Induction of transformational changes in normal endothelial cells by cultured human astrocytoma cells

Abstract

✓ Endothelial cell proliferation is a significant biological feature of malignant astrocytomas. The ability of the cells of these tumors to elaborate mitogenic angiogenesis factors has been well documented. However, less is known about the transformational effects that neoplastic astrocytes may have on the endothelial cells within malignant astrocytomas. In this study, the hypothesis that humoral factors elaborated by cells derived from malignant astrocytomas induce transformational changes in normal endothelial cells in vitro is investigated. Conditioned medium (CM) was prepared from exponentially growing cultures of a human glioblastoma cell line (UW18) and from two rat brain-tumor cell lines: an anaplastic astrocytoma (R175A) and a glioblastoma with sarcomatous elements (9L). Subconfluent target bovine aortic arch endothelial cells (BAEC's) were exposed for 48 hours to varying concentrations of CM prepared from each of these tumors, and then evaluated for transformational changes. Different molecular weight (MW) fractions of UW18 CM were prepared by molecular ultrafiltration, and each fraction was tested for transforming activity. Transformation endpoints included changes in cellular deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) content and distribution (measured by differential flow cytometry) and changes in de novo DNA synthesis determined by 3H-thymidine incorporation. Significant changes in the amount and distribution of DNA and RNA were observed in the BAEC's treated with UW18 CM compared to untreated BAEC's. At 10% concentrations of UW18 CM, changes in the RNA profile of target BAEC's were evident, and at 30% concentrations of UW18 CM, an irregular bimodal distribution was well established. Patterns of DNA were also altered in a concentration-dependent manner, with significant aneuploidy developing at UW18 CM concentrations of 20%. The DNA synthesis in BAEC's increased with increasing CM concentrations, up to a maximum of about 250% of control values at 30% concentrations of UW18 CM. The transformational changes induced after exposure of BAEC's to CM prepared from R175A and 9L were significantly less than those observed with UW18 CM. Molecular ultrafiltration was used to prepare UW18 CM fractions with MW cutoffs of less than 10 kD, 10 to 30 kD, and greater than 30 kD. Transformational activity was significant only in CM's with an MW of 10 to 30 kD. It is concluded that the UW18 human glioblastoma cell line elaborates a soluble factor, or group of factors, with an MW in the 10- to 30-kD range, capable of inducing transformational alterations in target normal endothelial cells, and that such transformation may account for some of the abnormal endothelial cell changes associated with malignant astrocytomas.