scholarly journals Adenovirus-Mediated Expression of Human Prorelaxin Promotes the Invasive Potential of Canine Mammary Cancer Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (8) ◽  
pp. 3683-3691 ◽  
Author(s):  
Josh D. Silvertown ◽  
Brad J. Geddes ◽  
Alastair J. S. Summerlee
Keyword(s):  
Cell Proliferation ◽  
Cell Migration ◽  
Mammary Cancer ◽  
Fluorescent Protein ◽  
Internal Ribosomal Entry Site ◽  
Cell Types ◽  
Entry Site ◽  
Monocytic Cell ◽  
Monocytic Cell Line

Abstract This study reports the characterization of a recombinant adenoviral vector containing a tetracycline-regulatable promoter, driving the bicistronic expression of the human H2 preprorelaxin (hH2) cDNA and enhanced green fluorescent protein, via an internal ribosomal entry site. An hH2 ELISA was used to measure the secreted levels of recombinant hH2 in transfected canine (CF33.Mt) and human (MDA-MB-435) mammary cancer cell lines over a 6-d period; secreted peptide peaked on d 2 and 4 for the canine and human cell types, respectively. An unprocessed hH2 immunoreactive form of approximately 18 kDa was identified by Western blotting analysis and confirmed by mass spectrometry, suggesting that prorelaxin remains unprocessed in these cell types. The biological activity of the adenovirally expressed human prorelaxin was measured in the established human monocytic cell line THP-1 cAMP ELISA and in an in vitro Transwell cell migration system. Exogenous recombinant hH2 and adenovirally-mediated delivery of prorelaxin to CF33.Mt cells conferred a significant migratory action in the cells, compared with controls. Cell proliferation assays were performed to discount the possibility that the effect of relaxin was mitogenic. Thus, we have demonstrated that prorelaxin has the ability to facilitate cell migration processes exclusive of its ability to stimulate cell proliferation. In validating this adenovirus-based system, we have created a potential tool for further exploration of the physiology of relaxin in mammalian systems.

scholarly journals Human herpesvirus-6 dysregulates monocyte-mediated anticryptococcal defences

2006 ◽  
Vol 55 (6) ◽  
pp. 695-702 ◽  
Author(s):  
Claudio Cermelli ◽  
Valeria Cenacchi ◽  
Francesca Beretti ◽  
Francesco Pezzini ◽  
Dario Di Luca ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Cell Types ◽  
Cell Contact ◽  
Target Cells ◽  
Human Herpesvirus 6 ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Herpesvirus 6 ◽  
Set Up

In order to investigate the interplay occurring between pathogens in the course of double infections, an in vitro model was set up in which the monocytic cell line THP-1 was exposed to Cryptococcus neoformans (Cn) and human herpesvirus 6 (HHV-6). Cn and HHV-6, both highly neurotropic, can cause serious diseases of the central nervous system and have monocytes, among other cell types, as target cells, causing alteration of their secretion pattern. Here, it was shown that unlike THP-1 cells exposed to cell-free virus inocula, THP-1 exposed to HHV-6-producing lymphocytes exhibited augmented phagocytosis against Cn. The phenomenon occurred after 24 h of monocyte/lymphocyte co-culture and was independent of direct cell-to-cell contact. Moreover, in the presence of HHV-6, THP-1 cells expressed enhanced secretory responses but reduced capability to counteract fungal infection: the enhanced ingestion by monocytes was followed by facilitated fungal survival and replication. These data provide initial in vitro evidence that HHV-6 may dysregulate monocyte-mediated anticryptococcal defences with an overall pro-cryptococcus result.

Purification of c-Kit-Negative Hematopoietic Stem Cells from Runx1-GFP Knock-In Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 135-135
Author(s):  
Robert B. Lorsbach ◽  
Sonny O. Ang ◽  
Weili Sun ◽  
Jennifer Moore ◽  
James R. Downing
Keyword(s):  
Bone Marrow ◽  
Cell Population ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Internal Ribosomal Entry Site ◽  
Entry Site ◽  
Hematopoietic Stem ◽  
Transcriptional Complex

Abstract The Runx1/Core Binding Factor- β (CBF β) transcriptional complex is required for the establishment of hematopoiesis during development. To permit the analysis of Runx1 expression in hematopoietic cell subsets, we have recently developed a novel murine line in which the expression of both full-length Runx1 and GFP is driven by the Runx1 promoter [Runx1-internal ribosomal entry site-green fluorescent protein (Runx1-GFP) knock-in mouse; Blood 103:2522]. Analysis of these mice has revealed that Runx1 is expressed in all hematopoietic lineages with the exception of erythroid cells. During our analysis, we identified in the bone marrow of these mice a cell population that expresses GFP at levels 2–5 fold higher than any other cell type (GFPhi). These cells have low forward and side scatter properties and do not express c-kit or several lineage-associated cell surface markers (lin−). In comparison to c-kit+lin− cells, these GFPhic-kit−lin− cells possess little colony forming activity in vitro. While they lack primary CFU-S activity in contrast to c-kit+lin− cells, GFPhic-kit−lin− cells possess secondary CFU-S activity. A c-kit−lin− hematopoietic stem cell (HSC) has been described by others that contributes to long-term, but not short-term, hematopoietic reconstitution in lethally irradiated recipients and may represent progenitors of c-kit+lin− HSCs in vivo (Ortiz et al. Immunity 10:173). The GFPhi bone marrow cells that we have identified share many of the properties of those c-kit−lin− cells identified by others; consequently, they likely represent the same cell population. Our ability to isolate these cells based on differential GFP expression should enable us to highly purify these GFPhic-kit−lin− cells and further characterize their immunophenotypic and biologic properties.

scholarly journals DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation, cell apoptosis, and cell migration

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Yixin Tong ◽  
Yuan Huang ◽  
Yuchao Zhang ◽  
Xiangtai Zeng ◽  
Mei Yan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Cell Apoptosis ◽  
Downstream Target ◽  
Normal Tissues ◽  
Physiological Processes ◽  
Mutual Regulation

AbstractAt present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

2009 ◽  
Vol 83 (6) ◽  
pp. 2540-2552 ◽  
Author(s):  
Michael H. Lehmann ◽  
Wolfgang Kastenmuller ◽  
Judith D. Kandemir ◽  
Florian Brandt ◽  
Yasemin Suezer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Viral Vector ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Human Monocytic Cell Line ◽  
Modified Vaccinia Virus Ankara ◽  
Ccl2 Expression ◽  
Human Monocytic Cell

ABSTRACT Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4+ lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.

scholarly journals Antifungal Prenylated Diphenyl Ethers from Arthrinium arundinis, an Endophytic Fungus Isolated from the Leaves of Tobacco (Nicotiana tabacum L.)

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3179 ◽  
Author(s):  
Peng Zhang ◽  
Xin Li ◽  
Xiao-Long Yuan ◽  
Yong-Mei Du ◽  
Bin-Gui Wang ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Cell Line ◽  
Endophytic Fungus ◽  
In Vitro Cytotoxicity ◽  
Ionization Mass ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Nicotiana Tabacum L ◽  
Diphenyl Ethers

An endophytic fungus Arthrinium arundinis TE-3 was isolated and purified from the fresh leaves of cultivated tobacco (Nicotiana tabacum L.). Chemical investigation on this fungal strain afforded three new prenylated diphenyl ethers (1−3) as well as three known analogues (4−6). Structure elucidation of the isolated compounds was carried out by analysis of 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) spectra, as well as by comparison of those data with literature data. The absolute configuration of the stereogenic center at C-8 in 1 was assigned by comparison of the experimental and calculated ECD spectra. Compounds 1 and 2 showed selective antifungal activity against Mucor hiemalis with minimum inhibitory concentration (MIC) values of 8 and 4 μg/mL, respectively. Compounds 5 and 6 exhibited inhibitory activity against Alteraria alternata with an MIC value of 8 μg/mL. In the cytotoxic assay, 2, 5, and 6 displayed moderate in vitro cytotoxicity against the human monocytic cell line (THP-1 cell line), with IC50 values of 40.2, 28.3, and 25.9 μM, respectively. This study indicated that endophytic fungi possess great potential for exploring new bioactive secondary metabolites.

