Inhibition of ribonucleotide reductase subunit M2 enhances the radiosensitivity of metastatic pancreatic neuroendocrine tumor.

Ribonucleotide Reductase (RNR) is a rate-limiting enzyme in the production of deoxyribonucleoside triphosphates (dNTPs), which are essential substrates for DNA repair after radiation damage. We explored the radiosensitization property of RNR and investigated a selective RRM2 inhibitor, 3-AP, as a radiosensitizer in the treatment of metastatic pNETs. We investigated the role of RNR subunit, RRM2, in pancreatic neuroendocrine (pNET) cells and responses to radiation in vitro. We also evaluated the selective RRM2 subunit inhibitor, 3-AP, as a radiosensitizer to treat pNET metastases in vivo. Knockdown of RNR subunits demonstrated that RRM1 and RRM2 subunits, but not p53R3, play significant roles in cell proliferation. RRM2 inhibition activated DDR pathways through phosphorylation of ATM and DNA-PK protein kinases but not ATR. RRM2 inhibition also induced Chk1 and Chk2 phosphorylation, resulting in G1/S phase cell cycle arrest. RRM2 inhibition sensitized pNET cells to radiotherapy and induced apoptosis in vitro. In vivo, we utilized pNET subcutaneous and lung metastasis models to examine the rationale for RNR-targeted therapy and 3-AP as a radiosensitizer in treating pNETs. Combination treatment significantly increased apoptosis of BON (human pNET) xenografts and significantly reduced the burden of lung metastases. Together, our results demonstrate that selective RRM2 inhibition induced radiosensitivity of metastatic pNETs both in vitro and in vivo. Therefore, treatment with the selective RRM2 inhibitor, 3-AP, is a promising radiosensitizer in the therapeutic armamentarium for metastatic pNETs.

A Novel Protozoa Parasite-Derived Protein Adjuvant Is Effective in Immunization with Cancer Cells to Activate the Cancer-Specific Protective Immunity and Inhibit the Cancer Growth in a Murine Model of Colorectal Cancer.

Cancer-specific CD8+ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8+ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable (either treated with mitomycin C or irradiated by X-ray) MC38 CRC cells in combination with rGRA6Nt adjuvant and received a challenge implantation of replication-capable MC38 tumor cells, those mice markedly inhibited the growth of the implanted tumors in association with a two-fold increase in CD8+ T cell density within the tumors. In addition, CD8+ T cells of the immunized mice secreted significantly increased amounts of granzyme B, a key mediator of the cytotoxic activity of CD8+ T cells, and IFN-γ in response to MC38 CRC cells in vitro when compared to the T cells from unimmunized mice. Notably, the protective effects of the immunization were specific to MC38 CRC cells, as the immunized mice did not exhibit a significantly inhibited growth of EL4 lymphoma tumors. These results indicate that rGRA6Nt is a novel and effective protein adjuvant when used in immunizations with non-replicable cancer cells to potently activate the protective immunity specifically against the cancer cells employed in the immunization.

Magnetic Resonance Imaging of Macrophage Response to Radiation Therapy.

BACKGROUND: Magnetic resonance imaging (MRI) is a non-invasive imaging modality which, in conjunction with biopsies, provide a qualitative assessment of tumor response to treatment. Intravenous injection of contrast agents such as fluorine (19F) nanoemulsions labels systemic macrophages, which can, then, be tracked in real time with MRI. This method can provide quantifiable insights into the behavior of tumor-associated macrophages (TAMs) in the tumor microenvironment and macrophage recruitment during therapy. METHODS: Female mice received mammary fat pad injections of murine breast or colon cancer cell lines. The mice then received an intravenous 19F nanoemulsion injection, followed by a baseline 19F MRI. For each cancer model, half of the mice then received 8 Gy of localized radiation therapy (RT), while others remained untreated. The mice were monitored for two weeks for tumor growth and 9F signal using MRI. RESULTS: Across both cohorts, the RT-treated groups presented significant tumor growth reduction or arrest, contrary to the untreated groups. Similarly, the fluorine signal in treated groups increased significantly as early as four days post therapy. The fluorine signal change correlated to tumor volumes irrespective of time. CONCLUSION: These results demonstrate the potential of 19F MRI to non-invasively track macrophages during radiation therapy and its prognostic value with regard to tumor growth.

