Umbilical cord endothelial cells expressing large T antigen: Comparison with primary cultures and effect of cell age

Summary A number of human endothelial cell lines from umbilical cord cells (HUVECs) have been generated by transfection with SV40 large T and small t antigen sequences. Comparison of these lines with primary cultures of HUVECs has been carried out by monitoring the expression of a number of endothelial cell markers with specific regard to cell age. The secreted levels of the protein plasminogen activator inhibitor (PAI) was found to be significantly reduced in SV40-transfected cells when compared to untransfected controls. Tissue plasminogen activator (tPA) and urokinase (uPA) levels were unchanged. As cells entered crisis, there was a rapid and significant increase in the levels of tPA, uPA, and PAI and this was observed for all clones screened. The endothelial cell marker von Willebrand Factor (vWF) was found intracellularly and was also secreted into the medium. The levels were not altered between transfected and untransfected cells. Angiotensin converting enzyme (ACE) activity was maintained in cell lines at levels found in nonimmortalized HUVECs. Both isoforms (α and β) of IL-1 (interleukin-1) increased as cells approached crisis, and the presence of these cytokines may be responsible for the increased levels of tPA, PAI, and uPA. With one exception, the ability of the transfected cells to produce prostacyclin (PGI₂) was lost by all clones.     

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.     

Transient gene expression using valproic acid in combination with co-transfection of SV40 large T antigen and human p21CIP/p27KIP

Transient gene expression (TGE) in HEK293 cells was optimized by Vink et al. by co-expression of human cell cycle inhibitors p21^CIP/p27^KIP and Simian virus 40 large T antigen (SVLT). In this study, we investigated the effect of this enhancer protein complex on the TGE experiments in a cell-cycle arrested condition of HEK293F cells induced by valproic acid. Growth profiles, consumptions of nutrients, formations of waste products, and product titers of recombinant human antibodies (huAb) were monitored during the 7-day cultivation time. Our results showed that the use of enhancer proteins increased the product yields in a growth arrest condition as well. During the growth phase, no differences were detected regarding viable cell densities (VCDs), viabilities, growth rates, and cell diameters between the TGE experiments with and without enhancer proteins. However, during the declining phase VCD and viability showed slightly higher values at day 6 and 7 in the presence of enhancers. Furthermore, we could not detect any differences in glucose and glutamine metabolism during batch cultivations with co-expression of enhancer proteins. Taken together, the special complex of enhancer proteins did not contribute to further enhancement of growth arrest and shift in the main cell metabolisms, but resulted in higher cell viability during the decline phase. Our observations suggest that the human cell cycle inhibitors p21^CIP/p27^KIP together with very low amount of SVLT antigen may induce alternative functional activities than growth arrest to further improve the yield of recombinant proteins.     

SV40 T抗原与人U_1和U_2 snRNA基因的特异结合

利用DNA结合免疫分析证明,编码人U_1和U_2 snRNA基因的5′端区域含有与SV40 T抗原特异结合的序列。SV40 T抗原与U_2基因的亲和力大于U_1基因。DNasel足印(footprinting)分析取得与DNA结合免疫分析一致的结果。U_1和U_2基因的5′端区域含有能被T抗原所保护的,免于DNasel降解的序列。这两个基因的两条链上都含有约30bp长的DNA被T抗原所保护。U_1基因被保护的区域在-11bp—-42bp之间,而U_2基因被保护区域是在-58bp—-90bp之间。     

Establishment and characterization of chondrocyte cell lines from the costal cartilage of SV40 large T antigen transgenic mice

Complete understanding of the physiology and pathology of the cartilage is essential to establish treatments for a variety of cartilage disorders and defects such as rheumatoid arthritis, congenital malformations, and tumors of cartilage. Although synthetic materials have been used in many cases, they possess inherent problems including wear of the materials and low mechanical strength. Autograft has been considered very effective to overcome these problems. However, the limitation of the transplant volume is a major problem in autograft to be overcome. The costal cartilage is the most serious candidate for donor site transplantation, since it is the largest permanent hyaline cartilage in the body. To investigate the possibility using the costal cartilage as a transplant source, we have established and characterized three mouse chondrocyte cell lines (MCC-2, MCC-5, and MCC-35) derived from the costal cartilage of 8-week-old male SV40 large T-antigen transgenic mice. At confluence, all the cell lines formed nodules that could be positively stained with alcian blue (pH 2.5). The size of nodules gradually increased during culturing time. After 2 and 6 weeks of culture, RT-PCR analysis demonstrated that all three cell lines expressed mRNA from the cartilage-specific genes for type II collagen, type XI collagen, aggrecan, and link protein. Furthermore, type X collagen expression was detected in MCC-5 and MCC-35 but not in MCC-2. Any phenotypic changes were not observed over 31 cell divisions. Immunocytochemistry showed further that MCC-2, MCC-5, and MCC-35 produced cartilage-specific proteins type II collagen and type XI collagen, while in addition MCC-5 and MCC-35 produced type X collagen. Treatment with 1alpha, 25-dihydroxyvitamin D(3) inhibited cell proliferation and differentiation of the three cell lines in a dose-dependent manner. These phenotypic characteristics have been found consistent with chondrocyte cell lines established from cartilage tissues other than costal cartilage. In conclusion, costal cartilage shows phenotypic similarities to other cartilages, i.e., articular cartilage and embryonic limbs, suggesting that costal cartilage may be very useful as the donor transplantation site for the treatment of cartilage disorders. Furthermore, the cell lines established in this study are also beneficial in basic research of cartilage physiology and pathology.     

