Analysis of N-Ethyl-N-nitrosourea-Induced Brain Carcinogenesis by Sequential Culturing During the Latent Period. I. Morphology and Tumorigenicity of the Cultured Cells and Their Growth in Agar

Pregnant inbred BD IX rats were given injections of buffer or N-ethyl-N-nitrosourea (ENU) at 40–50 mg/kg on day 15 or 16 of gestation. The average latent period for cerebral tumors was 246 days. Brain cultures were prepared 2, 3, 5, 34–35, 57–60, 90–91, 111–112, and 138–145 days post injection (PI) and examined for morphology, tumorigenicity, and growth in agar. Cultures in the last 3 groups derived from animals exposed transplacentally to ENU contained cells characteristic of brain tumor cultures; these have been called “basal layer cells” (BLC). They were not observed in control cultures. BLC persisted in several cultures belonging to the last 2 groups, and these cultures were tumorigenic and grew in agar. BLC did not persist in cultures prepared 90–91 days PI, which did not produce tumors or grow in agar. Three morphologic types of BLC were observed: “fried eggs,” crescents, and pyramids. These occurred in different proportions in the cultures. Predominance of the pyramidal type was associated with a shorter latency for cell-induced tumors compared with those cultures in which other types of BLC were in the majority. The “fried-egg” type was predominant in cultures in which BLC disappeared on passaging. Cultures prepared at earlier times did not contain BLC and did not grow in agar or in animals at early passages. However, cultures derived 2 days PI became tumorigenic after prolonged culturing, which indicated that they had already acquired the malignant potential.