Copy Number Variation That Influences the Ionizing Radiation Sensitivity of Oral Squamous Cell Carcinoma.

Genome instability in cancer cells causes not only point mutations but also structural variations of the genome, including copy number variations (CNVs). It has recently been proposed that CNVs arise in cancer to adapt to a given microenvironment to survive. However, how CNV influences cellular resistance against ionizing radiation remains unknown. PRMT5 (protein arginine methyltransferase 5) and APE1 (apurinic/apyrimidinic endonuclease 1), which enhance repair of DNA double-strand breaks and oxidative DNA damage, are closely localized in the chromosome 14 of the human genome. In this study, the genomics data for the PRMT5 and APE1 genes, including their expression, CNVs, and clinical outcomes, were analyzed using TCGA's data set for oral squamous cell carcinoma patients. The two genes were found to share almost identical CNV values among cancer tissues from oral squamous cell carcinoma (OSCC) patients. Levels of expression of PRMT5 and APE1 in OSCC tissues are highly correlated in cancer but not in normal tissues, suggesting that regulation of PRMT5 and APE1 were overridden by the extent of CNV in the PRMT5-APE1 genome region. High expression levels of PRMT5 and APE1 were both associated with poor survival outcomes after radiation therapy. Simultaneous down-regulation of PRMT5 and APE1 synergistically hampered DNA double-strand break repair and sensitized OSCC cell lines to X-ray irradiation in vitro and in vivo. These results suggest that the extent of CNV in a particular genome region significantly influence the radiation resistance of cancer cells. Profiling CNV in the PRMT5-APE1 genome region may help us to understand the mechanism of the acquired radioresistance of tumor cells, and raises the possibility that simultaneous inhibition of PRMT5 and APE1 may increase the efficacy of radiation therapy.

Analysis of Copy Number Variation of DNA Repair/Damage Response Genes in Tumor Tissues.

Cells experience increased genome instability through the course of disease development including cancer initiation and progression. Point mutations, insertion/deletions, translocations, and amplifications of both coding and noncoding regions all contribute to cancer phenotypes. Copy number variation (CNV), i.e., changes of the number of copies of nuclear DNA, occurs in the genome of even normal somatic cells. Studies to understand the effects of CNV on tumor development, especially aspects concerning tumor aggressiveness and the influence on outcomes of therapeutic modalities, have been reignited by the breakthrough technologies of the molecular genomics. This section discusses the significance of analyzing CNVs that cause simultaneous increase/decrease of clusters of genes, using the expression profile of XRCC1 with its neighbor genes LIG1, PNKP, and POLD1 as an example. Methods for CNV assay at the individual gene level on formalin-fixed, paraffin-embedded (FFPE) tissues using the NanoString nCounter technology will then be described.

A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Peroxiredoxin IV plays a critical role in cancer cell growth and radioresistance through the activation of the Akt/GSK3 signaling pathways.

High levels of redox enzymes have been commonly observed in various types of human cancer, although whether and how the enzymes contribute to cancer malignancy and therapeutic resistance have yet to be understood. Peroxiredoxin IV (Prx4) is an antioxidant with bona fide peroxidase and molecular chaperone functions. Here, we report that Prx4 is highly expressed in prostate cancer patient specimens, as well as established prostate cancer cell lines, and that its levels can be further stimulated through the activation of androgen receptor signaling. We used lentivirus-mediated shRNA knockdown and CRISPR-Cas9 based KO techniques to establish Prx4-depleted prostate cancer cells, which showed delayed cell cycle progression, reduced rate of cell proliferation, migration, and invasion compared to control cells. In addition, we used proteome profiler phosphokinase arrays to identify signaling changes in Prx4-depleted cells; we found that loss of Prx4 results in insufficient phosphorylation of both Akt and its downstream kinase GSK3α/ß. Moreover, we demonstrate that Prx4-depleted cells are more sensitive to ionizing radiation as they display compromised ability to scavenge reactive oxygen species and increased accumulation of DNA damage. In mouse xenograft models, we show depletion of Prx4 leads to significant suppression of tumor growth, and tumors formed by Prx4-depleted cells respond more effectively to radiation therapy. Our findings suggest that increased levels of Prx4 contribute to the malignancy and radioresistance of prostate cancer through the activation of Akt/GSK3 signaling pathways. Therefore, strategies targeting Prx4 may be utilized to potentially inhibit tumor growth and overcome radioresistance in prostate cancer.

