Characterization of initiated cells in N-methylnitrosourea-induced carcinogenesis of the CNS in the adult rat

Glial tumors may originate from the malignant transformation of multipotent glial progenitor cells, but tools to study malignant transformation leadin g to gliomas are limited by the lack of biological systems that represent e arly stages of this disease in adult animals. In order to characterize the initiated cells that give rise to gliomas, we have employed the N-methylnit rosourea (MNU) model for induction of brain tumors in adult rats (Rushing e t al,, 1998), Specifically, we have isolated and cultured transformed (prem alignant) cells from normal-appearing brains of rats exposed to MNU for 10 weeks and from histologically abnormal brains of rats exposed to MNU for 15 weeks, We compared them with cells cultured from control animals under ide ntical conditions. Cultured cells were classified according to their morpho logy, immunophenotype, karyotype, proliferation capacity, and tumorigenicit y in athymic mice. Cultures from untreated normal rat brains grew as monola yers and had normal karyotypes (42 X,Y), epithelioid morphology, and slow p roliferative capacity (doubling time >120 h). In contrast, cultured cells f rom brains of MNU-exposed animals had karyotypes that ranged from normal to highly aneuploid, Aneuploid lines grew rapidly in multilayers (doubling ti me <24 h), had differentiated astrocytic or oligodendroglial morphology and immunohistochemical staining profile, and yielded tumors in athymic mice. Initiated cells with minor chromosomal aberrations assumed mixed bipolar or tripolar morphologies in high density cultures, proliferated rapidly, but showed contact inhibition and failed to induce tumors when injected s.c. in athymic mice, In general, lines showing no evidence of chromosomal aberrat ions had the most epithelioid morphology, proliferated slowly (doubling tim e >72 h), and retained strict contact growth inhibition. The presumed undif ferentiated glial progenitor cells in culture from either control or MNU-tr eated rats variably expressed markers such as vimentin, nestin, and NG2 pro teoglycan, and they weakly expressed the mature astrocytic or oligodendrogl ial markers glial fibrillary acidic protein or galactocerbroside, respectiv ely. These cultures differentiated to bipolar-tripolar morphology with conc omitant maturation to a GFAP+ or GalC+ phenotype upon exposure to secondary messengers such as dibutyryl-cyclic-AMP and/or growth factors such as basi c fibrillary growth factor. Continuous stimulation with these messengers re sulted in terminal differentiation and consequent death upon withdrawal of the stimulus. These results provide information that could lead to detailed characterization of initiated, premalignant cells in the adult brain and t o a better understanding of glial carcinogenesis.