 2 weeks. To analyze how the dose responses for each strain of mouse differed 2 weeks after X-irradiation, the TCR MF dose responses were fitted to a linear–quadratic or a quadratic curve. The coefficients of the quadratic terms in both models for BALB/c mice were significantly higher than those for the other two strains. These findings suggest that some genetic factor(s) may control the susceptibility of somatic genes to both spontaneous and radiation-induced mutagenesis. Establishing an animal model for in vivo TCR mutations will contribute to the clarification of certain unresolved aspects of TCR mutagenesis in humans and will further advance knowledge of screening for environmental mutagens.

Three types of siRNAs and three types of left-overhang siRNAs (LoRNAs) were synthesized along with their conjugations with palmitic acid (C16) to investigate the correlation between Dicer recognition and gene-silencing potency. The siRNA types were composed of 21-nucleotide (nt), 23-nt, and 25-nt lengths of sense and antisense strands with a 2-nt overhang at each 3'-end. The three LoRNA types were composed of a 21-nt, a 23-nt, and a 25-nt length of sense strand with a 2-nt DNA at the 3'-blunt-end and a 23-nt, a 25-nt, and a 27-nt length of antisense strand with a 2-nt overhang at the 3'-end. Additionally, each of these siRNAs and LoRNAs was modified with a C16 at the 5'- or 3'-end of the sense strand; these were named C16-siRNAs and C16-LoRNAs, respectively. The siRNAs and C16-siRNAs were barely cleaved by Dicer, and their gene-silencing efficacies were not excellent, contrary to our expectations. In contrast, most of the LoRNAs and C16-LoRNAs became substrates of Dicer, and they showed both strong gene-silencing efficacies and high nuclease resistance. Among the LoRNAs, the 25D-C16/27-nt LoRNA, which is composed of a 25-nt sense strand with a 2-nt DNA conjugated with C16 at the 3'-end and a 27-nt antisense strand with a 2-nt overhang at the 3'-end, showed an excellent gene-silencing effect with high cell membrane permeability and strong resistance against nuclease degradation. Additionally, the Lo25D-C16/27RNA excelled in all three aspects, nuclease resistance, cell membrane permeability, and RNAi efficacy, compared with the cholesterol conjugation. We are certain that Lo25D-C16/27RNA can be useful as a new generation of RNAi molecules with which to overcome some of the limitations of RNAi technology.

In the present study, we engrafted normal colonic epithelia and histologically diagnosed colonic adenomas from a familial adenomatous polyposis (FAP) patient into severe combined immunodeficient (SCID) mice and subsequently examined them histologically and molecular biologically. Successful engraftment and metastasis was observed. The facts that human normal colonic epithelium and adenomatous polyps can take in SCID mice indicates the possibility that this human SCID mouse system will be useful for investigating the dynamics of human carcinogenesis in various tissues.

We established a novel severe combined immunodeficient (SCID) mouse model for the study of human lung cancer metastasis to human lung. Implantation of both human fetal and adult lung tissue into mammary fat pads of SCID mice showed a 100% rate of engraftment, but only fetal lung implants revealed normal morphology of human lung tissue. Using these chimeric mice, we analyzed human lung cancer metastasis to both mouse and human lungs by subcutaneous inoculation of human squamous cell carcinoma and adenocarcinoma cell lines into the mice. In 60 to 70% of SCID mice injected with human-lung squamous-cell carcinoma, RERF-LC-AI, cancer cells were found to have metastasized to both mouse lungs and human fetal lung implants but not to human adult lung implants 80 days after cancer inoculation. Furthermore, human-lung adenocarcinoma cells, RERF-LC-KJ, metastasized to the human lung implants within 90 days in about 40% of SCID mice, whereas there were no metastases to the lungs of the mice. These results demonstrate the potential of this model for the in vivo study of human lung cancer metastasis.