Extracellular vesicles released after cranial radiation: An insight into an early mechanism of brain injury.

Cranial radiation is important for treating both primary brain tumors and brain metastases. A potential delayed side effect of cranial radiation is neurocognitive function decline. Early detection of CNS injury might prevent further neuronal damage. Extracellular vesicles (EVs) have emerged as a potential diagnostic tool because of their unique membranous characteristics and cargos. We investigated whether EVs can be an early indicator of CNS injury by giving C57BJ/6 mice 10 Gy cranial IR. EVs were isolated from sera to quantify: 1) number of EVs using nanoparticle tracking analysis (NTA); 2) Glial fibrillary acidic protein (GFAP), an astrocyte marker; and 3) protein-bound 4-hydroxy-2-nonenal (HNE) adducts, an oxidative damage marker. Brain tissues were prepared for immunohistochemistry staining and protein immunoblotting. The results demonstrate: 1) increased GFAP levels (p < 0.05) in EVs, but not brain tissue, in the IR group; and 2) increased HNE-bound protein adduction levels (p < 0.05). The results support using EVs as an early indicator of cancer therapy-induced neuronal injury.

PI3K/mTOR Dual Inhibitor PF-04691502 Is a Schedule-Dependent Radiosensitizer for Gastroenteropancreatic Neuroendocrine Tumors.

Patients with advanced-stage gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have a poor overall prognosis despite chemotherapy and radiotherapy (e.g., peptide receptor radionuclide therapy (PRRT)). Better treatment options are needed to improve disease regression and patient survival. The purpose of this study was to examine a new treatment strategy by combining PI3K/mTOR dual inhibition and radiotherapy. First, we assessed the efficacy of two PI3K/mTOR dual inhibitors, PF-04691502 and PKI-402, to inhibit pAkt and increase apoptosis in NET cell lines (BON and QGP-1) and patient-derived tumor spheroids as single agents or combined with radiotherapy (XRT). Treatment with PF-04691502 decreased pAkt (Ser473) expression for up to 72 h compared with the control; in contrast, decreased pAkt expression was noted for less than 24 h with PKI-402. Simultaneous treatment with PF-04691502 and XRT did not induce apoptosis in NET cells; however, the addition of PF-04691502 48 h after XRT significantly increased apoptosis compared to PF-04691502 or XRT treatment alone. Our results demonstrate that schedule-dependent administration of a PI3K/mTOR inhibitor, combined with XRT, can enhance cytotoxicity by promoting the radiosensitivity of NET cells. Moreover, our findings suggest that radiotherapy, in combination with timed PI3K/mTOR inhibition, may be a promising therapeutic regimen for patients with GEP-NET.

Technical Note: Vendor miscalibration of preclinical orthovoltage irradiator identified through independent output check.