Genetic networks in nonpermissive temperature-induced cell differentiation of Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen

To identify the molecular basis by which nonpermissive temperature (NPT) induces cell differentiation in Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen, we performed global scale microarray and computational gene network analyses. In TTE3 cells, inactivation of the large T-antigen by a NPT at 39 degrees C led to cell differentiation accompanying elevation of transferrin, a marker for differentiation of Sertoli cells, and CDKN1A, a cyclin-dependent kinase inhibitor. Of the 22,690 probe sets analyzed, NPT down-regulated 498 probe sets and up-regulated 432 probe sets by >2.0-fold. Hierarchical clustering analysis showed six gene clusters. In the down-regulated cluster I, a significant genetic network including fibronectin 1 was associated with cellular growth and proliferation. In up-regulated cluster IV, a significant genetic network including CDKN1A was associated with cellular differentiation. The present results provide additional novel insights into the molecular basis of cell differentiation induced by NPT in cells.     

Immortalization of neuronal progenitors using SV40 large T antigen and differentiation towards dopaminergic neurons

Transplantation is common in clinical practice where there is availability of the tissue and organ. In the case of neurodegenerative disease such as Parkinson's disease (PD), transplantation is not possible as a result of the non‐availability of tissue or organ and therefore, cell therapy is an innovation in clinical practice. However, the availability of neuronal cells for transplantation is very limited. Alternatively, immortalized neuronal progenitors could be used in treating PD. The neuronal progenitor cells can be differentiated into dopaminergic phenotype. Here in this article, the current understanding of the molecular mechanisms involved in the differentiation of dopaminergic phenotype from the neuronal progenitors immortalized with SV40 LT antigen is discussed. In addition, the methods of generating dopaminergic neurons from progenitor cells and the factors that govern their differentiation are elaborated. Recent advances in cell‐therapy based transplantation in PD patients and future prospects are discussed.     

Down-modulation of erbB2 activity is necessary but not enough in the differentiation of 3T3-L1 preadipocytes

The high incidence of obesity-related pathologies, led to the study of the mechanisms involved in preadipose cell proliferation and differentiation. Here, we demonstrate that modulation of erbB2, plays a fundamental role during proliferation and adipogenic induction of preadipocytes. Using 3T3-L1 cells as model, we demonstrate that EGF (10 nM, 5 min) in addition to stimulate receptor tyrosine phosphorylation of both erbB2 and EGFR, is able to induce the heterodimer erbB2-EGFR. We treated proliferating 3T3-L1 cells with two inhibitors, AG 825 (IC(50) 0.35 microM, 54 times more selective for erbB2 than for EGFR, IC(50) 19 microM), and AG 879 (IC(50) of 1 microM for erbB2 versus 500 microM for EGFR). We found that both inhibited the proliferation on a dose-dependent basis, reaching a 30% maximal inhibition at 100 microM (P < 0.001) for AG825, and a 20% maximal inhibition at 10 microM (P < 0.001) for AG 879. These results involve erbB2 in 3T3-L1 proliferation. When studying the differentiation process, we found that the action of MIX-Dexa immediately activates MEK, JNK and p38 kinases. We observed that PD98059 and SP600125 (MEK-ERK and JNK inhibitors, respectively) added 1 h prior to the MIX-Dexa induction produced a decrease in erbB2 expression after 6 h, which is even greater than the one produced by the inducers, MIX-Dexa. This work supports erbB2 as a key factor in 3T3-L1 adipogenesis, acting mostly and not only during the proliferative phase but also during the differentiation through modulation of both its expression and activity.     

Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.     

Neoplastic transformation and tumorigenesis associated with overexpression of IMUP-1 and IMUP-2 genes in cultured NIH/3T3 mouse fibroblasts

Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.     

