A Knockout of the Tsg101 Gene Leads to Decreased Expression of ErbB Receptor Tyrosine Kinases and Induction of Autophagy Prior to Cell Death

The Tumor Susceptibility Gene 101 (Tsg101) encodes a multi-domain protein that mediates a variety of molecular and biological processes including the trafficking and lysosomal degradation of cell surface receptors. Conventional and conditional knockout models have demonstrated an essential requirement of this gene for cell cycle progression and cell viability, but the consequences of a complete ablation of Tsg101 on intracellular processes have not been examined to date. In this study, we employed mouse embryonic fibroblasts that carry two Tsg101 conditional knockout alleles to investigate the expression of ErbB receptor tyrosine kinases as well as stress-induced intracellular processes that are known to be associated with a defect in growth and cell survival. The conditional deletion of the Tsg101 gene in this well-controlled experimental model resulted in a significant reduction in the steady-state levels of the EGFR and ErbB2 but a stress-induced elevation in the phosphorylation of mitogen activated protein (MAP) kinases independent of growth factor stimulation. As part of an integrated stress response, Tsg101-deficient cells exhibited extensive remodeling of actin filaments and greatly enlarged lysosomes that were enriched with the autophagy-related protein LC3. The increase in the transcriptional activation and expression of LC3 and its association with Lamp1-positive lysosomes in a PI3K-dependent manner suggest that Tsg101 knockout cells utilize autophagy as a survival mechanism prior to their ultimate death. Collectively, this study shows that a knockout of the Tsg101 gene causes complex intracellular changes associated with stress response and cell death. These multifaceted alterations need to be recognized as they have an impact on defining particular functions for Tsg101 in processes such as signal transduction and lysosomal/endosomal trafficking

Simultaneous Detection of EGFR and VEGF in Colorectal Cancer using Fluorescence-Raman Endoscopy

Fluorescence endomicroscopy provides quick access to molecular targets, while Raman spectroscopy allows the detection of multiple molecular targets. Using a simultaneous fluorescence-Raman endoscopic system (FRES), we herein demonstrate its potential in cancer diagnosis in an orthotopically induced colorectal cancer (CRC) xenograft model. In the model, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were targeted with antibody-conjugated fluorescence and surface-enhanced Raman scattering (F-SERS) dots. FRES demonstrated fast signal detection and multiplex targeting ability using fluorescence and Raman signals to detect the F-SERS dots. In addition, FRES showed a multiplex targeting ability even on a subcentimeter-sized CRC after spraying with a dose of 50 µg F-SERS dots. In conclusion, molecular characteristics of tumor cells (EGFR in cancer cell membranes) and tumor microenvironments (VEGF in the extracellular matrix) could be simultaneously investigated when performing a colonoscopy

Vitamin C promotes the early reprogramming of fetal canine fibroblasts into induced pluripotent stem cells

Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process

Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection

Detection of Pig Cells Harboring Porcine Endogenous Retroviruses in Non-Human Primate Bladder After Renal Xenotransplantation

Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome

A conditional knockout of Tsg101 results in reduced expression of EGFR and ErbB2 but a stress-induced activation of Erk1/2 in a growth factor independent manner.

<p><b>A</b>., <b>B</b>. Western blot analyses to assess the steady-state levels of endogenous EGFR and ErbB2 (A) as well as exogenous human EGFR in Tsg101 (B) conditional knockout fibroblasts (−/−) and their isogenic controls expressing Tsg101 (fl/fl). <b>C</b>. Semi-quantitative RT-PCR to examine the transcriptional activation of the endogenous <i>EGFR</i> gene in response to Tsg101 ablation. Genomic DNA (gDNA) was used as a control for the PCR with exon-specific primers that span intronic sequences of the mouse <i>EGFR</i>. <b>D</b>. EGFR and Erk1/2 expression and activation in response to EGF stimulation (0, 30, and 60 min) in cells lacking Tsg101 and their isogenic controls. <b>E</b>. Western blot analysis to assess immediate and long-term changes in EGFR expression and activation of Erk1/2 between one and four days following the conditional deletion of <i>Tsg101</i>. <b>F</b>. Analysis of Erk1/2 phosphorylation in the presence and absence of growth factors (GF) in response to Tsg101 deficiency.</p

Tsg101-deficient cells initiate autophagy to prolong their survival prior to cell death.

<p><b>A</b>. Confocal images of wildtype cells and Tsg101 conditional knockout cells that were grown in the presence or absence of 3-Methyladenine (3MA) to inhibit the fusion and maturation of LC3 containing autophagosomes with lysosomes. Tsg101-deficient cells were maintained in the presence of the inhibitor following one day after deletion of <i>Tsg101</i> using an adenovirus-based delivery of Cre recombinase; bar represents 10 µm. <b>B</b>. Quantitative analysis of the colocalization of LC3 and Lamp1 following 2 and 3 days of treatment with 3MA (<i>P</i> value, <i>t</i> test). <b>C</b>. Counts of Tsg101-deficient cells and their controls following treatment with 1.5 mM 3MA. The inhibitor was administered 24 hrs after infection with AdCre and deletion of <i>Tsg101</i>.</p