PURPOSE: Accurate radiation dosimetry in radiobiological experiments is crucial for preclinical research in advancement of cancer treatment. Vendors of cell irradiators often perform calibration for end-users. However, calibration accuracy remains unclear due to missing detailed information on calibration equipment and procedures. In this study, we report our findings of a vender miscalibration of the radiation output and our investigation on the root cause of the discrepancy. METHODS: Independent calibration verification for a commercial preclinical orthovoltage irradiator was conducted. Initially, in the absence of ionization chambers calibrated at kV energy, radiochromic films (EBT3) was first calibrated at MV energy. Energy correction factors from literature were used to create an in-house kV dosimetry system. The miscalibration identified with the in-house kV EBT3 dosimetry was later confirmed by ADCL calibrated ionization chambers (Exradin A1SL and PTW 30013) at kV energy. Ionization chambers were suspended in-air following TG-61 recommendation for output calibration. To investigate the root cause of the miscalibration, additional measurements were performed with ionization chambers placed on the shelf. A validated Monte Carlo simulation code was also used to investigate the impact of placing the ionization chamber on the shelf instead of suspending it in air during the vendor-performed calibration process. RESULTS: Up to a 6% dosimetry error was observed when comparing the vendor calibrated output of the preclinical irradiator with our independent calibration check. Further investigation showed incorrect setups in the vendor's calibration procedure which may result in dose errors up to 11% from the backscatter of the shelf board during calibration, and up to 5% from omitting temperature and pressure corrections to ionization chamber readings. CONCLUSION: Our study revealed large dose calibration errors caused by incorrect setup and the omission of temperature/pressure correction in the vendor's calibration procedure. The findings also highlighted the importance of performing an independent check of the dose calibration for preclinical kV irradiators. More absolute dosimetry training is needed for both vendors and end users for establishing accurate absolute dosimetry.

Evaluation of antioxidant network proteins as novel prognostic biomarkers for head and neck cancer patients.

OBJECTIVES: Recurrence rates for head and neck squamous cell carcinoma (HNSCC) approach 50% at 5 years. Current staging fails to identify patients with a worse prognosis who might benefit from intensified treatment, which warrants improved prognostic biomarkers. The purpose of this retrospective case study is to identify potential prognostic biomarkers in patients with HNSCC including APE1 (DNA repair/redox gene regulator), NRF2 and PPARGC1A (redox gene regulators), SOD3 and DCN (antioxidant proteins). MATERIALS AND METHODS: Differential protein expression between benign, carcinoma in situ (CIS), and invasive HNSCC tissue specimens from 77 patients was assessed using immunohistochemistry. Protein expression was analyzed with multivariate, pair-wise, and Kaplan-Meier survival analyses to identify potential prognostic biomarkers. Utilizing The Cancer Genome Atlas's transcriptome database, pair-wise and survival analysis was performed to identify potential prognostic biomarkers. RESULTS: APE1, NRF2, PPARGC1A, SOD3, and DCN expression in HNSCC in relation to, lymph node invasion, and patient survival were examined. Elevated APE1 protein expression in CIS corresponded with reduced survival (p = 0.0243). Increased APE1 gene expression in stage T4a HNSCC was associated with reduced patient survival (p < 0.015). Increased PPARGC1A in invasive tumor correlated with reduced survival (p = 0.0281). Patients with lymph node invasion at diagnosis had significantly increased APE1 protein in the primary sites (p < 0.05). Patients with poorly differentiated invasive tumors had reduced PPARGC1A in CIS proximal to the invasive tumor and had elevated DCN and SOD3 in proximal benign tissue (p < 0.05). CONCLUSIONS: The expression of APE1, DCN, and SOD3 is a potential prognostic signature that identifies patients with worsened survival.

Impact of backscatter material thickness on the depth dose of orthovoltage irradiators for radiobiology research.

The orthovoltage x-ray energy frequently used in radiation research is prone to dosimetry errors due to insufficient backscatter conditions. In many radiobiology studies, especially for cell irradiations, precise dose calculation algorithms such as Convolution-Superposition or Monte Carlo are impractical and as such, less accurate hand calculation methods are used for dose estimation. These dose estimation methods typically assume full backscatter conditions. The purpose of this study is to demonstrate the magnitude of the dose error that results from insufficient backscatter, and to provide lookup tables to account this issue. The beam spectra of several widely used commercial systems (XRAD-225, XRAD-320, SARRP) were used in Monte Carlo (MC) simulations on a series of phantom setups to investigate the impact of varying backscatter conditions on dosimetry. The depth dose curves for different field sizes, water phantom thicknesses and beam qualities were generated. In addition, depth dependent backscatter factors for different field sizes and different beam qualities were calculated. It is demonstrated that as much as a 50% dose difference exists for different backscatter conditions at the beam qualities studied. The choice of cell dish size as well as other changes in the experiment setup can have more than 10% impact on the dose. The impact of backscatter is reduced with a decrease in field size. Further, the thickness needed to provide full backscatter can be approximated as being equal to the field size. It is imperative to ensure full backscatter conditions during system and dosimeter calibration, or to use the look-up table provided in this study.

MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays.

Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(l-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P' caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'<2PD<3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay.

Polyubiquitination of apurinic/apyrimidinic endonuclease 1 by Parkin.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein crucial for repair of oxidized DNA damage not only in genomic DNA but also in mitochondrial DNA. Parkin, a tumor suppressor and Parkinson's disease (PD) associated gene, is an E3 ubiquitin ligase crucial for mitophagy. Although DNA damage is known to induce mitochondrial stress, Parkin's role in regulating DNA repair proteins has not been elucidated. In this study, we examined the possibility of Parkin-dependent ubiquitination of APE1. Ectopically expressed APE1 was degraded by Parkin and PINK1 via polyubiquitination in mouse embryonic fibroblast cells. PD-causing mutations in Parkin and PINK1 abrogated APE1 ubiquitination. Interaction of APE1 with Parkin was observed by co-immunoprecipitation, proximity ligation assay, and co-localization in the cytoplasm. N-terminal deletion of 41 amino acid residues in APE1 significantly reduced the Parkin-dependent APE1 degradation. These results suggested that Parkin directly ubiquitinated N-terminal Lys residues in APE1 in the cytoplasm. Modulation of Parkin and PINK1 activities under mitochondrial or oxidative stress caused moderate but statistically significant decrease of endogenous APE1 in human cell lines including SH-SY5Y, HEK293, and A549 cells. Analyses of glioblastoma tissues showed an inverse relation between the expression levels of APE1 and Parkin. These results suggest that degradation of endogenous APE1 by Parkin occur when cells are stressed to activate Parkin, and imply a role of Parkin in maintaining the quality of APE1, and loss of Parkin may contribute to elevated APE1 levels in glioblastoma. © 2016 Wiley Periodicals, Inc.

Analysis of RNA expression of normal and cancer tissues reveals high correlation of COP9 gene expression with respiratory chain complex components.

BACKGROUND: The COP9 signalosome, composed of eight subunits, is implicated in cancer genetics with its deneddylase activity to modulate cellular concentration of oncogenic proteins such as IkB and TGFß. However, its function in the normal cell physiology remains elusive. Primarily focusing on gene expression data of the normal tissues of the head and neck, the cancer genome atlas (TCGA) database was used to identify groups of genes that were expressed synergistically with the COP9 genes, particularly with the COPS5 (CSN5), which possesses the catalytic activity of COP9. RESULTS: Expressions of seven of the COP9 genes (COPS2, COPS3, COPS4, COPS5, COPS6, COPS7A, and COPS8) were found to be highly synergistic in the normal tissues. In contrast, the tumor tissues decreased the coordinated expression pattern of COP9 genes. Pathway analysis revealed a high coordination of the expression of the COPS5 and the other COP9 genes with mitochondria-related functional pathways, including genes encoding the respiratory chain complex. CONCLUSIONS: The results indicate that mRNA expression data for the matched normal tissues available in TCGA are statistically reliable, and are highly useful to assess novel associations of genes with functional pathways in normal physiology as well as in the cancer tissues. This study revealed the significant correlation between the expressions of the COP9 genes and those related to the mitochondrial activity.

Protective Polymer Coatings for High-Throughput, High-Purity Cellular Isolation.

Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <$2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale.

Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice.

Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.

Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease.

The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEF(la) (MEF(lowAPE1)), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEF(la) cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEF(la) was lower than that in the wild-type MEF (MEF(wt)), indicating the low DNA damage stress in MEF(la) under the normal growth condition. Oxidative phosphorylation activity in MEF(la) was lower than in MEF(wt), while the glycolysis rates in MEF(la) were higher than in MEF(wt). In addition, we observed decreased intracellular oxidative stress in MEF(la). These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability.