Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods: We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results: We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Apolipoprotein E Isoform-Dependent Effects on Human Amyloid Precursor Protein/Aß-Induced Behavioral Alterations and Cognitive Impairments and Insoluble Cortical Aß42 Levels.

Mice expressing human amyloid precursor protein (APP) containing the dominant Swedish and Iberian mutations (AppNL-F ) or also Arctic mutation (AppNL-G-F ) show neuropathology and hippocampus-dependent cognitive impairments pertinent to Alzheimer's disease (AD) in mouse models at 18 and 6 months of age, respectively. Apolipoprotein E, involved in cholesterol metabolism, plays an important role in maintaining the brain. There are three human apolipoprotein E isoforms: E2, E3, and E4. Compared to E3, E4 increases while E2 protects against AD risk. At 6 months of age, prior to the onset of plaque pathology, E3, but not E4, protected against hAPP/Aß-induced impairments in spatial memory retention in the Morris water maze. However, these earlier studies were limited as hapoE was not expressed outside the brain and E3 or E4 was not expressed under control of an apoE promotor, E2 was often not included, hAPP was transgenically overexpressed and both mouse and hAPP were present. Therefore, to determine whether apoE has isoform-dependent effects on hAPP/Aß-induced behavioral alterations and cognitive impairments in adult female and male mice at 6 and 18 months of age, we crossed AppNL-G-F and AppNL-F mice with E2, E3, and E4 mice. To distinguish whether genotype differences seen at either time point were due to main effects of hAPP, hapoE, or hAPP × hapoE genetic interactions, we also behavioral and cognitively tested E2, E3, and E4 female and male mice at 6 and 18 months of age. We also compared behavioral and cognitive performance of 18-month-old AppNL-G-F and AppNL-F female and male mice on a murine apoE background along with that of age-and sex-matched C57BL/6J wild-type mice. For many behavioral measures at both time points there were APP × APOE interactions, supporting that apoE has isoform-dependent effects on hAPP/Aß-induced behavioral and cognitive performance. NL-G-F/E3, but not NL-G-F/E2, mice had lower cortical insoluble Aß42 levels than NL-G-F/E4 mice. NL-F/E3 and NL-F/E2 mice had lower cortical insoluble Aß42 levels than NL-F/E4 mice. These results demonstrate that there are apoE isoform-dependent effects on hAPP/Aß-induced behavioral alterations and cognitive impairments and cortical insoluble Aß42 levels in mouse models containing only human APP and apoE.

Longitudinal Analysis of Human Pancreatic Adenocarcinoma Development Reveals Transient Gene Expression Signatures.

Previous transcriptome studies of human pancreatic ductal adenocarcinoma (PDAC) compare non-cancerous pancreatic intraepithelial neoplasias (PanIN) with late-stage PDAC obtained from different patients, thus have limited ability to discern network dynamics that contribute to the disease progression. We demonstrated previously that the 10-22 cell line, an induced pluripotent stem cell-like line reprogrammed from late-stage human PDAC cells, recapitulated the progression from PanINs to PDAC upon transplantation into NOD/LtSz-scid/IL2R-gammanull mice. Herein, we investigated the transition from precursor to PDAC using the isogenic model. We analyzed transcriptomes of genetically tagged 10-22 cells progressing from PanINs to PDAC in mice and validated the results using The Cancer Genome Atlas PDAC dataset, human clinical PanIN and PDAC tissues, and a well-established murine PDAC model. We functionally studied candidate proteins using human normal (H6C7) and cancerous (Miapaca2, Aspc1) pancreatic ductal epithelial cell lines. 10-22 cell-derived PDAC displayed the molecular signature of clinical human PDAC. Expression changes of many genes were transient during PDAC progression. Pathways for extracellular vesicle transport and neuronal cell differentiation were derepressed in the progression of PanINs to PDAC. HMG-box transcription factor 1 (HBP1) and BTB domain and CNC homolog 1 (BACH1) were implicated in regulating dynamically expressed genes during PDAC progression, and their expressions inversely correlated with PDAC patients' prognosis. Ectopic expression of HBP1 increased proliferation and migration of normal and cancerous pancreatic cells, indicating that HBP1 may confer the cell dissemination capacity in early PDAC progression. This unique longitudinal analysis provides insights into networks underlying human PDAC progression and pathogenesis. IMPLICATIONS: Manipulation of HBP1, BACH1, and RUN3 networks during PDAC progression can be harnessed to develop new targets for treating PDAC.

Effects of Six Sequential Charged Particle Beams on Behavioral and Cognitive Performance in B6D2F1 Female and Male Mice.

The radiation environment astronauts are exposed to in deep space includes galactic cosmic radiation (GCR) with different proportions of all naturally occurring ions. To assist NASA with assessment of risk to the brain following exposure to a mixture of ions broadly representative of the GCR, we assessed the behavioral and cognitive performance of female and male C57BL/6J × DBA2/J F1 (B6D2F1) mice two months following rapidly delivered, sequential 6 beam irradiation with protons (1 GeV, LET = 0.24 keV, 50%), 4He ions (250 MeV/n, LET = 1.6 keV/µm, 20%), 16O ions (250 MeV/n, LET = 25 keV/µm 7.5%), 28Si ions (263 MeV/n, LET = 78 keV/µm, 7.5%), 48Ti ions (1 GeV/n, LET = 107 keV/µm, 7.5%), and 56Fe ions (1 GeV/n, LET = 151 keV/µm, 7.5%) at 0, 25, 50, or 200 cGy) at 4-6 months of age. When the activity over 3 days of open field habituation was analyzed in female mice, those irradiated with 50 cGy moved less and spent less time in the center than sham-irradiated mice. Sham-irradiated female mice and those irradiated with 25 cGy showed object recognition. However, female mice exposed to 50 or 200 cGy did not show object recognition. When fear memory was assessed in passive avoidance tests, sham-irradiated mice and mice irradiated with 25 cGy showed memory retention while mice exposed to 50 or 200 cGy did not. The effects of radiation passive avoidance memory retention were not sex-dependent. There was no effect of radiation on depressive-like behavior in the forced swim test. There was a trend toward an effect of radiation on BDNF levels in the cortex of males, but not for females, with higher levels in male mice irradiated with 50 cGy than sham-irradiated. Finally, sequential 6-ion irradiation impacted the composition of the gut microbiome in a sex-dependent fashion. Taxa were uncovered whose relative abundance in the gut was associated with the radiation dose received. Thus, exposure to sequential six-beam irradiation significantly affects behavioral and cognitive performance and the gut microbiome.

Rapid Response and Slow Recovery of the H3K4me3 Epigenomic Marker in the Liver after Light-mediated Phase Advances of the Circadian Clock.

Mammalian tissues display circadian rhythms in transcription, translation, and histone modifications. Here we asked how an advance of the light-dark cycle alters daily rhythms in the liver epigenome at the H3K4me3 (trimethylation of lysine 4 on histone 3) modification, which is found at active and poised gene promoters. H3K4me3 levels were first measured at 4 time points (zeitgeber time [ZT] 3, 8, 15, and 20) during a normal 12L:12D light-dark cycle. Peak levels were observed during the early dark phase at ZT15 and dropped to low levels around lights-on (ZT0) between ZT20 and ZT3. A 6-h phase advance at ZT18 (new lights-on after only 6 h of darkness) led to a transient extension of peak H3K4me3 levels. Although locomotor activity reentrained within a week after the phase advance, H3K4me3 rhythms failed to do so, with peak levels remaining in the light phase at the 1-week recovery time point. Eight weekly phase advances, with 1-week recovery times between each phase advance, further disrupted the H3K4me3 rhythms. Finally, we used the mPer2Luc knockin mouse to determine whether the phase advance also disrupted Per2 protein expression. Similar to the results from the histone work, we found both a rapid response to the phase advance and a delayed recovery, the latter in sync with H3K4me3 levels. A model to explain these results is offered.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/µm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/µm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Charged particle mutagenesis at low dose and fluence in mouse splenic T cells.

High-energy heavy charged particles (HZE ions) found in the deep space environment can significantly affect human health by inducing mutations and related cancers. To better understand the relation between HZE ion exposure and somatic mutation, we examined cell survival fraction, Aprt mutant frequencies, and the types of mutations detected for mouse splenic T cells exposed in vivo to graded doses of densely ionizing (48)Ti ions (1GeV/amu, LET=107 keV/µm), (56)Fe ions (1GeV/amu, LET=151 keV/µm) ions, or sparsely ionizing protons (1GeV, LET=0.24 keV/µm). The lowest doses for (48)Ti and (56)Fe ions were equivalent to a fluence of approximately 1 or 2 particle traversals per nucleus. In most cases, Aprt mutant frequencies in the irradiated mice were not significantly increased relative to the controls for any of the particles or doses tested at the pre-determined harvest time (3-5 months after irradiation). Despite the lack of increased Aprt mutant frequencies in the irradiated splenocytes, a molecular analysis centered on chromosome 8 revealed the induction of radiation signature mutations (large interstitial deletions and complex mutational patterns), with the highest levels of induction at 2 particles nucleus for the (48)Ti and (56)Fe ions. In total, the results show that densely ionizing HZE ions can induce characteristic mutations in splenic T cells at low fluence, and that at least a subset of radiation-induced mutant cells are stably retained despite the apparent lack of increased mutant frequencies at the time of harvest.

