Search Results (45)

Synthetic biology has fundamentally advanced cell engineering and helped to develop effective therapeutics such as chimeric antigen receptor (CAR)-T cells. For these applications, the detection, localization, and quantification of heterologous fusion proteins assembled from interchangeable building blocks is of high importance. The V5 tag, a 14-residue epitope tag, offers promising characteristics for these applications but has only rarely been used in this context. Thus, we have systematically evaluated the murine anti-V5 tag antibody mu_SV5-Pk1 as well as its humanized version, hu_SV5-Pk1, to analyze cells expressing V5-tagged receptors in samples from various in vitro and in vivo experiments. We found that the V5 tag signal on cells is affected by certain fixation and detachment reagents. Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) mouse tissue samples was performed to sensitively detect cells in tissue. We improved IHC by applying the hu_SV5-Pk1 monoclonal antibody (mAb) to avoid cross-reactivity within and unspecific background signals arising on fixed mouse tissue. Conversely, the absence of unspecific binding by the mu_SV5-Pk1 mAb was evaluated on 46 human normal or cancer tissues. Our findings present a robust toolbox for utilizing the V5 tag and cognate antibodies in synthetic biology applications. Full article

Background: Alternative NTRK1/TrkA splicing resulting in TrkAIII expression, originally discovered in advanced-stage metastatic neuroblastomas, is also pronounced in prostate, medullary thyroid, glioblastoma multiforme, MCPyV-positive Merkel cell, cutaneous malignant melanoma, and pituitary neuroendocrine tumor subsets. In tumor models, TrkAIII exhibits actionable oncogenic activity equivalent to the TrkT3-fused oncogene, and in tumor cell lines, alternative TrkAIII splicing is promoted by hypoxia, nutrient deprivation, endoplasmic reticulum stress, and SV40 large T antigen, implicating tumor microenvironmental conditions and oncogenic polyoma viruses in tumor-associated TrkAIII expression. Collectively, these observations characterize TrkAIII as a potentially frequent, actionable oncogenic alternative to TrkA gene fusion in different tumor types. Currently, therapeutic approval for efficacious Trk inhibitors is restricted to Trk-fused gene positive tumors and not for tumors potentially driven by TrkAIII. Methods: With the therapeutically relevant aim of improving the identification of tumors potentially driven by TrkAIII, we have developed a TaqMan-based qRT-PCR assay for evaluating TrkAIII expression in tumor cDNAs. Results: This assay, validated using gel-purified fs-TrkA and TrkAIII cDNAs alone and in complex cDNA mixtures, employs primers and probes designed from fs-TrkA and TrkAIII sequences, with specificity provided by a TaqMan probe spanning the TrkAIII exon 5–8 splice junction. It is highly efficient, reproducible, and specific and can detect as few as 10 TrkAIII copies in complex RNAs extracted from either fresh or FFPE tumor tissues. Conclusions: Inclusion of this assay into precision oncology algorithms, when paired with fs-TrkA qRT-PCR and TrkA immune histochemistry, will make it easier to identify patients with therapy-resistant, advanced-stage metastatic Trk-fused gene-negative tumors potentially driven by TrkAIII, for whom approval of third-line effective Trk inhibitors could be extended. Full article

Drug development for human disease relies on preclinical model systems such as human cell cultures and animal experiments before therapeutic treatments can ultimately be tested on humans in clinical studies. We here describe the generation of a novel human cell line (HLMVEC/SVTERT289) that we generated by transfection of microvascular endothelial cells from healthy donor lung tissue with the catalytic domain of telomerase and the SV40 large T/small t-antigen. These cells exhibited satisfactory growth characteristics and largely maintained their native characteristics, including morphology, cell surface marker expression, angiogenic potential and the protein composition of secreted extracellular vesicles. In order to test their suitability as a disease model, we simulated mechanical stress induced by cyclic stretch as encountered in ventilator-induced lung injury using the FlexCell^® system and compared their performance to primary lung endothelial cells. In this setting, HLMVEC/SVTERT289 cells exhibited significantly higher neprilysin activity on the cell surface and extracellular vesicles secreted from the cell line exhibited higher Tissue Factor and ACE2 expression but lower ACE expression and ACE activity than vesicles released from the primary cells. This study provides an unprecedented and detailed characterization of the HLMVEC/SVTERT289 cell line, which should help to appraise its suitability in different molecular studies. Full article

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), has a significant impact on the global pork industry. It results in reproductive failure in sows and respiratory issues in pigs of all ages. Despite the availability of vaccines, controlling the PRRSV remains challenging, partly owing to the limitations of cell culture systems. Current methods largely rely on primary porcine alveolar macrophages (PAMs), which must be harvested from piglets and have limited proliferative capacity. Although some simian cell lines support PRRSV replication, their inability to express porcine CD163, which is a key receptor for PRRSV entry, compromises their effectiveness, because the virus replicates differently in these non-target cells. To address these issues, we established an immortalized PAM cell line, PAM-T43, using SV40 large T antigen for immortalization and porcine serum as a culture supplement. PAM-T43 cells maintain essential macrophage functions, including CD163 expression and phagocytic activity, and exhibit high sensitivity to the PRRSV, efficiently supporting viral replication. This novel cell line offers significant potential for advancing PRRSV research, particularly in vaccine development and field strain isolation, by overcoming the limitations of current systems. Full article

