Using the allergic immune system to target cancer: activity of IgE antibodies specific for human CD20 and MUC1

Monoclonal antibodies are widely used in the treatment of many B cell lymphomas and certain solid tumors. All currently approved therapeutic monoclonal antibodies are of the immunoglobulin G (IgG) isotype. We hypothesized that tumor-specific monoclonal antibodies of the IgE isotype may serve as effective cancer therapeutics. To test this hypothesis, we produced mouse?Chuman chimeric IgE antibodies specific for the human B cell antigen CD20 and the epithelial antigen MUC1. We demonstrate here that anti-hCD20 IgE antibodies have in vitro cytotoxic activity when used with purified allergic effector cells derived from umbilical cord blood. At an effector-tumor ratio of 2:1, mast cells and tumor-specific IgE induced a 2.5-fold increase in tumor cell death, as compared to control IgE. Similar results were observed when eosinophils were used as effector cells. In an in vivo murine model of breast carcinoma, administration of anti-hMUC1 IgE reduced the growth of MUC1⁺ tumors by 25?C30?% in hFc??RI transgenic mice. In contrast, local production of IgE and cytokines chemotactic for macrophages, eosinophils and mast cells led to complete tumor eradication. These results suggest that allergic effector cells activated by IgE and cell surface antigens have the capacity to induce tumor cell death in vitro and in vivo. The use of chimeric antibodies and hFc??RI transgenic mice will greatly enhance investigations in the nascent field of allergo-oncology.     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

Cyclic tetrapyrrolic photosensitizers from Cladophora patentiramea (Cladophoraceae, Chlorophyta) and Turbinaria conoides (Sargassaceae, Phaeophyta) for photodynamic therapy

In screening for novel photosensitizers for photodynamic therapy, 14 seaweed samples from Port Dickson in Malaysia were collected. Methanolic extracts of these samples were prepared and evaluated for phototoxicity using a short-term cell viability assay, where promyelocytic leukemia cells, HL60 were incubated with the extracts prior to irradiation with a broad spectrum light at 9.6?J?cm^?2 (equivalent to 10.5?mW?cm^?2 for 10?min). Four of the methanolic extracts demonstrated moderate to strong phototoxicity and bioassay-guided isolation of photosensitizers was carried out on two selected seaweeds to yield a total of eight cyclic tetrapyrrolic compounds which are derivatives of chlorophyll-a and -b. Seven of these compounds showed >50% phototoxicity at 5?μg?mL^?1 while exhibiting minimal cytotoxicity in the dark, which is an important characteristic of an ideal photosensitizer.     

Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant

The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.     

MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme

Glioblastoma multiforme (GBM) is a heterogeneous disease despite its seemingly uniform pathology. Deconvolution of The Cancer Genome Atlas''s GBM gene expression data has unveiled the existence of distinct gene expression signature underlying discrete GBM subtypes. Recent conflicting findings proposed that microRNA (miRNA)-10b exclusively regulates glioma growth or invasion but not both. We showed that silencing of miRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subtype of GBM reduces its growth, invasion and angiogenesis while promoting apoptosis in vitro. In an orthotopic human glioma mouse model, inhibition of miRNA-10b diminishes the invasiveness, angiogenicity and growth of the mesenchymal subtype-like glioma cells in the brain and significantly prolonged survival of glioma-bearing mice. We demonstrated that the pleiotropic nature of miRNA-10b was due to its suppression of multiple tumor suppressors, including TP53, FOXO3, CYLD, PAX6, PTCH1, HOXD10 and NOTCH1. In particular, siRNA-mediated knockdown experiments identified TP53, PAX6, NOTCH1 and HOXD10 as invasion regulatory genes in our mesenchymal subtype-like glioma cells. By interrogating the REMBRANDT, we noted that dysregulation of many direct targets of miRNA-10b was associated with significantly poorer patient survival. Thus, our study uncovers a novel role for miRNA-10b in regulating angiogenesis and suggests that miRNA-10b may be a pleiotropic regulator of gliomagenesis.     

Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10–20 kb and compared their characteristics with short insert (1 kb) libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.     

The interactive effect of alcohol and folic acid on genome stability in human WIL2-NS cells measured using the cytokinesis-block micronucleus cytome assay

Chromosomal mutations are commonly found in cancer cells, and can be caused by several factors including dietary insufficiency and exposure to environmental and life-style genotoxins. Folate (vitamin B9), one of the essential micronutrients, is required for DNA repair and synthesis and to maintain genome stability. Since excessive alcohol (ethanol) consumption may alter folate status and low folate might alter susceptibility to alcohol toxicity, a study was performed to investigate the individual and interactive impacts of folic acid (FA) and ethanol on genome stability in vitro. The experiments were performed using WIL2-NS cells cross-tested at three FA (20, 200 and 2000 nM) and four ethanol concentrations (0, 0.09, 0.36 and 1.34%, v/v) over a two-week culture time. Chromosomal damage and cytotoxicity were measured using the cytokinesis-block micronucleus cytome assay. The present study showed dose-related genotoxic effects of both decreasing folic acid concentration and increased ethanol on day 15 resulting in significant induction of micronuclei, nuclear buds and nucleoplasmic bridges which are biomarkers of chromosome breakage or loss, gene amplification and chromosomal rearrangement, respectively. Increased ethanol and FA deficiency interacted to further significantly increase micronuclei and nucleoplasmic bridges. However there was no evidence showing alcohol's ability to cleave FA. The findings from this study suggest a protective effect of FA against alcohol-induced DNA damage and that FA deficiency in the physiological range has a stronger impact on genome stability than exposure to cytotoxic doses of ethanol achievable in binge drinking.