Accelerated (48)Ti Ions Induce Autosomal Mutations in Mouse Kidney Epithelium at Low Dose and Fluence.

Exposure to high-energy charged particles (HZE ions) at low fluence could significantly affect astronaut health after prolonged missions in deep space by inducing mutations and related cancers. We tested the hypothesis that the mutagenic effects of HZE ions could be detected at low fluence in a mouse model that detects autosomal mutations in vivo. Aprt heterozygous mice were exposed to 0.2, 0.4 and 1.4 Gy of densely ionizing (48)Ti ions (1 GeV/amu, LET = 107 keV/µm). We observed a dose-dependent increase in the Aprt mutant fraction in kidney epithelium at the two lowest doses (an average of 1 or 2 particles/cell nucleus) that plateaued at the highest dose (7 particles/cell nucleus). Mutant cells were expanded to determine mutation spectra and translocations affecting chromosome 8, which encodes Aprt. A PCR-based analysis for loss of heterozygosity (LOH) events on chromosome 8 demonstrated a significant shift in the mutational spectrum from Ti ion exposure, even at low fluence, by revealing "radiation signature" mutations in mutant cells from exposed mice. Likewise, a cytogenetic assay for nonreciprocal chromosome 8 translocations showed an effect of exposure. A genome-wide LOH assay for events affecting nonselected chromosomes also showed an effect of exposure even for the lowest dose tested. Considered in their entirety, these results show that accelerated (48)Ti ions induce large mutations affecting one or more chromosomes at low dose and fluence.

Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

Autosomal mutations in mouse kidney epithelial cells exposed to high-energy protons in vivo or in culture.

Proton exposure induces mutations and cancer, which are presumably linked. Because protons are abundant in the space environment and significant uncertainties exist for the effects of space travel on human health, the purpose of this study was to identify the types of mutations induced by exposure of mammalian cells to 4-5 Gy of 1 GeV protons. We used an assay that selects for mutations affecting the chromosome 8-encoded Aprt locus in mouse kidney cells and selected mutants after proton exposure both in vivo and in cell culture. A loss of heterozygosity (LOH) assay for DNA preparations from the in vivo-derived kidney mutants revealed that protons readily induced large mutational events. Fluorescent in situ hybridization painting for chromosome 8 showed that >70% of proton-induced LOH patterns resembling mitotic recombination were in fact the result of nonreciprocal chromosome translocations, thereby demonstrating an important role for DNA double-strand breaks in proton mutagenesis. Large interstitial deletions, which also require the formation and resolution of double-strand breaks, were significantly induced in the cell culture environment (14% of all mutants), but to a lesser extend in vivo (2% of all mutants) suggesting that the resolution of proton-induced double-strand breaks can differ between the intact tissue and cell culture microenvironments. In total, the results demonstrate that double-strand break formation is a primary determinant for proton mutagenesis in epithelial cell types and suggest that resultant LOH for significant genomic regions play a critical role in proton-induced cancers.

Comparative analysis of cell killing and autosomal mutation in mouse kidney epithelium exposed to 1 GeV protons in vitro or in vivo.

Human exposure to high-energy protons occurs in space flight scenarios or, where necessary, during radiotherapy for cancer or benign conditions. However, few studies have assessed the mutagenic effectiveness of high-energy protons, which may contribute to cancer risk. Mutations cause cancer and most cancer-associated mutations occur at autosomal loci. This study addresses the cytotoxic and mutagenic effects of 1 GeV protons in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events. Results for kidneys irradiated in vivo are compared with the results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3-4 months postirradiation in vivo. Incubation in vivo for longer periods (8-9 months) further attenuates proton-induced cell killing. Protons are mutagenic to cells in vitro and for in vivo irradiated kidneys. The dose-response for Aprt mutation is curvilinear after in vitro or in vivo exposure, bending upward at the higher doses. While the absolute mutant fractions are higher in vivo, the fold-increase over background is similar for both in vitro and in situ exposures. Results are also presented for a limited study on the effect of dose fractionation on the induction of Aprt mutations in kidney epithelial cells. Dose-fractionation reduces the fraction of proton-induced Aprt mutants in vitro and in vivo and also results in less cell killing. Taken together, the mutation burden in the epithelium is slightly reduced by dose-fractionation. Autosomal mutations accumulated during clinical exposure to high-energy protons may contribute to the risk of treatment-associated neoplasms, thereby highlighting the need for rigorous treatment planning to reduce the dose to normal tissues. For low dose exposures that occur during most space flight scenarios, the mutagenic effects of protons appear to be modest.

