scholarly journals Characterization and Establishment of an Immortalized Rabbit Melanocyte Cell Line Using the SV40 Large T Antigen

2019 ◽  
Vol 20 (19) ◽  
pp. 4874 ◽  
Author(s):  
Chen ◽  
Hu ◽  
Wang ◽  
Zhao ◽  
Yang ◽  
...  
Keyword(s):  
Cell Line ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Limited Culture ◽  
Immortalized Cell ◽  
Primary Rabbit ◽  
Fur Animals

Melanocytes (MCs) are specialized cells that synthesize melanin within the melanosome. Cultured MCs are useful in order to study their role in relation to pigmentation. However, MC isolation is laborious and the obtained cells have a limited culture time. In this study, we transformed lentivirus-mediated simian virus 40 Large T (SV40-LT) into primary rabbit melanocytes (Pri RMCs) to establish an immortalized cell line. Morphologically, the immortalized RMCs (Im RMC) were indistinguishable from the Pri RMCs, and dendrites were visible following Dopa staining. No significant differences in cell proliferation or growth between immortalized and primary RMCs were observed. Based on melanocyte-specific markers, the expression of MITF, TYR, and TYRP1 were detected by PCR, immunofluorescence staining, and western blot analysis. Through karyotype, soft agar, and tumorigenesis assays, the immortalized RMCs did not undergo malignant transformation. Our results show that Im RMCs can be used as a tool cell for future MC studies on the pigmentation mechanisms of fur animals.

Immortalized human bladder cell line exhibits amiloride-sensitive sodium absorption

1996 ◽  
Vol 270 (1) ◽  
pp. F148-F153 ◽  
Author(s):  
R. D. Perrone ◽  
C. Johns ◽  
S. A. Grubman ◽  
E. Moy ◽  
D. W. Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Short Circuit ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sodium Absorption ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Human Bladder ◽  
Bladder Cell

We have produced a continuous cell line using retroviral transduction of simian virus 40 (SV40) large T antigen into epithelial cells grown from a cystoscopic bladder biopsy from a female patient with interstitial cystitis. Immortalized urothelial cells are grown in a hormonally supplemented medium in the presence of lethally irradiated NIH-3T3 fibroblast coculture. They maintain their epithelial appearance and are positive for cytokeratins. SV40 large T antigen is localized to the cell nucleus. When grown on Anocell permeable supports, the cells form a complex epithelium with scalloped luminal membranes, apical junctional complexes containing tight junctions, stratification, transepithelial resistance of 500–1,000 omega.cm2, amiloride-sensitive short-circuit current indicative of active transepithelial Na+ absorption, and functional evidence for basolateral Na-K-adenosinetriphosphatase. This immortalized bladder cell line will facilitate the study of human bladder epithelial function and the response to diverse physiological and pathophysiological stimuli.

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens

1986 ◽  
Vol 6 (4) ◽  
pp. 1204-1217
Author(s):  
P S Jat ◽  
C L Cepko ◽  
R C Mulligan ◽  
P A Sharp
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Vector System ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

scholarly journals T Antigens of Simian Virus 40: Molecular Chaperones for Viral Replication and Tumorigenesis

2002 ◽  
Vol 66 (2) ◽  
pp. 179-202 ◽  
Author(s):  
Christopher S. Sullivan ◽  
James M. Pipas
Keyword(s):  
Cell Cycle ◽  
Viral Replication ◽  
Molecular Chaperones ◽  
Tumor Antigens ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen

SUMMARY Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen—a most amazing molecule.

scholarly journals Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

2007 ◽  
Vol 81 (17) ◽  
pp. 9162-9174 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Caroline Vincent ◽  
Simon Joubert ◽  
Peter A. Bullock ◽  
Jacques Archambault
Keyword(s):  
Specific Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sequence Specificity ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin ◽  
Single Stranded Dna ◽  
Terminal Domain

ABSTRACT SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

scholarly journals Multiple DNA Damage Signaling and Repair Pathways Deregulated by Simian Virus 40 Large T Antigen

2010 ◽  
Vol 84 (16) ◽  
pp. 8007-8020 ◽  
Author(s):  
Sergei Boichuk ◽  
Liang Hu ◽  
Jennifer Hein ◽  
Ole V. Gjoerup
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Viral Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Productive Infection ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin

ABSTRACT We demonstrated previously that expression of simian virus 40 (SV40) large T antigen (LT), without a viral origin, is sufficient to induce the hallmarks of a cellular DNA damage response (DDR), such as focal accumulation of γ-H2AX and 53BP1, via Bub1 binding. Here we expand our characterization of LT effects on the DDR. Using comet assays, we demonstrate that LT induces overt DNA damage. The Fanconi anemia pathway, associated with replication stress, becomes activated, since FancD2 accumulates in foci, and monoubiquitinated FancD2 is detected on chromatin. LT also induces a distinct set of foci of the homologous recombination repair protein Rad51 that are colocalized with Nbs1 and PML. The FancD2 and Rad51 foci require neither Bub1 nor retinoblastoma protein binding. Strikingly, wild-type LT is localized on chromatin at, or near, the Rad51/PML foci, but the LT mutant in Bub1 binding is not localized there. SV40 infection was previously shown to trigger ATM activation, which facilitates viral replication. We demonstrate that productive infection also triggers ATR-dependent Chk1 activation and that Rad51 and FancD2 colocalize with LT in viral replication centers. Using small interfering RNA (siRNA)-mediated knockdown, we demonstrate that Rad51 and, to a lesser extent, FancD2 are required for efficient viral replication in vivo, suggesting that homologous recombination is important for high-level extrachromosomal replication. Taken together, the interplay of LT with the DDR is more complex than anticipated, with individual domains of LT being connected to different subcomponents of the DDR and repair machinery.

Cyclic AMP regulates PDGF-stimulated signal transduction and differentiation of an immortalized optic-nerve-derived cell line

1999 ◽  
Vol 202 (4) ◽  
pp. 461-473
Author(s):  
R.I. Cohen ◽  
R. Mckay ◽  
G. Almazan
Keyword(s):  
Signal Transduction ◽  
Optic Nerve ◽  
Growth Factor ◽  
Cyclic Amp ◽  
Cell Line ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature

To facilitate the study of the molecular events underlying the development of optic-nerve-derived oligodendrocytes and their growth-factor-related signal transduction events, we immortalized perinatal rat optic nerve cells with a temperature-sensitive simian virus 40 large T-antigen, carrying the tsA58 and U19 mutations, via a retrovirus vector. The line, tsU19-9, was selected on the basis of the expression of the neural precursor marker nestin. At the permissive temperature, 33 degreesC, tsU19-9 cells had a flat epithelial morphology. In contrast, following exposure to platelet-derived growth factor (PDGF), a factor important in the lineage progression of oligodendrocytes, or in the presence of dibutyryl cyclic AMP at 39 degreesC (the non-permissive temperature), the cells underwent morphological and antigenic differentiation to cells characteristic of the oligodendrocyte lineage. We used this cell line to investigate the binding characteristics of PDGF and related signalling cascades. Competition binding, phosphoinositide hydrolysis and intracellular Ca2+ mobilization assays all demonstrated that the three different isoforms of PDGF (AA, AB and BB) bound to and acted on the cell line. Overnight exposure to forskolin, a treatment that initiated morphological and phenotypic progression into an oligodendrocyte lineage, decreased PDGF-BB-induced intracellular Ca2+ mobilization and inhibited basal and PDGF-stimulated [3H]thymidine incorporation. Our results demonstrate that tsU19-9 may serve as a resource to study early optic-nerve oligodendrocyte development.

Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1

1990 ◽  
Vol 10 (12) ◽  
pp. 6664-6673
Author(s):  
T E Riley ◽  
A Follin ◽  
N C Jones ◽  
P S Jat
Keyword(s):  
Cell Line ◽  
Growth Arrest ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Growth Defect ◽  
Primary Cells ◽  
Nonpermissive Temperature ◽  
Large T Antigen ◽  
Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

scholarly journals Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1.

1990 ◽  
Vol 10 (12) ◽  
pp. 6664-6673 ◽  
Author(s):  
T E Riley ◽  
A Follin ◽  
N C Jones ◽  
P S Jat
Keyword(s):  
Cell Line ◽  
Growth Arrest ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Growth Defect ◽  
Primary Cells ◽  
Nonpermissive Temperature ◽  
Large T Antigen ◽  
Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

scholarly journals Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

1986 ◽  
Vol 6 (4) ◽  
pp. 1204-1217 ◽  
Author(s):  
P S Jat ◽  
C L Cepko ◽  
R C Mulligan ◽  
P A Sharp
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Vector System ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

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