COMPLEMENTARY TUMOR-INDUCTION IN NEURAL GRAFTS EXPOSED TO N-ETHYL-N-NITROSOUREA AND AN ACTIVATED MYC GENE

Using a combination of transplacental carcinogen exposure and retrovir us-mediated oncogene transfer into fetal brain transplants, we have st udied complementary transformation by N-ethyl-N-nitrosourea (NEU) and the v-myc oncogene in the nervous system. Previous experiments had dem onstrated that both agents will not induce tumors independently wherea s simultaneous expression of v-H-ras and v-gag/myc exerted a powerful transforming potential in neural grafts. In order to identify other ge netic alterations that co-operate with an activated myc gene, the neur otropic carcinogen NEU was used to generate mutations of cellular gene s. On embryonic day 14 (ED14), pregnant donor animals (F344 rats) rece ived a single i.v. dose of NEU (50 mg/kg). Twenty-four hours later (ED 15), the fetal brains were removed, triturated and incubated with a re troviral vector carrying the v-gag/myc oncogene. Subsequently, these p rimary cell suspensions were transplanted stereotactically into the ca udate-putamen of syngenic adult recipients. After latency periods of 3 -6 months, 5 of 10 recipients harboring ED15 fetal brain transplants d eveloped malignant, poorly differentiated neuroectodermal tumors in th e grafts. No tumor development was observed in seven recipients harbor ing ED16 neural grafts. Cell lines were established from three tumors and the 110 kd gag/myc fusion protein encoded by the retroviral constr uct was identified in the tumors by Western blotting. Several candidat e genes for mutational activation by NEU including the H-ras, K-ras an d neu oncogenes were analyzed for specific point mutations by polymera se chain reaction (PCR) and direct DNA sequencing of the PCR products. However, no mutations were found in any of these genes. These finding s lend further support to the multistep hypothesis of neoplastic trans formation in the brain. The tumors induced in this model provide an in teresting tool for the identification of genes that co-operate with an activated myc gene in neurocarcinogenesis.