Yak is a special free-ranging cattle breed in the plateau areas of Qinghai and Tibet. Pasture withering in cold-season pastures results in energy deficiency in yaks, which undermines the rumen epithelial barrier. However, the leading factor causing rumen epithelial injury remains unknown. Glutamine (Gln), a conditionally essential amino acid, is insufficient under pathological conditions. Glucose (GLU) is an important energy source. Thus, we explored the effects of Gln or GLU deprivation on the barrier function of yak rumen epithelial cells and investigated the underlying mechanisms, as well as the differences in rumen epithelial barrier function between Gln deprivation (Gln-D) and GLU deprivation (GLU-D). In previous work, we constructed the yak rumen epithelial cells (YRECs) line by transferring the human telomerase reverse transcriptase gene (hTERT) and simian virus 40 large T antigen (SV40T) into primary YRECs. The YRECs were exposed to normal, Gln-D, GLU-D, and serum replacement (SR) media for 6, 12, and 24 h. Our data displayed that cell viability and tight junction protein expression in the SR group were not significantly changed compared to the normal group. Whereas, compared with the SR group, Gln-D treated for more than 12 h reduced cell viability and proliferation, and GLU-D treated for more than 12 h damaged the cell morphology and reduced cell viability and proliferation. The cell proliferation and cell viability were decreased more in GLU-D than in Gln-D. In addition, Gln-D treated for more than 12 h disrupted YREC cellular partially tight junctions by inducing oxidative stress and inflammation, and GLU-D treated for more than 12 h disrupted YREC cellular tight junctions by inducing apoptosis, oxidative stress, and inflammation. Compared with Gln-D, GLU-D more significantly induced cell injury and reduced tight junction protein levels. Our results provided evidence that GLU-D induced damage through the p38 mitogen-activated protein kinase (p38 MAPK)/c-junN-terminal kinase (JNK) signaling pathway, which was more serious than Gln-D treated for more than 12 h. Full article

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. Full article

The adult mammalian heart has been demonstrated to be endowed with low but real turnover capacity, especially for cardiomyocytes, the key functional cell type. The source, however, of that turnover capacity remains controversial. In this regard, we have defined and characterized a resident multipotent cardiac mouse progenitor population, Bmi1+DR (for Bmi1+ Damage-Responsive cells). Bmi1+DR is one of the cell types with the lowest ROS (Reactive Oxygen Species) levels in the adult heart, being particularly characterized by their close relationship with cardiac vessels, most probably involved in the regulation of proliferation/maintenance of Bmi1+DR. This was proposed to work as their endothelial niche. Due to the scarcity of Bmi1+DR cells in the adult mouse heart, we have generated an immortalization/dis-immortalization model using Simian Vacuolating Virus 40-Large Antigen T (SV40-T) to facilitate their in vitro characterization. We have obtained a heterogeneous population of immortalized Bmi1+DR cells (Bmi1+DR^IMM) that was validated attending to different criteria, also showing a comparable sensitivity to strong oxidative damage. Then, we concluded that the Bmi1-DR^IMM population is an appropriate model for primary Bmi1+DR in vitro studies. The co-culture of Bmi1+DR^IMM cells with endothelial cells protects them against oxidative damage, showing a moderate depletion in non-canonical autophagy and also contributing with a modest metabolic regulation. Full article

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC), but preclinical studies to delineate the mechanisms underlying the obesity-TNBC link as well as strategies to break that link are constrained by the lack of tumor models syngeneic to obesity-prone mouse strains. C3(1)/SV40 T-antigen (C3-TAg) transgenic mice on an FVB genetic background develop tumors with molecular and pathologic features that closely resemble human TNBC, but FVB mice are resistant to diet-induced obesity (DIO). Herein, we sought to develop transplantable C3-TAg cell lines syngeneic to C57BL/6 mice, an inbred mouse strain that is sensitive to DIO. We backcrossed FVB-Tg(C3-1-TAg)cJeg/JegJ to C57BL/6 mice for ten generations, and spontaneous tumors from those mice were excised and used to generate four clonal cell lines (B6TAg1.02, B6TAg2.03, B6TAg2.10, and B6TAg2.51). We characterized the growth of the four cell lines in both lean and DIO C57BL/6J female mice and performed transcriptomic profiling. Each cell line was readily tumorigenic and had transcriptional profiles that clustered as claudin-low, yet markedly differed from each other in their rate of tumor progression and transcriptomic signatures for key metabolic, immune, and oncogenic signaling pathways. DIO accelerated tumor growth of orthotopically transplanted B6TAg1.02, B6TAg2.03, and B6TAg2.51 cells. Thus, the B6TAg cell lines described herein offer promising and diverse new models to augment the study of DIO-associated TNBC. Full article

