Graduate Thesis Or Dissertation
 

Production of peptide growth factors by oncogene-transformed balb serum-free mouse embryo cells

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  • DNA from the human squamous carcinoma cell line, SK-MES-1, and the plasmids, pUC EJ6.6 containing H-ras oncogene, pSVc-mycl containing cellular myc gene, pNRSac containing N-ras oncogene, pBR SV containing SV40 virus DNA, pSV2-neu containing neu oncogene and pSV2-neo containing neomycin resistant gene were used to transform Balb serum-free derived mouse embryo (Balb SFME) cells by the calcium phosphate-DNA precipitation technique. Some of the transformed cell lines were tested for growth rate under the serum-free condition without the epidermal growth factor (EGF) and for tumorigenic activity in subcutaneously injected mice. The concentrated conditioned media obtained from the oncogene-transformed cell lines were analyzed by the growth stimulation assay and the EGF-receptor binding competition assay. The myc/H-ras-, the H-ras- and the neu-transformed Balb SFME cells grew in the minus EGF serum-free condition and developed tumorigenic potential; this was not true of the neo/myc-, the neo- and the nontransformed-Balb SFME cells. Concentrated conditioned media from the myc/H-ras- and the H-ras-transformed Balb SFME cells noticeably stimulated the growth of Balb SFME cells and competed for binding to EGFreceptor of Baib SFME cells. However, the neu-transformed Baib SFME cells did not produce detectable amounts of type alpha-transforming growth factor (TGF-alpha), showing no growth stimulation activity and no competition with labeled EGF in binding to EGF-receptor. Conditioned media from the N-ras-, the SV40-, and the SK-MES-1-transformed Balb SFME cells stimulated the growth of Balb SFME cells in the minus EGF serum-free culture, substituting for the EGF-dependence. The ras oncogene-transformed cells produced TGF-alpha, which produces a mitogenic response through binding to the EGF-receptor. However, the neu oncogene-transformed cells failed to produce TGF-alpha, but their receptor-like product autonomously produced a mitogenic signal. The presence of myc oncogene, when cotransformed with ras oncogene, increased the rapid growth ability of the ras oncogene-transformed cells in the minus EGF serum-free culture, developing earlier tumor formation in mice and producing more TGF-alpha into the conditioned media. By itself, the myc oncogene failed to produce either of these effects.
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