Characterization of activated proto‐oncogenes in chemically transformed syrian hamster embryo cells

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Abstract
The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto‐oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen‐induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK‐A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact‐inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha‐ras proto‐oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki‐ras, or N‐ras genes on Southern analysis or altered p21ras protein. The transforming gene in this cell line appears to be a non‐ras oncogene. These observations indicate that ∼40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha‐ras oncogenes. The activation of Ha‐ras proto‐oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non‐ras oncogene was detected in the NIH 3T3 focus assay, and ∼60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.