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig’s muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-β-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation. Full article

Protein phosphorylation and dephosphorylation are the most common post-translational modifications mediated by protein kinases and protein phosphatases, respectively. These reversible processes can modulate the function of the target protein, such as its activity, subcellular localization, stability, and interaction with other proteins. Phosphorylation of viral proteins plays an important role in the life cycle of a virus. In this review, we highlight biological implications of the phosphorylation of the monkey polyomavirus SV40 large T and small t antigens, summarize our current knowledge of the phosphorylation of these proteins of human polyomaviruses, and conclude with gaps in the knowledge and a proposal for future research directions. Full article

Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25–2.5 μM cisplatin (CisPt) or 12.5–100 μM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 μM CisPt from 3 to 96 h expressing 2000–5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity. Full article

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract and originate from the interstitial cells of Cajal (ICC), which is the pacemaker for peristaltic movement in the gastrointestinal tract. Existing GIST cell lines are widely used as cell models for in vitro experimental studies because the mutation sites are known. However, the immortalization methods of these cell lines are unknown, and no Chinese patient-derived GIST cell lines have been documented. Here, we transfected simian virus 40 large T antigen (SV40LT) into primary GIST cells to establish an immortalized human GIST cell line (ImGIST) for the first time. The ImGIST cells had neuronal cell-like irregular radioactive growth and retained the fusion growth characteristics of GIST cells. They stably expressed signature proteins, maintained the biological and genomic characteristics of normal primary GIST cells, and responded well to imatinib, suggesting that ImGIST could be a potential in vitro model for research in GIST to explore the molecular pathogenesis, drug resistance mechanisms, and the development of new adjuvant therapeutic options. Full article

The yak lives in harsh alpine environments and the rumen plays a crucial role in the digestive system. Rumen-associated cells have unique adaptations and functions. The yak rumen fibroblast cell line (SV40T-YFB) was immortalized by introducing simian virus 40 large T antigen (SV40T) by lentivirus-mediated transfection. Further, we have reported the effects of lipopolysaccharide (LPS) of different concentrations on cell proliferation, extracellular matrix (ECM), and proinflammatory mediators in SV40T-YFB. The results showed that the immortalized yak rumen fibroblast cell lines were identified as fibroblasts that presented oval nuclei, a fusiform shape, and positive vimentin and SV40T staining after stable passage. Chromosome karyotype analysis showed diploid characteristics of yak (n = 60). LPS at different concentrations inhibited cell viability in a dose-dependent manner. SV40T-YFB treated with LPS increased mRNA expression levels of matrix metalloproteinases (MMP-2 and MMP-9), inflammatory cytokines (TNF-α, IL-1β, IL-6), and urokinase-type plasminogen activator system components (uPA, uPAR). LPS inhibits the expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), plasminogen activator inhibitor-2 (PAI-2), fibronectin (FN), anti-inflammatory factor IL-10, and collagen I (COL I) in SV40T-YFB. Overall, these results suggest that LPS inhibits cell proliferation and induces ECM degradation and inflammatory response in SV40T-YFB. Full article

Calorie restriction (CR) inhibits triple-negative breast cancer (TNBC) progression in several preclinical models in association with decreased insulin-like growth factor 1 (IGF1) signaling. To investigate the impact of CR on microRNAs (miRs) that target the IGF1/IGF1R pathway, we used the spontaneous murine model of TNBC, C3(1)/SV40 T-antigen (C3-TAg). In C3-TAg mice, CR reduced body weight, IGF1 levels, and TNBC progression. We evaluated the tumoral expression of 10 miRs. CR increased the expression of miR-199a-3p, miR-199a-5p, miR-486, and miR-15b. However, only miR-15b expression correlated with tumorigenicity in the M28, M6, and M6C C3-TAg cell lines of TNBC progression. Overexpressing miR-15b reduced the proliferation of mouse (M6) and human (MDA-MB-231) cell lines. Serum restriction alone or in combination with low levels of recombinant IGF1 significantly upregulated miR-15b expression and reduced Igf1r in M6 cells. These effects were reversed by the pharmacological inhibition of IGFR with BMS754807. In silico analysis using miR web tools predicted that miR-15b targets genes associated with IGF1/mTOR pathways and the cell cycle. Our findings suggest that CR in association with reduced IGF1 levels could upregulate miR-15b to downregulate Igf1r and contribute to the anticancer effects of CR. Thus, miR-15b may be a therapeutic target for mimicking the beneficial effects of CR against TNBC. Full article

In their influential reviews, Hanahan and Weinberg coined the term ‘Hallmarks of Cancer’ and described genome instability as a property of cells enabling cancer development. Accurate DNA replication of genomes is central to diminishing genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase (Pol-prim), during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair are discussed. Full article