A cell culture line of human small cell (oat cell) carcinoma of the lung (NCI-H69) was studied for growth factor requirements for replication in serum-free medium. By a series of addition experiments, the combination of selenium (3 × 10-8m), hydrocortisone (10-8m), insulin (5 µg/ml), transferrin (100 µg/ml), and 17β-estradiol (10-8m) (SHITE) added to Roswell Park Memorial Institute Medium 1640 without serum was found to allow optimum replication when cells were transferred from serum-containing medium. NCI-H69 cells have replicated continuously in SHITE-supplemented Roswell Park Memorial Institute Medium 1640 for periods of more than 12 months. The cells replicate with approximately the same doubling times as in medium supplemented with 10% fetal calf serum, but they exhibited a lower saturation density and longer lag phase in SHITE-supplemented medium compared to serum-supplemented medium. When individual components of SHITE were deleted, immediate and dramatic differences in growth were seen only with deletion of transferrin while minimal decreases were noted with deletion of the other factors. After 6 months of growth in SHITE, deletion of insulin or transferrin from the formula resulted in cessation of cell growth in serum-free medium while deletion of hydrocortisone or estradiol (alone or together) did not. A variety of other hormones and growth factors did not add significantly to SHITE in promoting replication. In addition, a series of hormones was found to inhibit replication or saturation density achieved in serum-free, hormone-supplemented medium including epidermal growth factor, luteinizing hormone-releasing factor, parathyroid hormone, blood meal, and the tripeptide glycyl-l-histidyl-l-lysine acetate. Six other small cell carcinoma lines were tested and also were able to replicate in SHITE-supplemented serum-free medium indicating the generality of this formula for small cell carcinoma, NCI-H69 cells cultured for 4 months in SHITE medium retained their amine precursor uptake and decarboxylation properties, characteristic small cell carcinoma histology by light microscopy, dense core (neurosecretory) granules by electron microscopy, and tumorigenicity in nude mice. The identification of this growth formula should allow selection of tumor cells directly from patient samples, study of hormone production and regulation by these cell lines, and identification of new growth and therapeutic agents, and it should provide a way of selecting for normal amine handling cells related histogenetically to small cell carcinoma.

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