Chemically and virally transformed cells able to grow without anchorage in serum‐free medium: Evidence for an autocrine growth factor
Authors: Xin Li Wen , Jullien Pierre , Lawrence David A. , Pironin Martine , Vigier Philippe 
Published: February 2005

BA 10‐IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7‐methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serumfree (S−) synthetic medium, without insulin and transferrin, and form anchorage‐independent (AI) colonies in this same, but semi‐solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA 10‐IR cells; but HEF do not respond, in S− medium, to their factor, or that of BA10‐IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3‐Ki cells) also form AI colonies in semi‐solid S− medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10‐IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin.

BA10‐IR cells and FR3T3‐Ki cells also release β‐TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable β‐TGF. However, the factors shed by BA10‐IR cells or HEF which stimulate AI growth of BA10‐IR and FR3T3‐Ki cells are proteins which seem unrelated to known transforming growth factors.

Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi‐solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980).

The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage‐independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum. The action of serum is presumably the result of the cooperation between various serum mitogens, such as PDGF and EGF (Heldin and Westermark, 1984), and growth factors which synergize with them, such as insulin and transferrin, which are required for the growth of most cells (cf., Barnes and Sato, 1980).

In a number of cases, the capacity to form AI colonies has been related to the secretion by the transformed cells of proteins, operationally called transforming growth factors (or TGF), which reversibly confer the capacity to grow without anchorage in serum‐containing medium and also other parameters of transformation to certain established, but untransformed, fibroblastic cells (Roberts et al., 1983; Brown and Blakeley, 1984; Lawrence, 1985). These TGFs fall into at least two distinct classes:(1)α‐TGF, which binds to the EFG receptors and is structurally and functionally related to EFG; and (2) β‐TGF, which is unrelated to EFG and acts synergistically with α‐TGF or EGF to promote the Al growth of rat NRK (49F clone) indicator cells (Anzano et al., 1982). However, β‐TGF antagonizes the mitogenic activity of EGF on attached NRK cells and inhibits the Al growth of many neoplastic cells. It can also either stimulate or inhibit this growth, depending on the mitogenic factors in the medium (Roberts et al., 1985). Furthermore, it is found in the culture medium or extracts of both untransformed and transformed cells (Roberts et al., 1983; Lawrence, 1985), and is shed by these cells in a high molecular weight latent form, activated by acidification (Lawrence et al., 1984; Lawrence, Pircher, and Jullien, 1985; Pircher et al., 1984; Krycève‐Martinerie et al., 1985).

The frequent correlation observed between anchorage‐independence, reduced serum‐dependence, and TGF production has led to the suggestion that transformed cells displaying these phenotypic characters may be autocrine systems stimulated by their TGFs (Sporn and Todaro, 1980; Sporn and Roberts, 1985). In line with this model, it has been shown that the Al growth of virally or chemically transformed cells, or explanted cancer cells, was stimulated by α‐TGF (De Larco and Todaro, 1978; Ozanne, Fulton, and Kaplan, 1980; Todaro, Fryling, and De Larco, 1980), or β‐ or unclassified TGF (Krycève‐Martinerie et al., 1982; Kaplan and Ozanne, 1982; Tucker et al., 1983) shed by these cells. The TGFs (or accompanying factors) shed by some of these cells were also shown to stimulate their growth, in monolayer culture, in serum‐free (S−) medium containing insulin and/or transferrin (Kaplan, Anderson, and Ozanne, 1982; Kaplan and Ozanne, 1982). McClure (1983) further observed that mouse BALB/c3T3 cells transformed by simian virus 40 were able to form Al colonies in semi‐solid S− medium containing insulin and transferrin, in response to an autocrine growth factor which was not characterized. More recently, Sauvaigo et al. (1986) observed the autonomous proliferation of a human melanoma cell line and a metastatic variant of this line in S− medium without additives, also in response to an uncharacterized autocrine factor.

We report here data obtained in the study of a line of chemically tranformed hamster embryo fibroblasts (HEF), called BA 10‐IR (Leavy et al., 1976), which grow in S− medium without insulin and transferrin, both as attached cells and unattached aggregates (spheroids). These cells form Al colonies in semi‐solid S− medium without additives, although this capacity is enhanced by insulin and transferrin. This phenotype is accompained by the secretion of an autocrine factor which stimulates Al growth of BA10‐IR cells, in S− medium, and is potentiated by insulin and transferrin. Untransformed HEF and rat and mouse embryo fibroblasts secrete factors which display a similar activity on BA10‐IR cells, but not on themselves. We have further observed that rat FR3T3 fibroblasts transformed by Kristen murine sarcoma virus (Ki‐MSV) also form Al colonies in semi‐solid S− medium, shed an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10‐IR cells. But their colning efficiency without additives is much lower. BA10‐IR cells and Ki‐MSV‐transformed FR3T3 cells also secrete active as well as latent β‐TGF activated by acidification, and HEF latent, activable β‐TGF. However, the factors stimulating Al growth in S− medium appear to be unrelated to known TGFs.