Sex omission and male bias are still widespread in cell experiments.

Integrating sex as an important biological variable is imperative to enhance the accuracy and reproducibility of cell-based studies, which provide basic information for subsequent preclinical and clinical study designs. Recently, international funding agencies and renowned journals have been attempting to integrate sex as a variable in every research step. To understand what progress has been made in reporting of cell sex in the articles published in AJP-Cell Physiology since the analysis in 2013, we examined the sex notation of the cells in relevant articles published in the same journal in 2018. Of the 107 articles reporting cell experiments, 53 reported the sex of the cells, 18 used both male and female cells, 23 used male cells only, and 12 used female cells only. Sex omission was more frequent when cell lines were used than when primary cells were used. In the articles describing experiments performed using rodent primary cells, more than half of the studies used only male cells. Our results showed an overall improvement in sex reporting for cells in AJP-Cell Physiology articles from 2013 (25%) to 2018 (50%). However, sex omission and male bias were often found still. Furthermore, the obtained results were rarely analyzed by sex even when both male and female cells were used in the experiments. To boost sex-considerate research implementation in basic biomedical studies, cooperative efforts of the research community, funders, and publishers are urged.

Sex Chromosomes Are Severely Disrupted in Gastric Cancer Cell Lines.

Sex has not received enough attention as an important biological variable in basic research, even though the sex of cells often affects cell proliferation, differentiation, apoptosis, and response to stimulation. Knowing and considering the sex of cells used in basic research is essential as preclinical and clinical studies are planned based on basic research results. Cell lines derived from tumor have been widely used for proof-of-concept experiments. However, cell lines may have limitations in testing the effect of sex on cell level, as chromosomal abnormality is the single most characteristic feature of tumor. To examine the status of sex chromosomes in a cell line, 12 commercially available gastric carcinoma (GC) cell lines were analyzed using several different methods. Loss of Y chromosome (LOY) accompanied with X chromosome duplication was found in three (SNU-484, KATO III, and MKN-1) out of the six male-derived cell lines, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Two (SNU-5 and MKN-28) out of six female-derived cell lines showed a loss of one X chromosome, while SNU-620 gained one extra copy of the X chromosome, resulting in an XXX karyotype. We found that simple polymerase chain reaction (PCR)-based sex determination gives a clue for LOY for male-derived cells, but it does not provide detailed information for the gain or loss of the X chromosome. Our results suggest that carefully examining the sex chromosome status of cell lines is necessary before using them to test the effect of sex on cell level.

Cell Cycle Dysregulation Is Associated With 5-Fluorouracil Resistance in Gastric Cancer Cells.

BACKGROUND/AIM: 5-Fluorouracil (5-FU) is an anticancer drug commonly used to treat gastric cancer; however, continuous 5-FU chemotherapy causes drug resistance. MATERIALS AND METHODS: We established five sublines of 5-FU-resistant AGS gastric cancer cells to investigate changes that may have occurred in the development of 5-FU resistance. Drug resistance to other chemotherapeutic reagents, proliferation, cell-cycle changes, and wound healing ability were assessed for each subline. RESULTS: Retarded cell growth, G0/G1 phase arrest, up-regulation of p57, and down-regulation of cyclin D1 were commonly observed in all five sublines. Resistance to paclitaxel and cisplatin was also observed in most of the sublines. CONCLUSION: Our data support the notion that G0/G1 arrest due to changes in p57 and cyclin D1 expression may confer drug resistance, while EMT seems non-essential to 5-FU resistance in AGS gastric carcinoma cells.

Epstein-Barr Virus miR-BART17-5p Promotes Migration and Anchorage-Independent Growth by Targeting Kruppel-Like Factor 2 in Gastric Cancer.

Epstein-Barr virus (EBV) infects more than 90% of the global population and is associated with a variety of tumors including nasopharyngeal carcinoma, Hodgkin lymphoma, natural killer/T lymphoma, and gastric carcinoma. In EBV-associated gastric cancer (EBVaGC), highly expressed EBV BamHI A rightward transcripts (BART) miRNAs may contribute to tumorigenesis with limited viral antigens. Despite previous studies on the targets of BART miRNAs, the functions of all 44 BART miRNAs have not been fully clarified. Here, we used RNA sequencing data from the Cancer Genome Atlas to find genes with decreased expression in EBVaGC. Furthermore, we used AGS cells infected with EBV to determine whether expression was reduced by BART miRNA. We showed that the expression of Kruppel-like factor 2 (KLF2) is lower in AGS-EBV cells than in the AGS control. Using bioinformatics analysis, four BART miRNAs were selected to check whether they suppress KLF2 expression. We found that only miR-BART17-5p directly down-regulated KLF2 and promoted gastric carcinoma cell migration and anchorage-independent growth. Our data suggest that KLF2 functions as a tumor suppressor in EBVaGC and that miR-BART17-5p may be a valuable target for effective EBVaGC treatment.

Epstein-Barr virus miR-BART1-3p suppresses apoptosis and promotes migration of gastric carcinoma cells by targeting DAB2.

Although Epstein-Barr virus (EBV) is known to encode over 40 different miRNAs of its own, the roles of most EBV miRNAs remain unknown. Disabled homolog 2 (DAB2) is a putative tumor suppressor, but its role in gastric carcinoma (GC), especially in EBV-associated GC, needs to be clarified. Our qRT-PCR and mRNA microarray results showed that DAB2 expression was down-regulated in EBV-positive GC cells compared to EBV-negative cells. Four BART miRNAs that might target DAB2 were predicted, and we found, using a luciferase reporter assay, that miR-BART1-3p directly targeted the 3'-UTR of DAB2. The miR-BART1-3p transfection decreased DAB2 expression at both mRNA and protein levels, while transfection of an inhibitor of miR-BART1-3p, miR-BART1-3p(i), increased DAB2 expression. In addition, miR-BART1-3p as well as siDAB2 increased migration and decreased apoptosis. Meanwhile, miR-BART1-3p(i) or pcDNA3.1-DAB2 transfection decreased migration and increased apoptosis in EBV-infected GC cells. Furthermore, decreased migration by miR-BART1-3p(i) was abrogated by co-transfected siDAB2, while decreased migration by miR-BART1-3p(i) was further suppressed by a co-transfected DAB2 over-expression vector. Our data suggest that miR-BART1-3p plays an important role in the tumorigenesis of EBV-associated GC by directly targeting DAB2.

EBV miR-BART10-3p Promotes Cell Proliferation and Migration by Targeting DKK1.

In Epstein-Barr virus (EBV)-infected epithelial cancers, BamHI A rightward transcript (BART) miRNAs are highly expressed. However, only a few target genes of BART miRNAs have been investigated. Our mRNA microarray data showed that DKK1 was markedly down-regulated in EBV-associated gastric carcinoma (EBVaGC) cells. Using luciferase reporter assay we tested whether miR-BART10-3p regulates DKK1 by directly targeting the 3'-UTR of DKK1 mRNA. We observed that miR-BART10-3p transfection decreased DKK1 expression, while an LNA inhibitor of miR-BART10-3p (LNA-miR-BART10-3p(i)) increased DKK1 expression. Furthermore, miR-BART10-3p and siDKK1 promoted cell proliferation and migration. In contrast, transfecting GC cells with LNA-miR-BART10-3p(i) or DKK1 over expression vector suppressed cell proliferation and migration. Our results suggest that miR-BART10-3p may be involved in the tumor progression of EBVaGC by targeting DKK1.

Efficient and wash-free labeling of membrane proteins using engineered Npu DnaE split-inteins.

An efficient and wash-free method to conjugate a fluorescent tag to a target membrane protein is developed, using engineered Npu DnaE split-inteins. This approach allowed fast labeling while avoiding the strenuous synthesis of a long polypeptide. Two different modes of labeling, namely specific binding and covalent conjugation, are observed. The covalent labeling was monitored within 5 min, without background staining.

Photo-triggered fluorescent labelling of recombinant proteins in live cells.

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed. The approach is based on photo-caged split-intein mediated conditional protein trans-splicing reaction and enabled background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Macrophage-derived TGF-beta1 induces IgA isotype expression.

TGF-beta1 is a potent IgA isotype switching factor. However the cell population that generates the TGF-beta is not known. In this study, we examined the origin of the TGF-beta1 that is secreted by LPS-activated murine total spleen cell cultures and that is responsible for IgA isotype switching. Treatment with anti-TGFbeta1 antibody decreased IgA secretion 2 fold in these cultures and caused a 5-fold decrease in the number of IgA secreting cells. In Mphi-depleted spleen cell populations, this IgA response was markedly reduced and anti-TGFbeta1 antibody had no additional effect on IgA production. The inference that Mphi-derived TGF-beta1 is responsible for the isotype switching is supported by observations with the macrophage line, P388D1. LPS, particularly in the presence of IFN-gamma, induced P388D1 cells to secrete active TGF-beta1. The supernatant from such an activated P388D1 culture, in combination of IL-2, stimulated IgA secretion and this effect was abolished by anti-TGFbeta1 antibody.