Detergent solubilization and partial purification of tumor specific surface and transplantation antigens from SV40‐virus‐transformed mouse cells

Chang, Chia Ju; Pancake, Samuel J.; Luborsky, Samuel W.; Mora, Peter T.

doi:10.1002/ijc.2910190216

Cited by 24 publications

(12 citation statements)

References 26 publications

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“…Note that, as has been observed before, the molecular weight value deduced from density gradient sedimentation is lower than that derived from gel electrophoresis experiments, 78.1K vs 91K, the difference being ascribed to anomalies in the SDS-PAGE technique Schaffhausen et al, 1978). Table IV summarizes sedimentation data and includes for comparison results from earlier work in this laboratory in which T antigen preparations were purified by DEAE-and DNA-cellulose chromatography before analyses on sucrose gradients (Chang et al, 1977;Mora el al., 19776). As the present work started with fractionation of crude cells extracts by sucrose gradient sedimentation, followed by immunoprecipitation of fractions, these T antigen preparations might be less dissociated than the earlier ones since they had less exposure to denaturing conditions.…”

Section: Discussionmentioning

confidence: 80%

Subcellular distribution of simian virus 40 T antigen species in various cell lines: The 56K protein

Luborsky

Chandrasekaran

1980

Intl Journal of Cancer

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The subcellular distribution of SV40 anti-T serum-specific species was examined in SV40-transformed, T-antigen-positive tissue culture cell lines of rat and of AL/N and BALB/c mouse origin. Cells were labelled with [35S]methionine. The cytoplasm, nuclear and membrane fractions were obtained, and their radioimmunoprecipitates analyzed by gel electrophoresis. Tests were performed to determine the purity of these subcellular fractions, and negligible cross-contamination was found. The cytoplasm fractions lacked detectable anti-T serum reactivity. Large amounts of both large T antigen and a 56K protein were always present together both in the nuclear fractions and, in a somewhat lesser amount, in the plasma membrane fractions of all cell lines examined. Analysis of density gradient sedimentation profiles of the immunoprecipitates of whole-cell extracts indicated these species were associated in some fashion, probably with each other. The activity of the 56K protein may be associated with its presence on the cell surface where, either alone or acting together with the large T antigen, it might provide the surface activity responsible for tumor-specific surface and/or transplantation antigen activities.

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Section: Discussionmentioning

confidence: 80%

Subcellular distribution of simian virus 40 T antigen species in various cell lines: The 56K protein

Luborsky

Chandrasekaran

1980

Intl Journal of Cancer

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“…t The presence of T, TSTA, and TSSA antigens in cells was determined by published methods (4,15,(22)(23)(24)(25).…”

Section: Methodsmentioning

confidence: 99%

Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences

Kuster

Mora

Brown

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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A clonal line of highly oncogenic “spontaneously transformed” mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10 4 -10 5 ) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.

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“…The second, with a pI of 4.7, overlaps, by chemical analysis, a component present also in normal membranes. These immunochemical analyses were carried out on neutral-detergent extracts which, according to recent work can be expected to preserve SV4O-specific U-antigen (3) as well as transplantation (12) and surface-antigen (12,13). We therefore suggest that the two categories of antigen identified in (4,5) correspond to the SV4O-specific surface-and U-antigenicities described herein by immunofluorescence.…”

Section: Discussionmentioning

confidence: 53%

Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope

Lin¹,

Schmidt‐Ullrich²,

Wallach³

1977

Proc. Natl. Acad. Sci. U.S.A.

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Nucleus-and mitochondrion-free membranes from hamster lymphocytes transformed by simian virus 40 (SV40), GD248 cells,'cause guinea pigs to produce immune sera that reveal the presence in GD248 plasma membranes and mitochondria of two types of glycoprotein that are not detected SV40-transformed T19 hamster reticulum cells, Balb/c 3T3 mouse fibroblasts, and W18 VA2 human fibroblasts, using the antisera against GD248 membrane, at 40 produced a distinct cell surface fluorescence; however, above 200, staining at the nuclear perimeter, the SV40 U-antigen reaction, becomes equally prominent. In SV40-transformed cells that had been fixed in cold acetone, as well as in purified GD248 nuclei, thermostable U-antigen staining is dramatic, but there is no reaction for nuclear T-antigen. Rabbit antisera against T19 cells gave immunofluorescence reactions equivalent to those obtained with the antisera against GD248 cells. Normal guinea pig or rabbit sera and cells that had not been transformed by SV40 gave no reaction. Our sera from tumor-bearing hamsters gave only nuclear T-antigen fluorescence. The results indicate the presence of related, SV40-specific antigens in the surface membranes, nuclear envelope, and possibly other intracellular organelles of SV40-transformed cells. The U-antigen of simian virus 40 (SV40) was discovered during work on cells infected by Ad2+NDl, an adenovirus 2-SV40 hybrid virus (1, 2). Indirect immune fluorescence tests, using sera of hamsters bearing SV40-induced tumors, define the U-antigen of the infected cells by its perinuclear location and its stability to heating at 500 (1, 2). However, Robb (3), using an immunoprecipitation approach, has recently documented the existence of U-antigenic, 94,000-dalton proteins in SV40-transformed cells.Immunofluorescence has hitherto not succeeded in localizing the U-antigen of SV40-transformed cells, whether the sera of hamsters bearing tumors or monkeys injected with Ad2+NDl-infected cells are used (2). However, we now observe that hyperimmune guinea pig sera raised against purified, nucleus-free membranes from SV40-transformed hamster lymphocytes (GD248 cells) (4, 5) yield a strong, thermostable, perinuclear U-antigen immunofluorescence, not only with GD248 cells or their isolated nuclei, but also with T19, a reticulum cell sarcoma derived from GD248 tumors,t and with SV40-transformed Balb/c 3T3 mouse fibroblasts. Rabbit antisera against.T19 cells produced equivalent results. In addition, both sera produce distinct surface immunofluorescence not only with viable GD248 and T19 cells, but also with SV40-transformed mouse fibroblasts and SV4O-transformed human W18 VA2 fibroblasts.Abbreviation: SV40, simian virus 40.

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Detergent solubilization and partial purification of tumor specific surface and transplantation antigens from SV40‐virus‐transformed mouse cells

Cited by 24 publications

References 26 publications

Subcellular distribution of simian virus 40 T antigen species in various cell lines: The 56K protein

Subcellular distribution of simian virus 40 T antigen species in various cell lines: The 56K protein

Immunologic selection against simian virus-40 transformed cells: Concomitant loss of viral antigens and early viral gene sequences

Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope

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