Using a combination of transplacental carcinogen exposure and retrovirus-mediated oncogene transfer into fetal brain transplants, we have studied complementary transformation by N-ethyl-N-nitrosourea (NEU) and the v-myc oncogene in the nervous system. Previous experiments had demonstrated that both agents will not induce tumors independently whereas simultaneous expression of v-H-ras and v-gag/myc exerted a powerful transforming potential in neural grafts. In order to identify other genetic alterations that co-operate with an activated myc gene, the neurotropic carcinogen NEU was used to generate mutations of cellular genes. On embryonic day 14 (ED14), pregnant donor animals (F344 rats) received a single i.v. dose of NEU (50 mg/kg). Twenty-four hours later (ED15), the fetal brains were removed, triturated and incubated with a retroviral vector carrying the v-gag/myc oncogene. Subsequently, these primary cell suspensions were transplanted stereotactically into the caudate-putamen of syngenic adult recipients. After latency periods of 3-6 months, 5 of 10 recipients harboring ED15 fetal brain transplants developed malignant, poorly differentiated neuroectodermal tumors in the grafts. No tumor development was observed in seven recipients harboring ED16 neural grafts. Cell lines were established from three tumors and the 110 kd gag/myc fusion protein encoded by the retroviral construct was identified in the tumors by Western blotting. Several candidate genes for mutational activation by NEU including the H-ras, K-ras and neu oncogenes were analyzed for specific point mutations by polymerase chain reaction (PCR) and direct DNA sequencing of the PCR products. However, no mutations were found in any of these genes. These findings lend further support to the multistep hypothesis of neoplastic transformation in the brain. The tumors induced in this model provide an interesting tool for the identification of genes that co-operate with an activated myc gene in neurocarcinogenesi