Two different sizes of siRNAs, of which one type was 21-nucleotide (nt) siRNA containing 2-nt dangling ends and the other type was 27-nt siRNA with blunt ends, were conjugated with a nuclear export signal peptide of HIV-1 Rev at the 5'-sense end. Processing by Dicer enzyme, cell membrane permeability, and RNAi efficiency of the peptide-conjugated siRNAs were examined. Dicer cleaved the peptide-conjugated 27-nt siRNA leading to the release of 21-nt siRNA, whereas the peptide-conjugated 21-nt siRNA was not cleaved. High membrane permeability and cytoplasmic localization was found in the conjugates. Moreover, the peptide-conjugated 27-nt siRNA showed increased potency of RNAi in comparison with the nonmodified 21-nt and 27-nt siRNAs, whereas the peptide-conjugated 21-nt siRNA showed decreased RNAi efficacy. This potent RNAi efficacy is probably owing to acceleration of RISC through recognition by Dicer, as well as to the improvement of cell membrane permeability and intracellular accumulation.

Novel anti-HIV lectin family which shows a strict binding specificity for high mannose glycans has been found in lower organisms. The bacterial orthologue has been identified in the genome of Pseudomonas fluorescens Pf0-1 and the gene coding a putative lectin was cloned, expressed in Escherichia coli and purified by one step gel filtration. Glycan array screening of the recombinant lectin, termed PFL, has revealed that PFL preferentially recognizes high mannose glycans with α1-3 Man that was highly exposed at the D2 position. In contrast, masking of this α1-3 Man with α1-2 Man dramatically impaired lectin-carbohydrate interactions. Reducing terminal disaccharide, GlcNAc-GlcNAc of high mannose glycans was also essential for PFL-binding. PFL showed a potent anti-influenza virus activity by inhibiting the virus entry into cells at doses of low nanomolar concentration. At micromolar concentration or higher, PFL showed a cytotoxicity accompanying loss of the cell adhesion against human gastric cancer MKN28 cells. The cell surface molecule to which PFL bound was co-precipitated with biotin-labeled PFL and identified as integrin α2 by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Intriguingly, upon treatment with exogenous PFL, integrin α2 on the cell surface underwent rapid internalization to the cytoplasm and accumulated to perinuclear region, together with the bound PFL. The resulting loss of cell adherence would trigger a signaling pathway that induced anoikis-like cell death. These events were effectively inhibited by pretreatment of PFL with mannnan, indicating the involvement of high mannose glycans on PFL-induced cell death that was triggered by PFL-integrin α2 interactions.

The development of Dicer-substrate small interfering RNAs (DsiRNAs) has been pursued in recent years because these molecules exhibit a much more potent gene-silencing effect than 21-nucleotide (nt) siRNAs. In the present study, we designed eight different types of amino-modified DsiRNAs and a palmitic acid-conjugated DsiRNA expected to result in improved biological properties of siRNAs, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. The DsiRNAs were modified with an amine at the 5'- and/or 3'-end of the sense and/or antisense strand. Dicer enzyme cleaved most of the amino-modified DsiRNAs to lead to the release of 21-nt siRNA; some of them, however, were not or partly cleaved. All amino-modified DsiRNAs exhibited strong resistance against nuclease degradations. Among the amino-modified DsiRNAs, the DsiRNA modified with an amine restricted at the 3'-end of the sense strand showed the most enhanced gene-silencing effect and maintained its potent gene suppression after one week of cell transfection against Renilla luciferase activity. For further improvement, palmitic acid was conjugated to DsiRNA at the 3'-end of the sense strand (C16-DsiRNA) to facilitate the membrane permeability and potent gene-silencing activity. The C16-DsiRNA showed enhanced membrane permeability to HeLa cells. The C16-DsiRNA exhibited extremely high inhibition of Renilla luciferase activity.

Short interfering RNA (siRNA) technology is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with palmitic acid at the 5'-end of the sense strand (C16-siRNAs) using our novel synthesis strategy in order to improve the potency of siRNA. The C16-siRNAs exhibited enhanced nuclease stability. In addition, they showed potent gene-silencing efficacy against exogenous Renilla luciferase in HeLa cells compared with a nonmodified siRNA in the presence of Lipofectamine 2000. The C16-siRNAs also had a more potent inhibitory effect on Renilla luciferase activity than the other siRNA conjugated with lipids at the 5'-end and the 3'-end by palmitoyl conjugation. For further improvement, the gene silencing potency of the C16-siRNAs against the endogenous vascular endothelial growth factor (VEGF) gene in HeLa cells was investigated. In this investigation, the siRNAs were prepared not only with the normal RNA sequence but also coupled with an inverted thymidine (idT) at the 3'-ends of both the sense and antisense strands (siRNA-idT), including palmitic acid conjugations at the 5'-end of the sense strand, to improve stability. The C16-siRNA including idT modifications exhibited a significantly greater inhibitory effect on the VEGF gene in the presence of Lipofectamine 2000. It is noteworthy that C16-siRNA-idT demonstrated long-term gene-silencing efficacy of up to 5 days. Interestingly, the C16-siRNAs, including that with idT modifications, exhibited strong RNAi potency in the absence of any transfection reagents, although only at high concentrations. Both the C16-siRNAs and C16-siRNA-idT induced a high level of membrane permeability in HeLa cells. Our developed C16-siRNAs, particularly C16-siRNA-idT, are thus among the promising candidates for a new generation of modified siRNAs that can solve the many problems associated with siRNA technology.

To identify the genetic events that must be involved in thyroid tumor progression, we initially investigated p53 gene alterations in 10 papillary adenocarcinomas, 4 follicular adenocarcinomas, and 8 undifferentiated carcinomas. Base substitutional mutations in exons 5 to 8 and loss of heterozygosity (LOH) of the p53 gene were not detected in papillary or follicular adenocarcinomas. However, 7 of 8 undifferentiated carcinomas were carrying base substitutional mutations, and LOH was detected in 3 of 5 informative cases. Furthermore, to verify that the p53 gene alterations are truly involved in tumor progression, DNA from individual foci of the four undifferentiated carcinomas coexisting with a differentiated focus and from one follicular adenocarcinoma with an undifferentiated focus was analyzed by direct sequencing and polymerase‐chain‐reaction‐restriction‐fragment‐length polymorphism (PCR‐RFLP). Base substitutional mutations in the p53 gene from exons 5 to 8 were identified exclusively in the undifferentiated foci, but not in the differentiated foci. LOH was observed in 3 of 4 informative undifferentiated foci. In one of these positive cases, LOH was observed in both papillary adenocarcinoma and undifferentiated carcinoma. However, a p53 gene mutation at codon 248 was detected in the undifferentiated carcinoma but not in the papillary adenocarcinoma. The results imply that LOH occurs first in papillary adenocarcinoma followed by a p53 mutation during the transition from papillary adenocarcinoma to undifferentiated carcinoma. Maintenance of LOH during tumor progression excludes the possibility that these different histological foci are derived from different origins and represents molecular evidence that undifferentiated carcinoma is very likely derived from preexisting papillary adenocarcinoma. Furthermore, these results strongly suggest that the mutated p53 gene plays a crucial role in de‐differentiation during the progression of thyroid tumors.

The Philadelphia chromosome consists of a reciprocal translocation between the ABL oncogene at chromosome 9q34 and the BCR gene at chromosome 22q11, resulting in the expression of chimeric BCR-ABL mRNAs specific to chronic myelogenous leukemia (CML). Presence of the fusion gene can be detected with high specificity and sensitivity by means of reverse transcription and polymerase chain reaction. Using this assay, it was possible to detect BCR-ABL fusion genes induced among HL60 cells after 100 Gy of X-irradiation in vitro. In total, five fusion gene transcripts were obtained among 10(8) cells examined. These fusion genes contained not only CML-specific BCR-ABL rearrangements, but also other forms of BCR-ABL fusions. These latter genes had junctions of BCR exon 4/ABL exon 2 intervened by a segment of DNA of unknown origin, BCR exon 5/ABL exon 2, and BCR exon 4/ABL exon 2. The results appear to be direct evidence for the induction of the BCR-ABL fusion gene by X-irradiation. In terms of leukemogenesis, it appears that only those cells bearing certain CML-related BCR-ABL fusion genes are positively selected by virtue of a growth advantage in vivo.

Human dendritic cell (DC) precursors were engrafted and maintained in NOD/SCID- human chimeric mice (NOD/SCID-hu mice) implanted with human cord blood mononuclear cells, although no mature human DCs were detected in lymphoid organs of the mice. Two months after implantation, bone marrow (BM) cells of NOD/SCID-hu mice formed colonies showing DC morphology and expressing CD1a in methylcellulose culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The CD34-/CD4+/HLA-DR+ cell fraction in NOD/SCID-hu mouse BM generated CD1a(+) cells that were highly stimulatory in mixed leukocyte reactions in culture with GM-CSF and TNF-alpha. These results suggest a strong potential for NOD/SCID-hu BM to generate human DCs, although DC differentiation may be blocked at the CD34-/CD4+/HLA-DR+ stage. (Blood. 2001;97:3655-3657)

