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Mutational Activation of pp60^c-src Leads to a Tumorigenic Phenotype in a Preneoplastic Syrian Hamster Embryo Cell Line

Department of Cancer Biology, Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709 [T. J. L., B. F. T., T. M. G.]; and Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121 [S. B. K.]

Previous studies indicated that overexpression of wild-type avian c-src cannot induce neoplastic transformation of NIH 3T3 cells. In this study, we isolated and characterized novel spontaneously derived transforming mutants of avian pp60^c-src from a Syrian hamster embryo-derived cell line, 10W, transfected with the avian c-src gene. Seventeen independently derived transfected 10W cell clones were injected into athymic nude mice. After a latency period, tumors eventually arose and were established in culture. The tumorigenic phenotype was always accompanied by the presence of the avian c-src DNA and functional expression of pp60^c-src. However, most of the tumor-derived cell lines expressed an electrophoretically altered form of pp60^c-src, suggesting mutations in src. Consistent with this hypothesis, DNAs isolated from the tumor-derived lines, but not the parental 10W cell lines, morphologically transformed NIH 3T3 cells in a focus-forming assay. We characterized pp60^c-src in detail from three of the tumor-derived lines: 4AT, 4BT, and E2T. Two of these lines contained mutations within the exogenous c-src coding region. Line 4AT has an internal repeat of 29 amino acids immediately following Gln-513, which disrupts the spacing between the end of the kinase domain and Tyr-527, the negative regulatory site in pp60^c-src. Line 4BT has a 5-bp deletion following Phe-520, which results in loss of Tyr-527. However, the DNA sequence of the coding region of pp60^c-src from a third line, E2T, was completely wild type. Cyanogen bromide cleavage analyses of the altered pp60^c-src from lines 4AT and 4BT showed that Tyr-527, the site of negative regulation of c-src, is not phosphorylated, but Tyr-416, the site of in vitro autophosphorylation, is phosphorylated. However, in line E2T, Tyr-527 was phosphorylated, and Tyr-416 was phosphorylated to a lesser extent. Additionally, two proteins that indicate activation of src, p85 cortactin and p120^cas, are phosphorylated in at least six of the tumor-derived cell lines, although to a lesser extent in line E2T. These results suggest that dephosphorylation of Tyr-527 and phosphorylation of Tyr-416 correlate with activation of pp60^c-src in the tumor-derived lines 4AT and 4BT, respectively. However, in line E2T, the high levels of pp60^c-src, in combination with a partial activation of the pp60^c-src protein as indicated by phosphorylation of Tyr-416, appear to be involved in the neoplastic process, rather than mutation.

¹ To whom requests for reprints should be addressed, at RCII, Room 806, Department of Cancer Biology, Glaxo Wellcome Research and Development, Research Triangle Park, NC 27709. Phone: (919) 483-6335; Fax: (919) 315-8597.

Received 1/30/96. Accepted 3/24/97.

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