Morphological revertants of an avian sarcoma virus-transformed mammalian cell line exhibit tumorigenicity and contain pp60src.

Abstract

The biological and biochemical properties of Rous sarcoma virus-transformed and revertant field vole cells were investigated. Revertant vole cells appear morphologically similar to normal, uninfected cells, yet, like transformed vole cells, they are fully capable of growing in agar suspension and producing tumors in athymic nude mice. These highly tumorigenic, yet morphologically normal appearing, vole cells express viral-specific antigens such as the gag gene product (Pr76) but lack the env gene protein (gp85). Moreover, they contain the sre gene protein, pp6Osrc-These results support the concept of the pleiotropic nature of the src gene product and in addition suggest that pp6Osrc may have multiple mechanisms of action. With this revertant cell system it may be feasible to distinguish between those biochemical functions of the src gene product that are important for tumorigenicity in vivo and those that are related to in -vitro morphological transformation.The ability of Rous sarcoma virus (RSV) to transform avian and mammalian cells in culture and toinduce tumors in animals has been localized to one of the four genes of the RSV genome, designated src (1). Restriction of expression of the RSV genome in infected cells, particularly the src gene, has been examined by comparative molecular studies of transformed cells expressing the src gene product (pp6osrc) and their revertant counterparts that have lost the transformed phenotype and appear to be incapable of expressing this particular genetic sequence (2).For the past several years, we have been studying a mammalian fibroblast cell line (European field vole) that can not only be transformed by RSV but,-in some instances, has reverted morphologically to the normal phenotype (3,4). One of the interesting aspects of this particular cell system is that the reversion phenomenon, unlike that of other transformed mammalian cells, appears not to reflect a consequence of either an alteration in, or the loss of, the src gene sequence or its transcription (3). This suggested that posttkanscriptional changes must contribute to morphological reversion in these transformed vole cells. Moreover, viral-specific RNA, including src RNA, was detected in polyribosomes of revertant vole cells, suggesting that these viral mRNAs are actively translated in these cells. In this communication, we demonstrate that the viral proteins encoded for by these particular viral mRNAs, including pp60src, are present in revertant vole cells. Moreover, we demonstrate that although the presence of pp6osrc in these cells is not correlated with morphological transformation, it is correlated with tumorigenicity, because these normal-appearing vole cells are capable of producing tumors in athymic nude mice.MATERIALS AND METHODS Cells. Normal, uninfected field vole (Microtus agrestis) fibroblasts, established as described (5), were infected by the Schmidt-Ruppin (SR) (subgroup D) strain of RSV. Several transformed cell lines (clones 1, 21, 22) were isolated by cloning in agar. After four passage...

Correlation between phosphorylation of a 34,000-molecular-weight protein, pp60src-associated kinase activity, and tumorigenicity in transformed and revertant vole cells.

“…Our laboratory has been investigating the mechanism of reversion of RSV-transformed European vole (Microtius agrestis) fibroblast cells established in tissue culture (10,19). Recently, two revertant subclones of RSV-transformed cells have been isolated which may shed considerable light not only on the mechanism of reversion of these subclones but also on the mechanism of pp60src-mediated transformation and the specific cellular substrates involved in these events as well (21,22). One of these vole cell lines is a partial revertant, line 866-5RC, which appears normal with respect to its morphology, growth patterns, and levels of actin, cytoskeleton, and fibronectin, but has retained its ability to grow in soft agar and to induce tumors in nude mice (21).…”

“…Recently, two revertant subclones of RSV-transformed cells have been isolated which may shed considerable light not only on the mechanism of reversion of these subclones but also on the mechanism of pp60src-mediated transformation and the specific cellular substrates involved in these events as well (21,22). One of these vole cell lines is a partial revertant, line 866-5RC, which appears normal with respect to its morphology, growth patterns, and levels of actin, cytoskeleton, and fibronectin, but has retained its ability to grow in soft agar and to induce tumors in nude mice (21). Moreover, the amounts of pp60src and its kinase activity in these cells were essentially equivalent to those in transformed vole cells (5).…”

Intercellular communication and the control of growth: XI. Alteration of junctional permeability by thesrc gene in a revertant cell with normal cytoskeleton

“…This clone is a partial revertant that retained the high level of pp60 src kinase activity, the anchorage independence, and the tumorigenicity of the fully transformed cells. But it lost the cytoskeletal alteration: it is normal in respect to cell shape, cytoskeleton organization, and amounts of actin and fibronectin (Lau, Krzyzek, Brugge, Erikson, Schollmeyer & Faras, 1979;Collett, Brugge, Erikson, Lau, Krzyzek & Faras, 1979;Nawrocki, Lau & Faras, 1984). The analytical complement of this system is a subclone that lost 97-98% of the pp60 src kinase activity, the anchorage independence and the tumorigenicity, in addition--a full revertant (Lau, Krzyzek & Faras, 1981).…”

Polyomavirus middle T antigen downregulates junctional cell-to-cell communication.

“…This lack of correlation echoes the situation in transformation by pp6O-sr( (where the morphological changes are more prominent). There, with the aid of a cytoskeleton-mutant cell (19), the actions of pp6Ov-sr( on junctional permeability and morphology were shown to be independent (3).…”