Role of estrogen receptor—related antigen in initiating the growth of human glioma cells

Abstract

Object. The expression of estrogen receptor—related antigen (ER-D5) has been demonstrated in many tumors, including those of the brain, but the actual role of ER-D5 in cell growth is unknown. The authors evaluated the role of ER-D5 in the growth of gliomas in vitro. Methods. Human glioblastoma multiforme (GBM) cell lines A172, T98G, U87MG, and U118MG; rat C6 glioma and 9L gliosarcoma; AS human astrocytoma; GBM in primary culture and tumor tissues; and normal brain tissues were examined for ER-D5 by using immunohistochemical, Western immunoblot, flow cytometry, and enzyme-linked immunosorbent assays. The ER-D5 was detected in all tumor cell types of human origin, but not in rat cell lines and normal brain; the expression of ER-D5 was not related to cell cycle phase. Kinetic analysis of ER-D5 expression in cultured cell lines revealed that an enhanced and sharp accumulation of ER-D5 occurred during the first 24 hours of culture, followed by a sharp fall in the next 24 hours. Gradual decreases of ER-D5 during the subsequent days were demonstrated in all human cell lines, and in primary cultures of GBM. This accumulation pattern of ER-D5 was confirmed on Western blot analysis. The ER-D5 was also detected in cells cultured in serum-free medium. Culture cells were treated with D5 antibody against ER-D5 for 48 hours and the effects were evaluated using a monotetrazolium colorimetric assay; the result revealed that growth of cultured cells was inhibited in a dose-dependent manner, and that addition of a single median inhibitory concentration dose resulted in complete growth inhibition and arrest of cell growth at the G0/G1 phase at 96 hours posttreatment. Conclusions. These findings indicated that synthesis and accumulation of ER-D5 is an essential event in the very early phase of in vitro growth of human gliomas.