A novel polymerase chain reaction method was developed to preferentially amplify a segment of DNA containing a base substitution mutation. This technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. By virtue of a subtle difference in the melting temperature between the blocker-normal DNA and blocker-mutant DNA hybrids, the method allows preferential amplification of the mutant DNA. We used the human N-ras gene as a model. Two different types of N-ras mutations could be effectively amplified when they were present with an excess amount of normal DNA at a ratio of 1:10(3). Furthermore, the sensitivity was increased 10-fold by using single strand conformation polymorphism analysis for the amplified products, and mutant DNA was detected in the presence of a 10(4) times excess amount of normal DNA.

Human malignant pleural mesothelioma (HMPM) is an aggressive neoplasm that is highly resistant to conventional therapies. We established 3 HMPM cell lines (TCC-MESO-1, TCC-MESO-2 and TCC-MESO-3) from Japanese patients; the first 2 from the primary and metastatic tumors of a patient with the epithelioid type of HMPM, and the third from a patient with biphasic characteristics of the tumor (epithelioid and sarcomatous phenotypes). The 3 cell lines resembled the original HMPMs in their morphological and biological features, including the genetic alterations such as lack of p16 expression and mutation of p53. Their tumorigenicity was determined in SCID mice by orthotopic implantation (20-46%). The tumorigenicity of the HMPM cell lines, which was relatively low, was enhanced by repeated subcultures and orthotopic implantations, and 3 competent tumorigenic sublines were produced (Me1Tu, Me2Tu and Me3Tu sublines from the TCC-MESO-1, TCC-MESO-2 and TCC-MESO-3 cell lines, respectively). The resultant HMPM sublines efficiently generated tumors in the SCID mice (100%) following orthotopic implantation. SCID mice implanted with the competent sublines, into one of which the luciferase gene was introduced, displayed quantitative fluctuation of the bioluminescence for the tumor volume in vivo. Oral administration of S-1, an anticancer agent, suppressed the proliferation of the luciferase gene-expressing Me1Tu subline in the mouse models in vivo, with a treated-to-control ratio of the mean tumor volume of 0.2. The orthotopic implantation mouse model proved to be useful for quantitative evaluation of the efficacy of novel anticancer drugs and also for studying the biology of HMPMs in vivo.

Concerns over cancer development from exposure to environmental sources of densely ionizing, high linear energy transfer (LET) radiation, such as alpha-particles from radon, is a current public health issue. The study of tumors attributable to high LET irradiation would greatly augment our insights into the biological mechanisms of carcinogenesis. Chronic low-dose-rate internal exposure to alpha-radiation from thorium dioxide deposits following intravascular administration of the radiographic contrast agent Thorotrast is known to markedly increase the risk of cancer development, especially that of hepatic angiosarcomas and cholangiocarcinomas. Although the mechanism is hypothesized to be via cellular damage, DNA being a major target, wrought by the high LET alpha-particles, the specific genes and the actual sequence of events involved in the process of transforming a normal cell into a malignant one are largely unknown. To shed some light on the molecular mechanisms of cancer development during a lifetime exposure to alpha-radiation, we analyzed the most commonly affected tumor suppressor gene in humans, p53, in 20 Thorotrast recipients who developed cancer, mostly of hepatic bile duct and blood vessel origin. Of the 20 cases, 19 were found to harbor p53 point mutations. Moreover, the accompanying non-tumor tissues from these patients also had p53 mutations, albeit at lower frequency. The distribution pattern of the point mutations was significantly different between the non-tumor and tumor tissues, with most mutations in malignant tissues located in the highly conserved domains of the p53 gene. Our results support the idea that p53 mutations are important in the genesis of Thorotrast-induced tumors but that these point mutations are a secondary outcome of genomic instability induced by the irradiation. Additionally, non-tumor cells harboring p53 mutations may gain some survival advantage in situ but mutations in the domains responsible for the formation of structural elements critical in binding DNA may be necessary for a cell to reach full malignancy.

