scholarly journals The transcriptome of wild-type and immortalized corneal epithelial cells

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Kai Furuya ◽  
Tao Wu ◽  
Ai Orimoto ◽  
Eriko Sugano ◽  
Hiroshi Tomita ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Expression Pattern ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial

AbstractCellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products

1991 ◽  
Vol 11 (8) ◽  
pp. 4253-4265
Author(s):  
H G Wang ◽  
G Draetta ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Physical Interaction ◽  
Resting Cells ◽  
Wild Type ◽  
Quiescent Cells ◽  
Kinase Expression

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.

scholarly journals Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

1998 ◽  
Vol 18 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
Woo S. Joo ◽  
Henry Y. Kim ◽  
John D. Purviance ◽  
K. R. Sreekumar ◽  
Peter A. Bullock
Keyword(s):  
Structural Changes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
The Core ◽  
Structural Distortions ◽  
In The Wild ◽  
Sv40 Dna ◽  
Initial Assembly

ABSTRACT Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

scholarly journals Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050 ◽  
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation

1988 ◽  
Vol 8 (3) ◽  
pp. 1380-1384 ◽  
Author(s):  
V Cherington ◽  
M Brown ◽  
E Paucha ◽  
J St Louis ◽  
B M Spiegelman ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Adipocyte Differentiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lipid Binding ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Glycerophosphate Dehydrogenase ◽  
Triglyceride Accumulation

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells

1987 ◽  
Vol 7 (5) ◽  
pp. 2031-2034
Author(s):  
D E Brash ◽  
R R Reddel ◽  
M Quanrud ◽  
K Yang ◽  
M P Farrell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Primary Culture ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Bronchial Epithelial Cells ◽  
Large T Antigen ◽  
Bronchial Epithelial ◽  
Antigen Gene ◽  
Strontium Phosphate

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).

Immortalization of Rat Eustachian Tube Epithelial Cells by Adenovirus 12-Simian Virus 40 Hybrid Virus

2002 ◽  
Vol 111 (10) ◽  
pp. 919-925 ◽  
Author(s):  
Sunji Jin ◽  
Shigehiro Ueyama ◽  
Sung-Kyun Moon ◽  
Johng S. Rhim ◽  
Xin-Xing Gu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Otitis Media ◽  
Eustachian Tube ◽  
Primary Cultures ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Male Rat ◽  
Positive Staining

The eustachian tube epithelial cells play an important role in the initial pathogenesis of otitis media. In order to study the role of the eustachian tube epithelial cells in the pathogenesis of otitis media, we have established a rat eustachian tube epithelial cell line. The cell line was derived by infecting primary cultures of eustachian tube epithelial cells with the adenovirus 12-simian virus 40 (Ad12-SV40) hybrid virus. The immortalized cells have retained the morphological characteristics of the parental cells and show positive staining with anti-cytokeratin antibodies (a marker for epithelial cells), but not with anti-vimentin antibodies (a fibroblast marker). The cells have been in continuous culture for more than 10 months and have undergone 38 passages. Western blotting and cell staining have confirmed the expression of the SV40 T antigen and p53. Chromosomal analysis indicates that the cell line is aneuploid and derived from male rat epithelial cells. Together, our results suggest that the cell line originated from eustachian tube epithelial cells from a male rat and was successfully immortalized by the Ad12-SV40 virus.

scholarly journals Enterocyte Proliferation and Intestinal Hyperplasia Induced by Simian Virus 40 T Antigen Require a Functional J Domain

2007 ◽  
Vol 81 (17) ◽  
pp. 9481-9489 ◽  
Author(s):  
Abhilasha V. Rathi ◽  
M. Teresa Sáenz Robles ◽  
James M. Pipas
Keyword(s):  
Transgenic Mice ◽  
Family Members ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Morphological Examination ◽  
Enterocyte Proliferation ◽  
J Domain ◽  
Intestinal Hyperplasia

ABSTRACT Transgenic mice expressing the simian virus 40 large T antigen (TAg) in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. This induction requires TAg action on the retinoblastoma (Rb) family of tumor suppressors and is independent of the p53 pathway. In cell culture systems, the inactivation of Rb proteins requires both a J domain in TAg that interacts with hsc70 and an LXCXE motif that directs association with Rb proteins. Together these elements are sufficient to release E2Fs from their association with Rb family members. We have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type TAg, the D44N mutant is unable to induce enterocyte proliferation. Histological and morphological examination revealed that mice expressing the J domain mutant have normal intestines without loss of growth control. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. Furthermore, mice expressing D44N in a null p130 background are still unable to develop hyperplasia. These studies demonstrate that the ectopic proliferation of enterocytes by TAg requires a functional J domain and suggest that the J domain is necessary to inactivate all three pRb family members.

scholarly journals Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated ras Oncogene in Transforming Primary Rat Embryo Fibroblasts

2002 ◽  
Vol 76 (7) ◽  
pp. 3145-3157 ◽  
Author(s):  
Tina M. Beachy ◽  
Sara L. Cole ◽  
Jane F. Cavender ◽  
Mary J. Tevethia
Keyword(s):  
Amino Acids ◽  
Simian Virus 40 ◽  
Suppressive Effect ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Rat Embryo ◽  
Specific Amino Acid ◽  
Antigen Fragment ◽  
Transcriptional Transactivator

ABSTRACT Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.

scholarly journals Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

1994 ◽  
Vol 14 (4) ◽  
pp. 2686-2698 ◽  
Author(s):  
M T Sáenz Robles ◽  
H Symonds ◽  
J Chen ◽  
T Van Dyke
Keyword(s):  
Tumor Progression ◽  
Choroid Plexus ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
T Antigen ◽  
Cellular Proteins ◽  
Wild Type ◽  
Binding Region ◽  
Amino Terminal

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.

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