Carcinogenesis of Murine Astrocytes in Culture

ABSTRACT

Astrocytes play important roles in normal brain development and the physiological processes. In particular, 30% of the brain volume consists of astrocytes, and they are the primary target cell in the brain for cellular injuries from chemical exposures. The present study attempts to establish an immortalized murine astrocyte cell line to study the mechanisms of chemical-induced carcinogenesis of astrocytes. Primary astrocytes isolated from mice were transfected with plasmid carrying the SV40 T antigen. Clonal cells obtained after G418 selection were continuously subcultured to establish an immortalized astrocyte cell line. The cell line was positive on GFAP expression and was sensitive to exposure to such chemicals as MNNG. Cells were treated with MNNG for 5 days, with doses ranging from 0.001ug/ml to 1ug/ml. Dose-dependent cellular transformations of astrocytes were observed. Treatments at 0.01ug/ml showed the most distinct characteristics of neoplastic transformation. Subsequent treatment with TPA produced higher levels of neoplastic cell transformation than MNNG treatment alone, as evidenced by increases of saturation density, soft-agar colony formation and cell aggregation. Promotional effects of TPA on cell transformation was further demonstrated by the shortening duration of foci appearance. Addition of hydrocortisone to the culture media resulted in further promotion of cell transformation in astrocytes treated with MNNG and TPA, suggesting that glucocortocoid also plays a role in the promotion of chemical-induced astrocyte transformation. The present study demonstrates that astrocytes are susceptible to chemical-induced carcinogenicity and subject to mechanisms of multistage carcinogenesis. Analysis of MNNG-transformed astrocytes showed that, while the expression of TGF-beta was decreased, expression of GFAP, IL-1betaand fibronectin were increased. The results suggest that these factors are associated with mechanisms of MNNG-induced astrocyte transformation and may be used as potential candidates for biomarkers representing astrocyte-related tumors and cell toxicities. The study showed scientific evidence that growth factors, cytokine and the extracellular matrix are involved in processes of chemical-induced transformation of astrocytes. In addition, the present work provided an excellent opportunity to develop an immortalized astrocyte cell line that can be used for studying mechanisms of astrocyte-related diseases.