Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

Abstract

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [3S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

Homologous SV40 RNA trans-splicing

“…A dependence of 100 kD sT-ag formation on an active origin of replication was evidenced previously. 33 As to the SV40 virus, which is not being considered a human tumor virus so far, one cannot exclude that spontaneously occurring mutations triggering the formation of the 100 kD sT-ag or 1-3)/cellular RNa (lanes 4-6) of strongly (clones 2 and 7) and weakly (clone 5) expressing rat 2 cell clones. RT-PcR using primers g and w detected in all clones a 462 nt band resulting from the spliced T-ag mRNa as well as a 609 nt band related to the sT-ag mRNa, which was distinct in clones 2 and 7 and faint in clone 5.…”

“…28 They speculated that a part of the T-ag might be duplicated and the mRNA coding for that protein might be the product of abnormal splicing of an RNA transcribed from genomic tandem integration. The 100 kD sT-ag described by Chen and colleagues 33,34 in transformed mouse cells was found to be non-lytic, occurred independent of t-ag expression, was strongly associated with independence of anchorage requirement, was generated in cells transformed by molecularly cloned wild-type SV40 DNA, and required an intact SV40 origin of replication. The coding sequence of that 100 kD sT-ag was determined to include two separate partial repeats of the SV40 genome and the protein was speculated being generated from the spliced primary transcript.…”

“…sT-ags of 100 kD have been reported in SV40 transformed cells for more than 30 years. 27,28,33,34 Smith and colleagues describe a 100 kD sT-ag in transformed cells from rat and mouse, which exhibited a 35 S-methionine fingerprint virtually identical to that of normal T-ag with neither additional nor missing methionine tryptic peptides. 28 They speculated that a part of the T-ag might be duplicated and the mRNA coding for that protein might be the product of abnormal splicing of an RNA transcribed from genomic tandem integration.…”

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

“…All three of these revertants were previously found to express T-antigens (both 96,000 and 17,000 molecular weight) and to yield wild-type SV40 upon fusion with monkey cells (3,35). They have regained some but not necessarily all growth properties of Swiss 3T3 cells (Table 1).…”

“…MCDB 402 medium was prepared as described previously (26) from double glass-distilled water (2) with the following modifications for use with 10% CO, incubators: 4.6 g of NaCI per liter, 3.0 g of NaHCO3 per liter, and 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). The final concentrations of three components were changed to the following: biotin, 100 ng/ml; vitamin B12, 3 x 10-7 M: and sodium silicate, 10-6 M. We also added either 100 ,ug of streptomycin per ml and 100 U of penicillin per ml or 50 ,ug of gentamicin per ml.…”