Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

Kaplan, David R.; Bockus, B J; Roberts, Thomas M.; Bolen, Joseph B.; Israel, Mark A.; Schaffhausen, Brian

doi:10.1128/mcb.5.7.1795

Cited by 8 publications

(8 citation statements)

References 32 publications

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“…Since the bands were not seen when extracts from 293 cells infected with wild-type adenovirus or affinity gel lacking antibody was used (not shown), these polypeptides are likely to be specifically associated with middle T antigen. Another reasonable source of middle T antigen is tissue from nude mice carrying tumors expressing middle T antigen as a cDNA from the metallothionein promoter (11). Coomassie blue staining showed that the tumor tissue was less rich in middle T antigen and the extent of purification was much lower.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of middle T antigen expressed by using an adenovirus expression system

Schaffhausen

Bockus

Berkner

et al. 1987

J Virol

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The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60jCSrC. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60C src remained unassociated. Immunoaffinity purification resulted in approximately 100 ,ug of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74-and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.

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Section: Resultsmentioning

confidence: 99%

“…Rabbit serum was prepared from animals immunized with small t antigen (12a). Finally, monoclonal antibody pAB 815 directed against middle T antigen was used to prepare an affinity gel by being coupled to Affigel 10 (Biorad) as described previously (11). The * Corresponding author.…”

Section: Methodsmentioning

confidence: 99%

Characterization of middle T antigen expressed by using an adenovirus expression system

Schaffhausen

Bockus

Berkner

et al. 1987

J Virol

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“…Treatment of 14-2 cells with 1 ,M cadmium sulfate for 7 hr to induce the metallothionein promoter resulted in a 3-fold stimulation of immunoprecipitable PtdIns kinase activity (Fig. 2B, lanes 1 and 2), tyrosine kinase activity (data not shown), and MTAg (18).…”

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confidence: 83%

“…We used 14-2 cells, an NIH 3T3 cell line transformed with a MTAg cDNA expressed from a metallothionein promoter (18). 14-2 cells produce approximately the same amount of MTAg as do polyoma virus-infected cells and are morphologically transformed, as assayed by their ability to form foci and grow in soft agar (18). Immunoprecipitates of 14-2 cells using rabbit anti-T serum had 20 to 50-fold higher PtdIns kinase activity than parallel immunoprecipitates from nontransformed NIH 3T3 cells ( Fig.…”

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confidence: 84%

Phosphatidylinositol metabolism and polyoma-mediated transformation.

Kaplan¹,

Whitman²,

Schaffhausen³

et al. 1986

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

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The effect of polyoma middle-sized tumor antigen (MTAg) on phosphatidylinositol metabolism has been characterized in vivo and in vitro using polyoma-transformed and polyoma-infected cells. Cells infected with transformationcompetent polyoma virus exhibit increased levels of inositol phospholipids and the second messenger inositol trisphosphate. MTAg or pp6Ocsrc Immunoprecipitates from MTAg-transformed cells contain an activity that phosphorylates phosphatidylinositol and phosphatidylinositol 4-phosphate. This activity is induced in parallel with MTAg when the MTAg synthesis is regulated by hormonal or heavy metal inducers. Immunoprecipitates from one class of polyoma mutants defective in transformation have a reduced level of associated phosphatidylinositol kinase activity in vitro yet are capable of tyrosine phosphorylation on exogenous protein substrates at rates comparable to wild-type virus. Thus, for these mutants, phosphatidylinositol kinase activity is more tightly correlated with transformation than is protein kinase activity. These results suggest that alterations in phosphatidylinositol metabolism by MTAg play a role in transformation by polyoma virus.A possible mechanism for generating some of the phenotypic changes associated with transformation may involve altered phosphorylation and turnover of the membrane phospholipid phosphatidylinositol (PtdIns). Experiments with pp60v-src and pp68v-rs, the transforming gene products of the Rous sarcoma virus and UR2 viruses, respectively, suggest that PtdIns phosphorylation is regulated by viral oncogene products. These studies have identified PtdIns kinase activities associated with pp6Ovsrc and pp6ov-ros proteins and increased PtdIns turnover in vivo in cells transformed with these oncogenes (1, 2). Increased turnover of PtdIns and its phosphorylated derivatives, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bis(phosphate) (PtdInsP2), has been implicated in cellular responses to a wide variety of stimuli, including response to some growth factors (3, 4). PtdInsP2 breakdown generates two second messengers, diacylglycerol and inositol tris(phosphate) (InsP3). Diacylglycerol activates the calcium-and phospholipid-dependent protein kinase, which is the major cellular receptor for tumor promoting phorbol esters (5). InsP3 mobilizes calcium from internal stores (6).The roles oftyrosine and PtdIns phosphorylation in cellular growth regulation are relevant to the mechanism of transformation by the DNA tumor virus polyoma. Central to polyoma viral-mediated transformation is the activity of one of the polyoma virus early gene products, the middle-sized tumor antigen (MTAg) (7,8). Mutations that alter MTAg but do not affect the other viral proteins can either abolish or drastically diminish the ability of the virus to transform (7, 9). In established lines, MTAg alone is sufficient to cause transformation (10). MTAg is a membrane-bound protein anchored by a stretch of hydrophobic amino acids at its carboxyl terminus (11,12). A subpopul...

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“…pPyLT-1, pPyMT-1, and pPyST-1 (13) were obtained from R. Kamen (Genetics Institute, Boston, MA). pTR880 (T.M.R., unpublished data), pTR890 (14), and pTR841 (15), which carry cDNAs for LTAg, MTAg, and STAg, respectively, were developed in this laboratory. NIH 3T3 and BALB/3T3 clone A31 cells were obtained from C. Scher (The Children's Hospital, Philadelphia, PA), and the 3T3-F442A mouse preadipocyte cell line was obtained from stocks derived from those described by Green Control retrovirus stocks, lacking T-antigen coding region, contain titers of -106 Neor cfu/ml.…”

Section: Methodsmentioning

confidence: 99%

Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation.

Cherington¹,

Morgan²,

Spiegelman³

et al. 1986

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

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We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)l] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.The role of the individual polyoma tumor (T) antigens in tumorigenesis and in the polyoma virus replication cycle is currently the subject of intense study. Large T antigen (LTAg) is a nuclear protein with origin-specific DNA binding properties, and it is essential for viral DNA replication (1). In addition, this protein also negatively regulates early viral transcription (1) and is both necessary and sufficient to immortalize primary rodent fibroblasts (2). Middle-sized T antigen (MTAg) is sufficient to transform immortalized cell lines to form foci in monolayer cultures and colonies in soft agar and to become tumorigenic (3). MTAg immunoprecipitates contain tyrosine kinase activity (4), and recent results indicate that the tyrosine kinase activity in MTAg immunoprecipitates is pp60'csrc (5). An activity in MTAg immunoprecipitates will also phosphorylate the membrane phospholipid phosphatidylinositol (6), leading to the generation of two potent second messengers, diacylglycerol and inositol trisphosphate, which are important signals in normal cellular regulation (7) and may be important in MTAg transformation (8). Unlike LTAg and MTAg, small T antigen (STAg) has no known intrinsic or associated biochemical activity. Bastin and coworkers (9) have shown, however, that either STAg or LTAg must complement MTAg to cause tumor formation in newborn rats. In addition, Benj...

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Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

Cited by 8 publications

References 32 publications

Characterization of middle T antigen expressed by using an adenovirus expression system

Characterization of middle T antigen expressed by using an adenovirus expression system

Phosphatidylinositol metabolism and polyoma-mediated transformation.

Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation.

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