Effect of sodium and acetate ions on 8-hydroxyguanine formation in irradiated aqueous solutions of DNA and 2'-deoxyguanosine 5'-monophosphate.

PURPOSE: The aim of this work was to study the combined effect of sodium and acetate ions on the radiation yield of 8-hydroxyguanine (8-OHG), one of the major DNA base lesions induced by free radicals. MATERIALS AND METHODS: Aqueous solutions of DNA and 2'-deoxyguanosine 5'-monophosphate (dGMP) with various concentrations of sodium acetate and sodium perchlorate were γ-irradiated, enzymatically digested and analyzed by high-performance liquid chromatography (HPLC) methods. RESULTS: It was found that both salts decrease the 8-OHG radiation yield in the concentration range studied for both DNA and dGMP, except in the case of dGMP wherein an increase in yield occurs in the concentration range from 0.1-1 mM. The dependence of the 8-hydroxy-2'-deoxyguanosine radiation yield on the concentration of both sodium acetate and sodium perchlorate have different shapes and have steeper slopes for the DNA compared with the dGMP solutions. CONCLUSIONS: The observed decrease in the radiation yield of 8-OHG with increasing concentrations of sodium acetate is consistent with the hypothesis that sodium acetate produces two concentration-dependent effects in the DNA solutions: (1) A conformational change in the DNA caused by Na(+) counterions; and (2) free radical reactions related to the radiolysis of acetate ion.

Marked aneuploidy and loss of multiple chromosomes are common in autosomal mutants isolated from normal mouse kidney epithelium.

Marked aneuploidy and loss of multiple chromosomes are hallmarks of cancer, but whether these events are only present in malignant cells is not known. In prior work, we showed that approximately half of spontaneous autosomal mutants isolated directly from normal kidney epithelium arose from loss of a marker chromosome 8 containing the wild type Aprt gene. Chromosome loss was detected by loss of heterozygosity (LOH) for all chromosome 8 polymorphic loci examined. To determine whether loss of chromosome 8 reflected a larger mitotic event, LOH was examined for polymorphic loci on 11 nonselected chromosomes in Aprt mutants that lost the selected chromosome 8 homologue. LOH events were detected for one or more nonselected chromosomes in 38% of these mutants. The additional LOH events also reflected apparent chromosome loss based on the molecular analysis. Metaphase spreads from mutants that lost chromosome 8 were markedly aneuploid, and chromosome painting revealed reduced levels for any chromosome shown to be lost with the LOH analysis. In contrast, LOH on nonselected chromosomes was infrequent in Aprt mutants exhibiting intragenic events or mitotic recombination for chromosome 8, and marked aneuploidy was absent. These observations suggest that the mechanism leading to chromosome loss in somatic mammalian cells is often not a simple nondisjunction event and instead could result from a single catastrophic event. They also suggest that cells with characteristics of malignancy are present in normal appearing tissue.

Non-linear effects in the formation of DNA damage in medaka fish fibroblast cells caused by combined action of cadmium and ionizing radiation.

Ionizing radiation-induced formation of genomic DNA damage can be modulated by nearby chemical species such as heavy metal ions, which can lead to non-linear dose response. To investigate this phenomenon, we studied cell survival and formation of 8-hydroxyguanine (8-OHG) base modifications and double strand breaks (DSB) caused by combined action of cadmium (Cd) and gamma radiation in cultured medaka fish (Oryzias latipes) fibroblast cells. Our data show that the introduction of Cd leads to a significant decrease in the fraction of surviving cells and to increased sensitivity of cells to ionizing radiation (IR). Cd also appears to cause non-linear increases in radiation-induced yields of 8-OHG and DSB as dose-yield plots of these lesions exhibit non-linear S-shaped curves with a sharp increase in the yields of lesions in the 10-20 microM range of Cd concentrations. The combined action of ionizing radiation and Cd leads to increased DNA damage formation compared to the effects of the individual stressors. These results are consistent with a hypothesis that the presence of Cd modulates the efficiency of DNA repair systems thus causing increases in radiation-induced DNA damage formation and decreases in cell survival.