The validity of molecular studies using DNA and RNA extracted from decades-old formalin-fixed and paraffin-embedded tissue blocks has been demonstrated. The quality and usability of DNA and RNA from archival tissues are modified by various factors, such as the fixative, the fixation time, and the postmortem time. However, in contrast to DNA, there are no comprehensive studies quantitatively addressing the feasibility of RNA from old (more than 10 years) archival samples. This study examined the integrity of RNA extracted from 738 autopsy liver and 63 autopsy thyroid cancer tissue blocks procured during a span of nearly four decades, beginning in 1952 and ending in 1989, from the atomic bomb survivors. The integrity of RNA was assessed by amplification of c-BCR messenger RNA (mRNA) between two sequential exons with an intervening intron by reverse-transcription polymerase chain reaction (RT-PCR). The integrity of RNA was influenced by the age of the samples and the postmortem time, but not by the formalin-fixation period. It was possible to amplify more than 60% of the samples. Using these RNAs, the HCV genome in liver cancers and the H4-RET gene in thyroid cancers were detectable. This study illustrates the possibility of molecular studies using RNA from routinely prepared paraffin blocks stored for long periods and provides the statistics and critical factors to consider in assessing the feasibility of such contemplated studies.

The frequencies of mutant erythrocytes with loss of heterozygosity at the glycophorin A (GPA) locus and mutant CD4+ T cells lacking surface expression of the T-cell receptor alphabeta (TCR)/CD3 complex were measured by flow cytometry for Japanese Werner's syndrome (WRN) patients. The hemizygous and homozygous GPA mutant frequencies (GPA Mfs) and the TCR/CD3-defective mutant frequency (TCR Mf) in WRN patients were found to be significantly higher than those in normal controls in the same age range. However, because these Mfs in the patients are only about twice those in controls, it is difficult to conclude that the WRN gene mutations cause instability of somatic genes. This contrasts markedly with Bloom's syndrome (BLM) patients, whose GPA and TCR Mfs were previously reported to increase about 50- and 15-fold, respectively. The difference in Mfs is one aspect of the large variation in the phenotype observed between WRN and BLM patients, suggesting a different role of the responsible genes, both of which belong to the RecQ DNA helicase gene family, in the control of somatic mutagenesis.

Previous studies of blood cells from atomic bomb survivors have shown that frequencies of chromosome aberrations and somatic mutations are elevated in heavily exposed survivors and that T-cell functions and the number of mature T cells are decreased in the survivors who were exposed to radiation as adults. Current progress in flow cytometry allows a sophisticated analysis of various subsets of T, B and NK cells. In the present study, proportions of such subsets in peripheral blood lymphocytes from atomic bomb survivors (159 survivors estimated to be exposed to > or =1.5 Gy) and 252 controls were measured using multiple combinations of monoclonal antibodies to lymphocyte differentiation antigens to investigate whether the previous radiation exposure had altered the composition of the subsets. Among T-cell subsets, the proportion of CD4+ T-cell subsets was decreased significantly in the heavily exposed survivors; this tendency was apparent for the CD4+CD45RA+ naive T-cell subset. However, there were no significant differences in the proportions of CD8+ T-cell subsets between the exposed survivors and controls. As for the B-cell subsets, the proportion of both CD5+ and CD5 B cells as well as CD23+ and CD23- B cells increased in the heavily exposed survivors. Further, no effect of radiation was found in the proportion of NK-cell subsets. These results strongly suggest that previous radiation exposure altered the composition of T and B cells in the peripheral blood of atomic bomb survivors, and they raise the possibility that atomic bomb radiation may have affected the developmental processes of T and B cells.

Four primary cancers found at autopsy of a patient who received the thorium-based contrast agent Thorotrast 50 years ago and who was healthy up until a few months before his death from liver failure were analyzed for p53 mutations. The data suggest that the chronic α-irradiation may be a large causative factor. Multiple mutations were found in all the cancer tissues: two foci of a cholangiocellular carcinoma, a tubular adenocarcinoma of the stomach, a squamous cell carcinoma of the lung, and an adenocarcinoma of Vater's ampulla. The total number of point mutations detected were 13. Moreover, homozygous aberrations were detected in a large area of normal small intestine and noncancer liver tissues suggesting that nontumor cells which harbored p53 abnormalities gained a survival advantage and clonally expanded.

