Regulation of metastatic ovarian cell in serum‐free medium by epidermal growth factor and transferrin

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Abstract
A rat ovarian tumorous cell line (OV1N) has been isolated and established in tissue culture whose growth can be controlled by the addition of epidermal growth factor (EGF) and transferrin to serum‐free medium. EGF at 1 ng/ml (1.5 × 10−10M) stimulates growth about 300% in a six‐day incubation assay. A combination of 1 ng/ml EGF plus 1 μg/ml (1.25 × 10−8M) transferrin stimulates growth 1700% above controls in six days. EGF and transferrin at these concentrations completely replaced fetal calf serum (FCS), which maximally stimulates the cells at 2%. At maximal stimulation (either by FCS or EGF and transferrin) a doubling time of from 16–20 hours is obtained. The growth of OV1N cells was also studied in vivo and compared to another rat ovarian cell line, 31A‐F2. Cells from both cell lines were injected intrasplenically into normal, ovariectomized and hypophysectomized female Fischer 344 rats. Animal data have indicated that 31A‐F2 cells behave more like “normal” ovarian cells in that they grew only in ovariectomized hosts. OV1N cells, on the other hand, grew metastatically in all hosts and killed 36% of those individuals.The binding of 125I‐EGF was also studied in OV1N and 31A‐F2 cells. OV1N cells bound about eightfold more 125I‐EGF than did 31A‐F2 cells at 37°C. The dose of 125I‐EGF required to specifically saturate 31A‐F2 sites was 10 ng/ml (1.5 × 10−9M) or half‐maximally at 4 ng/ml (6 × 10−10M). Scatchard plots indicate that 31A‐F2 cells contain roughly fourfold less EGF‐specific sites than OV1N cells. Incubation of either OV1N or 31A‐F2 cells with a 100‐fold excess addition of various proteins plus a maximal or a half‐maximal addition of 125I‐EGF demonstrated that unlabeled EGF was most effective in reducing 125I‐EGF binding to both OV1N and 31A‐F2 cell sites. Insulin, ovarian growth factor, transferrin, or their combinations, had minimal, if any effect on 125I‐EGF binding.