Expression of c-fos and c-myc protooncogenes in an immortalized hepatocyte line harbouring SV40 T antigen and hGH as transgenes

A clonal hepatocyte line (FMH-202-2), derived from livers of fetal transgenic mice harbouring human growth hormone (hGH) and SV40 T antigen as transgenes, was used in the investigation of protooncogene expression involved in liver-specific growth control and/or in hepatocellular transformation. In this model system, representing an immortalized, yet untransformed phenotype, the transgenes hGH and SV40 T antigen were expressed constitutively. The c-fos protooncogene was induced by incubation with insulin, epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) in a transient manner comparable to its expression in primary murine hepatocytes. Elucidation of second messenger mechanisms demonstrated that c-fos induction by hepatotrophic growth factors was not mediated by protein kinase C. In contrast to primary hepatocytes, the c-myc protooncogene exhibited a constitutive expression pattern which was independent of growth factor stimulation. These results indicate that apart from hGH and SV40 T antigen, c-myc may play a role in cellular immortalization, but that constitutive expression of these genes, even in combined coexpression, does not suffice to induce the transformed phenotype.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58

Process extension was induced in cells of the N18-RE-105 neuroblastoma-retinal hybrid line by toxic agents, including glutamate and the p53-inducing anticancer agents adriamycin and etoposide. Both adriamycin and glutamate activated p53 as measured by a plasmid transfection assay. It was therefore hypothesized that SV40 large T antigen, which binds p53, would interfere with cellular differentiation. To test this hypothesis, the temperature-sensitive form of SV40 large T was transduced into N18-RE-105 cells by retroviral infection. SV40 large T-infected cells became de-differentiated, grew in tightly-packed colonies, lost expression of neurofilament, and lost the ability to differentiate in response to glutamate and adriamycin. The de-differentiating effect of SV40 large T antigen may be due to binding and inactivation of cellular proteins, such as p53, p107, p130, p300, and retinoblastoma protein, which are important in cellular growth and differentiation. It is suggested that p53 may play a role in cellular differentiation, perhaps under unusual circumstances involving stress or cytotoxicity. Received: 29 April 1997 / Accepted: 18 June 1997     

1α,25-Dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in Human Monocytes

CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1α,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-γ but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response.     

Phagocytosis of Fonsecaea pedrosoi conidia, but not sclerotic cells caused by Langerhans cells, inhibits CD40 and B7-2 expression

Fonsecaea pedrosoi is the major etiological agent of chromoblastomycosis, a chronic, suppurative, granulomatous mycosis usually confined to skin and subcutaneous tissues, presenting a worldwide distribution. The host defense mechanisms in chromoblastomycosis have not been extensively investigated. Langerhans cells (LC) are bone-marrow-derived, dendritic antigen-presenting cells of the epidermis, which constitutively express major histocompatibility complex (MHC) class II, and comprise 1-3% of total epidermal cells. LC are localized in suprabasal layers of the epidermis and in mucosa, where they play important roles in skin immune responses. The purpose of the present study was to evaluate the interaction of F. pedrosoi conidia or sclerotic cells with LC purified from BALB/c mice skin. We demonstrate here that LC phagocytose F. pedrosoi conidia but not sclerotic cells in the first 3 h of interaction, inhibiting hyphae formation during 12-hour coculture from both forms, internalized or not. Also, LC maturation, analyzed using CD40 and B7-2 expression, was inhibited by conidia, but not by sclerotic cells, indicating an important innate immunity function of LC against F. pedrosoi infection in these mice.     

Fluid shear stress induces Runx‐2 expression via upregulation of PIEZO1 in MC3T3‐E1 cells

Mechanically induced biological responses in bone cells involve a complex biophysical process. Although various mechanosensors have been identified, the precise mechanotransduction pathway remains poorly understood. PIEZO1 is a newly discovered mechanically activated ion channel in bone cells. This study aimed to explore the involvement of PIEZO1 in mechanical loading (fluid shear stress)‐induced signaling cascades that control osteogenesis. The results showed that fluid shear stress increased PIEZO1 expression in MC3T3‐E1 cells. The fluid shear stress elicited the key osteoblastic gene Runx‐2 expression; however, PIEZO1 silencing using small interference RNA blocked these effects. The AKT/GSK‐3β/β‐catenin pathway was activated in this process. PIEZO1 silencing impaired mechanically induced activation of the AKT/GSK‐3β/β‐catenin pathway. Therefore, the results demonstrated that MC3T3‐E1 osteoblasts required PIEZO1 to adapt to the external mechanical fluid shear stress, thereby inducing osteoblastic Runx‐2 gene expression, partly through the AKT/GSK‐3β/β‐catenin pathway.