scholarly journals Alkaline Comet Assay using the monocytic cell line THP-1

2019 ◽  
Author(s):  
Ana Neves-Costa ◽  
Dora Pedroso ◽  
Luis F Moita
Keyword(s):  
Comet Assay ◽  
Cell Line ◽  
Adherent Cell ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Strand Breaks ◽  
Human Monocytic Cell Line ◽  
Human Monocytic Cell ◽  
Dna Strand

Abstract This protocol details the experimental procedure for performing the comet assay, a very sensitive DNA break assay based on single cell gel electrophoresis.The analysis of DNA strand breaks, both single- and double-strand breaks (SSBs and DSBs, respectively), was performed in immune responsive cells. The cell line used was the human monocytic cell line THP-1, an adherent cell type with many known applications in in vitro studies of innate immunity. The comet assay is a robust procedure that allows the accurate and reproducible quantification of DNA damage. Here we describe not only the comet assay step-by-step protocol, but also some important aspects related to troubleshooting.

Kallistatin Inhibits Endothelial Cell Proliferation, Migration and Adhesion in Vitro and Angiogenesis in the Ischemic Hindlimb of Rats

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 706-707
Author(s):  
Robert Q Miao ◽  
Jun Agata ◽  
Lee Chao ◽  
Julie Chao
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Gene Delivery ◽  
Fluorescent Protein ◽  
Regional Blood Flow ◽  
Endothelial Cell Proliferation ◽  
Hek 293

P76 Kallistatin is a serine proteinase inhibitor (serpin) which has multifunctions including regulation of tissue kallikrein activity, blood pressure, inflammation and neointima hyperplasia. In this study, we investigated the potential role of kallistatin in vascular biology by studying its effects on the proliferation, migration and adhesion of cultured primary human endothelial cells in vitro, and angiogenesis in the ischemic hindlimb of rats. Purified kallistatin significantly inhibits cultured endothelial cell proliferation, migration and adhesion induced by VEGF or bFGF. To further investigate the role of kallistatin in vascular growth in vivo, we prepared adenovirus carrying the human kallistatin gene under the control of the cytomegalovirus promoter/enhancer (Ad.CMV-cHKBP). Expression of recombinant human kallistatin in HEK 293 cells transfected with Ad.CMV-cHKBP was identified by a specific ELISA. The effect of adenovirus-mediated kallistatin gene delivery on angiogenesis was evaluated in a rat model of hindlimb ischemia. Adenovirus carrying the human kallistatin or green fluorescent protein (GFP) gene were injected locally into the ischemic adductor at the time of surgery. Histological and morphometric analysis at 14 days post injection showed that adenovirus-mediated kallistatin gene delivery significantly reduced capillary density in the ischemic muscle as compared to that of control rats injected with GFP. The anti-angiogenic effect of kallistatin was associated with reduced regional blood flow in the ischemic hindlimb measured by microsphere assays. Expression of human kallistatin was identified in the injected muscle and immunoreactive human kallistatin levels were measured in the muscle and in the circulation of rats following kallistatin gene delivery. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and in vascular remodeling.

scholarly journals Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ethan P. Metz ◽  
Erin L. Wuebben ◽  
Phillip J. Wilder ◽  
Jesse L. Cox ◽  
Kaustubh Datta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Cycle Machinery

Abstract Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

Sign in / Sign up

Export Citation Format

Share Document