We developed an experimental model for studying the growth and epilation of the human hair follicle by implanting human scalp tissue onto immunodeficient C.B-17 scid/scid mice. The skin grafts showed continuous growth of black human hairs for at least 1 year and maintained the normal histological structure of a human hair follicle and other tissues associated with the skin. Using this in vivo model, we evaluated the effect of irradiation on the function of human hair follicles. Localized X irradiation (1 to 6 Gy) induced hair loss dose-dependently and synchronously in the third week after irradiation. The hairs undergoing epilation showed a gradual decrease in width toward the root. The minimum width at the thinnest portion of the surviving hair 4 weeks after irradiation suggested that epilation resulted from the breaking of hairs when the hair width decreased to less than 20 microm. After the highest-dose irradiation, the normal structure of the hair bulb was totally abrogated, and long and narrow epithelial tissues associated with regressed papillary cells remained. The surviving epithelia were morphologically similar to the outer epithelial sheath of the follicle associated with palisadic basal cell layers. In the third week some cells in the basal layers of the surviving epithelium in each follicle expressed proliferating cell nuclear antigen. By about 9 weeks after irradiation, the complete structure of the follicle regenerated, with hair growth activity even in the grafts irradiated at the highest dose, although about 30% of the hairs did not regrow. These findings suggest that follicular stem cells that survive high-dose exposure in the sheath-like epithelial tissue can reproduce the complete follicle structure. This animal model can be used to assess the effects of radiation exposure on human skin and to identify and characterize human follicular stem cells.

Many studies have suggested a relationship between certain alleles of the human leukocyte antigen (HLA) and the prevalence of some diseases or the immunological responsiveness to certain antigens. Furthermore, our studies in the past have demonstrated decreased immune function among atomic-bomb survivors who were exposed to high doses of radiation. However, no studies have addressed the possibility of various degrees of radiation-induced immune suppression being dependent on HLA type. To investigate the possibility of differing frequency distributions of HLA type in the Hiroshima atomic bomb survivors, HLA-DQA1 alleles and HLA-DR antigens were typed for 291 survivors in a high-dose group (>1.5 Gy), 339 survivors in an intermediate-dose group (0.005-1.5 Gy), and 388 in a distally exposed control group (

A new HaeIII polymorphism, which is found in the first intron of the human p53 gene, provides a genetic marker for tumor suppressor p53 gene alterations.

Epidemiological studies of the atomic bomb survivors have suggested dose-related increases in mortality from diseases other than cancer. Cardiovascular disease is one such noncancer disease for which increases in both mortality and incidence have been found to be associated with radiation dose. Immunological studies have revealed long-term impairment of T-cell-mediated immunity, especially involving deficiencies of CD4 helper T cells, in atomic bomb survivors. In the present study, we investigated whether decreases in CD4 T cells were associated with myocardial infarction in atomic bomb survivors. Of 1,006 survivors examined to determine the proportion of CD4 T cells in peripheral blood lymphocytes, 18 persons had a history of myocardial infarction. The proportion of CD4 T cells was significantly decreased with increased radiation dose [corrected]. Further, the prevalence of myocardial infarction was significantly greater in individuals with a lower proportion of CD4 T cells. These results suggest that myocardial infarction in atomic bomb survivors may be associated with defects in CD4 helper T cells.

Cryopreservation of lymphocytes has become increasingly important, especially when the cells are to be used in retrospective studies of selected and dwindling populations, such as A-bomb survivors. This report describes an efficient method for cryopreservation of human lymphocytes which does not significantly alter various immunological characteristics of these cells. The proportions of Leu-1+ cells (T cells), Leu-2a+ cells (suppressor-cytotoxic T cells), Leu-3a+ cells (helper-inducer T cells), HLA-DR+ cells, Mo2+ cells (monocytes), B1+ cells (B cells), and Leu-7+ cells (natural killer (NK) cells), as determined by monoclonal antibodies, were found to be stable following cryopreservation. NK cell activity against K-562 target cells showed a 40-60% decrease immediately after thawing, but recovered to approximate pre-freezing levels after preincubation for 18 h. Neither lymphocyte subsets nor cell viability significantly changed following preincubation after cryopreservation. However, the ratio of cells binding to K-562 cells increased after this preincubation and may account for the observed recovery of NK cell activity. NK cell activity remained relatively stable up to 14 months of storage which confirms that freezing damage depends on the freezing process rather than on the duration of cryopreservation.

Background Pseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated. Methods Cell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated. Results The strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth. Conclusion PFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2099-2) contains supplementary material, which is available to authorized users.