A macrophage-monocyte cell line from a dog with malignant histiocytosis

Summary The DH82 cell line was established from the neoplastic progenitor cells of canine MH and was characterized as histiocytic in origin based on light microcopic and ultrastructural morphology, positive staining reactions for alpha naphthyl acetate esterase and acid phosphatase, presence of Fc receptors, phagocytosis of latex beads, and plastic adherence in culture.     

Epithelial to mesenchymal transition in Madin-Darby canine kidney cells is accompanied by down-regulation of Smad3 expression,leading to resistance to transforming growth factor-beta-induced growth arrest

In normal epithelial cells, transforming growth factor-beta (TGF-beta) typically causes growth arrest in the G(1) phase of the cell cycle and may eventually lead to apoptosis. However, transformed cells lose these inhibitory responses and often instead show an increase in malignant character following TGF-beta treatment. In the canine kidney-derived epithelial cell line, MDCK, synergism between activation of the Raf/MAPK pathway and the resulting autocrine production of TGF-beta triggers transition from an epithelial to a mesenchymal phenotype. During this process, these cells become refractive to TGF-beta-induced cell cycle arrest and apoptosis. TGF-beta signals are primarily transduced to the nucleus through complexes of receptor-regulated Smads, Smad2 and Smad3 with the common mediator Smad, Smad4. Here we show that the transition from an epithelial to mesenchymal phenotype is accompanied by gradual down-regulation of expression of Smad3. Restoration of Smad3 to previous levels of expression restores the cell cycle arrest induced by TGF-beta without reverting the cells to an epithelial phenotype or impacting on the MAPK pathway. Regulation of apoptosis is not affected by Smad3 levels. These data attribute to Smad3 a critical role in the control of cell proliferation by TGF-beta, which is lost following an epithelial to mesenchymal transition.     

The enforced expression of c-Myc in pig fibroblasts triggers mesenchymal-epithelial transition (MET) via F-actin reorganization and RhoA/Rock pathway inactivation

In previous studies from other labs it has been well demonstrated that the ectopic expression of c-Myc in mammary epithelial cells can induce epithelial-mesenchymal transition (EMT), whereas in our pilot experiment, epithelial-like morphological changes were unexpectedly observed in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)] and pig mesenchymal stem cells, suggesting that the same c-Myc gene is entitled to trigger EMT in epithelial cells and mesenchymal-epithelial transition (MET) in fibroblasts. This prompted us to characterize the existence of a MET in c-Myc-expressing PEFs and PDFs at the molecular level. qRT-PCR, immunofluorescence and western blot analysis illustrated that epithelial-like morphological changes were accompanied by the increased expression of epithelial markers [such as cell adhesion proteins (E-cadherin, α-catenin and Bves), tight junction protein occludin and cytokeratins (Krt8 and Krt18)], the reduced expression of mesenchymal markers [vimentin, fibronectin 1 (FN1), snail1, collagen family of proteins (COL1A1, COL5A2) and matrix metalloproteinase (MMP) family (MMP12 and MMP14)] and the decreased cell motility and increased cell adhesion in c-Myc-expressing PEFs and PDFs. Furthermore, the ectopic expression of c-Myc in pig fibroblasts disrupted the stress fiber network, suppressed the formation of filopodia and lamellipodia, and resulted in RhoA/Rock pathway inactivation, which finally participates in epithelial-like morphological conversion. Taken together, these findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET, to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute.     

Booster effect of canine distemper, canine parvovirus infection and infectious canine hepatitis combination vaccine in domesticated adult dogs

Domesticated adult dogs with antibody titer classified as below 'high' to one or more of canine distemper virus (CDV), canine parvovirus type-2 (CPV-2) and canine adenovirus type-1 (CAdV-1) were then given an additional inoculation, and the effectiveness of this booster evaluated 2 months later. Consequently, CDV and CAdV-1 antibody titer experienced a significant increase, but the same effect was not observed in the antibody titer of CPV-2. These findings suggest that with additional inoculation, a booster effect may be expected in increasing antibody titers for CDV and CAdV-1, but it is unlikely to give an increase in CPV-2 antibody titer.     

Modulation of the EMT/MET process by pyrrole–imidazole polyamide targeting human transforming growth factor-β1

Transforming growth factor-β1 (TGF-β1) is a potent induction factor for epithelial–mesenchymal transition (EMT). Mesenchymal–epithelial transition (MET), as the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole–imidazole (PI) polyamide, a novel gene regulator that targets human TGF-β1, and investigated its effects on the EMT/MET process. PI polyamide targeted to TGF-β1 significantly inhibited the mRNA expression of TGF-β1 and SNAI1 as an EMT marker and increased mRNA and protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-β1 was applied to human fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline phosphatase-positive colonies. The expression of undifferentiated markers was also observed in these colonies. These results suggest that PI polyamide targeted to human TGF-β is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.     

Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and Stalls invasion

Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET). These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin) and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNA(MYOF) transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF.     

Transforming growth factor-β1 promotes lung adenocarcinoma invasion and metastasis by epithelial-to-mesenchymal transition

Lung cancer is a highly malignant carcinoma, and most deaths of lung cancer are caused by metastasis. The alterations associated with epithelial-to-mesenchymal transition (EMT) may be related to the cancer cell metastasis. Nevertheless, the mechanism of lung cancer metastasis remains unclear. We conducted a study in vitro to investigate whether transforming growth factor-β1 (TGF-β1) could induce changes of, such as cell morphology, expression of relative protein markers, and cellular motile and invasive activities. In this research, the changes of cell morphology were first investigated under a phase contrast microscope, then western blotting was employed to detect the expression of E-cadherin, vimentin, and fibronectin, and finally cell motility and invasion were evaluated by cell wound-healing as well as invasion assays. The data indicated that human lung adenocarcinoma cell lines, A-549 and PC-9 cells of epithelial cell characteristics, were induced to undergo EMT by TGF-β1. Following TGF-β1 treatment, cells showed dramatic morphological changes assessed by phase contrast microscopy, accompanied by decreased epithelial marker E-cadherin and increased mesenchymal markers vimentin and fibronectin. More importantly, cell motility and invasion were also enhanced in the EMT process. These results indicated that TGF-β1 may promote lung adenocarcinoma invasion and metastasis via the mechanism of EMT.     

犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬痘热病毒感染性cDNA克隆.对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒.酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM 2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础.     

Increase of O-Glycosylated Oncofetal Fibronectin in High Glucose-Induced Epithelial-Mesenchymal Transition of Cultured Human Epithelial Cells

Growing evidences indicate that aberrant glycosylation can modulate tumor cell invasion and metastasis. The process termed "epithelial-mesenchymal transition" (EMT) provides a basic experimental model to shed light on this complex process. The EMT involves a striking decline in epithelial markers, accompanied by enhanced expression of mesenchymal markers, culminating in cell morphology change and increased cell motility. Few recent studies have established the participation glycosylation during EMT. Studies now come into knowledge brought to light the involvement of a site-specific O-glycosylation in the IIICS domain of human oncofetal fibronectin (onfFN) during the EMT process. Herein we show that high glucose induces EMT in A549 cells as demonstrated by TGF-β secretion, cell morphology changes, increased cellular motility and the emergence of mesenchymal markers. The hyperglycemic conditions increased onfFN protein levels, promoted an up regulation of mRNA levels for ppGalNAc-T6 and FN IIICS domain, which contain the hexapeptide (VTHPGY) required for onfFN biosynthesis. Glucose effect involves hexosamine (HBP) biosynthetic pathway as overexpression of glutamine: fructose-6-phosphate amidotransferase increases mesenchymal markers, onfFN levels and mRNA levels for FN IIICS domain. In summary, our results demonstrate, for the first time that the metabolism of glucose through HBP promotes O-glycosylation of the oncofetal form of FN during EMT modulating tumorogenesis.     

TGF-β1对AGS细胞上皮-间充质转化及体外侵袭的影响

目的观察转化生长因子-β1(TGF-β1)对人胃癌细胞株AGS发生上皮-间充质转化(epithelial-mesenchymal transition,EMT)及体外侵袭的影响。方法将体外培养的AGS用TGF-β1干预后,倒置显微镜下观察细胞形态学的变化,MTT比色法检测TGF-β1对AGS增殖的影响,细胞划痕试验和Transwell侵袭试验检测细胞运动和侵袭力的改变;免疫荧光和Western blot检测snail、E-cadherin(上皮钙粘蛋白)、和N-cadherin(神经钙粘蛋白)表达的变化。结果TGF-β1诱导AGS向间充质细胞形态转化,低浓度促进细胞增殖,而高浓度时细胞增殖率逐步降低,且snail和间充质细胞表型N-cadherin表达上调,而上皮细胞表型E-cadherin表达下调,同时细胞运动和侵袭能力大大增强。结论TGF-β1可诱导AGS发生EMT,从而增加其侵袭、转移的能力。     

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.     

Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.     

R-cadherin influences cell motility via Rho family GTPases

Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity.