共查询到20条相似文献,搜索用时 0 毫秒
1.
Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen 总被引:3,自引:0,他引:3
Davies BR Steele IA Edmondson RJ Zwolinski SA Saretzki G von Zglinicki T O'Hare MJ 《Experimental cell research》2003,288(2):390-402
Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal-epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33 degrees C, but fail to increase in number at the nonpermissive temperature of 39 degrees C. Serum-deprived OSE-C2 cells are stimulated to grow at 33 degrees C by EGF, whereas they are growth inhibited at 33 degrees C by TGFbeta in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer. 相似文献
2.
Caroline Miller Jill Rulfs Stephen R. Jaspers Michael Buckholt Thomas B. Miller Jr 《Molecular and cellular biochemistry》1994,136(1):29-34
Freshly isolated ventricular myocytes have been used extensively as an adult cardiac model system. Due to their inability to undergo cytokinesisin vitro and their dedifferentiated properties in long-term culture, they can not be used for extended studies. Recent reports tell of the establishment of fetal and neonatal cardiac cell lines and the development of adult cardiomyocytes from transgenic animals. A recent report by Kirshenbaum [1], is the first to demonstrate insertion of genes in to adult ventricular myocytes using viral infection. This paper discusses the infection of primary adult differentiated cardiomyocytes with the SV40 large T antigen and subsequent proliferation under temperature sensitive control. Upon further characterization, the cells could be used as a model to study muscle differentiation and repair as well as adult cardiac cell physiology. 相似文献
3.
Philippe Merviel Armelle Degeorges Jacques Salat-Baroux Fabien Calvo 《Biology of the cell / under the auspices of the European Cell Biology Organization》1995,84(3):187-193
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. 相似文献
4.
Transcriptionally regulated immortalization overcomes side effects of temperature-sensitive SV40 large T antigen 总被引:4,自引:0,他引:4
May T Wirth D Hauser H Mueller PP 《Biochemical and biophysical research communications》2005,327(3):734-741
The temperature-sensitive mutant of the SV40 virus large T antigen (TAg) tsA58 is frequently employed for the conditional immortalization of primary cells. By increasing the temperature to 39 degrees C, the activity of the mutant TAg is reduced and the status of such cells may then resemble more closely that of primary cells. As an alternative, we used a novel immortalization vector with a tetracycline-regulated expression of the wild-type TAg. This enabled us to investigate the effects of the immortalizing gene expression and of temperature shifts independently of each other. Even for wild-type TAg-derived cell lines the elevated temperatures led to various clone-dependent phenotypes. This suggests that in freshly established cell lines temperature-sensitive growth phenotypes can arise spontaneously and independently of a temperature-sensitive immortalizing gene. Similar effects were observed with spontaneously immortalized cells. On the other hand, not all of the ts-TAg-derived cell lines were proliferation arrested at the non-permissive temperature. Therefore, the assumption that temperature-sensitive growth is solely due to the ts-TAg must be verified for each ts-TAg-derived cell line individually. This complexity could be avoided by using the autoregulatory immortalization vector expressing the wild-type TAg. 相似文献
5.
Petra Kiszel Sonja Fiesel Susanne Voit Beate Waechtler Thomas Meier Tobias Oelschlaegel Michael Schraeml Alfred M. Engel 《Biotechnology progress》2019,35(3):e2786
Transient gene expression (TGE) in HEK293 cells was optimized by Vink et al. by co-expression of human cell cycle inhibitors p21CIP/p27KIP 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 p21CIP/p27KIP together with very low amount of SVLT antigen may induce alternative functional activities than growth arrest to further improve the yield of recombinant proteins. 相似文献
6.
SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts 总被引:13,自引:0,他引:13
F A Ray D S Peabody J L Cooper L S Cram P M Kraemer 《Journal of cellular biochemistry》1990,42(1):13-31
To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death. 相似文献
7.
A. Alwin Prem Anand S. Gowri Sankar V. Kokila Vani 《Journal of cellular and molecular medicine》2012,16(11):2592-2610
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. 相似文献
8.
Infection of CV1 cells expressing the polyoma virus middle T antigen or the SV40 agnogene product with simian virus 40 host-range mutants 总被引:2,自引:0,他引:2
Susan L. Spence Lois C. Tack Jocelyn H. Wright Susan Carswell James M. Pipas 《In vitro cellular & developmental biology. Plant》1990,26(6):604-611
Summary SV40 viruses bearing mutations at the carboxy-terminus of large T antigen exhibit a host-range phenotype: such viruses are
able to grow in BSC monkey kidney cells at 37° C, but give at least 10 000-fold lower yields than wild type virus in BSC cells
at 32° C or in CV1 monkey kidney cells at either temperature. The block to infection in the nonpermissive cell type occurs
after the onset of viral DNA replication. Infectious progeny virions are produced at very low efficiency. Although capsid
proteins are synthesized at decreased levels, this does not account for the magnitude of the defect. Presumably some step
of virion assembly or maturation is affected in these mutants. We have previously reported that the viral agnogene product,
a protein throught to be involved in viral assembly or release, fails to accumulate in CV1 cells infected with host-range
mutants. In polyoma virus the middle T antigen plays a role in virion maturation by influencing the phosphorylation of capsid
proteins. In this communication we show that host-range mutants fail to undergo productive infection of CV1 cells expressing
middle T antigen. These mutants do form plaques on an agnoprotein-expressing cell line. However, the agnoprotein does not
seem to act by correcting the mutational block but rather increases the efficiency of plaque formation.
This work was supported by grants CA40586 and BRSG 2S07RR07084-23 to J. M. P. and grant CA33079 to L. T., from the National
Institutes of Health, Bethesda, MD. 相似文献
9.
Earnest H. L. Leung Peter C. K. Leung Nelly Auersperg 《In vitro cellular & developmental biology. Animal》2001,37(8):515-521
The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human USE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34 degrees C), these cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degrees C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin. varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24-43% of cells. At 39 degrees C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d. the cells assumed more epithelial morphologies, keratin expression reached 85-100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collage n III at both temperatures. As indicated by beta-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39 degrees C by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag. 相似文献
10.
11.
Takayama M Kim E Kidokoro M Shimamura K Shiroki K Yajima H Kosukegaw A Handa H Inokuchi AS 《In vitro cellular & developmental biology. Animal》2000,36(2):110-116
Summary We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive
T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen
conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration
at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the
same conditions. The serum/Ca++-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for
their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C.
The serum/Ca++-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes.
However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca++-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6
mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro. 相似文献
12.
13.
以P53蛋白与SV40大T抗原之间的相互作用作为药物分子靶标,利用酵母双杂交系统的α-半乳糖苷酶活力的测定方法,测定了两个系列的化合物对于P53蛋白与SV40大T抗原相互作用的影响。其中一个系列包括ZnCl2,MgCl2,CaCl2,和Cu-Cl2、BaCl2等二价金属盐;另一个系列是几种新疆特色植物的天然提取物,包括蒜氨酸、大蒜素、罗丹明B类物质AJ-8、黄酮、苦参碱、去氢骆驼蓬碱和骆驼蓬碱等。结果发现,ZnCl2能够特异使P53蛋白与SV40大T抗原之间的相互作用增强15%左右,而大蒜素和黄酮能够降低这两个蛋白的相互作用,降低程度分别为40%和30%,其它药物基本不影响两个蛋白的相互作用。 相似文献
14.
Fitzgerald U Hettle S MacDonald C McLean JS 《In vitro cellular & developmental biology. Animal》2000,36(4):222-227
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. 相似文献
15.
《Bioorganic & medicinal chemistry》2014,22(22):6490-6502
Polyomavirus infections are common and relatively benign in the general human population but can become pathogenic in immunosuppressed patients. Because most treatments for polyomavirus-associated diseases nonspecifically target DNA replication, existing treatments for polyomavirus infection possess undesirable side effects. However, all polyomaviruses express Large Tumor Antigen (T Ag), which is unique to this virus family and may serve as a therapeutic target. Previous screening of pyrimidinone–peptoid hybrid compounds identified MAL2-11B and a MAL2-11B tetrazole derivative as inhibitors of viral replication and T Ag ATPase activity (IC50 of ∼20–50 μM). To improve upon this scaffold and to develop a structure–activity relationship for this new class of antiviral agents, several iterative series of MAL2-11B derivatives were synthesized. The replacement of a flexible methylene chain linker with a benzyl group or, alternatively, the addition of an ortho-methyl substituent on the biphenyl side chain in MAL2-11B yielded an IC50 of ∼50 μM, which retained antiviral activity. After combining both structural motifs, a new lead compound was identified that inhibited T Ag ATPase activity with an IC50 of ∼5 μM. We suggest that the knowledge gained from the structure–activity relationship and a further refinement cycle of the MAL2-11B scaffold will provide a specific, novel therapeutic treatment option for polyomavirus infections and their associated diseases. 相似文献
16.
Precise conditional immortalization of mouse cells using tetracycline‐regulated SV40 large T‐antigen
Konstantinos Anastassiadis Maria Rostovskaya Sandra Lubitz Stefanie Weidlich A. Francis Stewart 《Genesis (New York, N.Y. : 2000)》2010,48(4):220-232
Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline‐regulated system for the expression of SV40 large T‐antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand‐binding domain (LBD) of the androgen receptor (irtTA‐ABD) or of a mutated glucocorticoid receptor (irtTA‐GBD*). Induction of T‐antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA‐ABD or dexamethasone for irtTA‐GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T‐antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T‐antigen. genesis 48:220–232 2010. © 2010 Wiley‐Liss, Inc. 相似文献
17.
Horst-Werner Stürzbecher Martin Mrike Mathias Montenarh Roland Henning 《FEBS letters》1985,180(2):285-290
The potential significance of the phosphorylation of SV40 large T antigen for oligomers and complexes with the cellular protein p53 was investigated. We observed that T antigen oligomers remain stable after enzymatic dephosphorylation by alkaline phosphatase up to 80%. Separate analysis of free and p53-bound T antigen revealed a considerably lower phosphorylation of the p53-bound subclass. Therefore, a simple correlation between the overall phosphorylation of T antigen and the formation of oligomers and T-p53 complexes is highly unlikely. 相似文献
18.
Florina Moldovan Houda Benanni Jean Fiet Olivier Cussenot Jacques Dumas Christian Darbord Hany R. Soliman 《In vitro cellular & developmental biology. Animal》1996,32(1):16-23
Summary The plasmid pMK16 containing-SV40 replicated origin defective gene was efficiently introduced into early-passage human umbilical
vein endothelial cells (HUVEC) using positively charged liposomes. The resulting cell line acquired an almost infinite lifespan,
was morphologically unchanged, expressed SV40-antigen, and coexpressed von Willebrand factor (vWF), tissue plasminogen activator
(t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin conversion enzyme (ACE), and endothelin converting enzyme (ECE).
In addition, these are the first immortalized human endothelial cells, to our knowledge, that biosynthesized and secreted
interleukins (IL-1β and IL-6) in both a constitutive and regulated fashion and endothelin-1 (ET-1), the most potent vasoactive
peptide, which has been suggested to be implicated in the pathogenesis of hypertension. Interestingly enough, both of the
immortalized cells and the early-passage HUVEC from which the immortalized cells were obtained biosynthesized and secreted
the same levels of ET-1 suggesting full maintenance of its biosynthetic pathway including the presence of active ECE, which
cleaves big endothelin-1 (big-ET-1) to ET-1 and regulation factors. Moreover, the immortalized cells retained the ability
to express the functional specific amino acid Na+-independent system Y+ transporter, which mediates L-arginine transport into endothelial cells from which endothelium-derived relaxing factor (EDRF,
nitric oxide) is formed via the action of nitric oxide-synthase. Obtaining these immortalized human endothelial cells without
alteration of the differentiated characteristics constitutes a useful model: (a) to study ET-1 secretion, gene regulation,
and human ECE, which may be an important therapeutic target in disease conditions in which ET-1 is to be implicated; (b) to
study L-arginine transport, which is a key step in the formation of EDRF; (c) to study IL-1β and IL-6 secretions, and gene
regulations; (d) to substitute large quantities of HUVEC; and, finally, (e) to reproduce, starting with different primary
endothelial cells both from human and animal origin. 相似文献
19.
B Schwartz P Vicart C Delouis D Paulin 《Biology of the cell / under the auspices of the European Cell Biology Organization》1991,73(1):7-14
The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides -878 to +93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained characteristics of differential cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells. 相似文献
20.
Ito M Yokouchi K Yoshida K Kano K Naito K Miyazaki J Tojo H 《Development, growth & differentiation》2006,48(1):41-47
Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells. 相似文献