In Vitro Chemical Mutagenesis and Viral Transformation of a Human Endothelial Cell Strain

Abstract We have established and characterized a diploid cell strain of normal human endothelial cells, RuBa 7E. RuBa 7E cells have an average cloning efficiency of 20% during early passages and undergo approximately 50 doublings in vitro before senescing spontaneously. At confluence, RuBa 7E cells form a homogeneous monolayer of flat polygonal cells. RuBa 7E cells react positively with antibody to human endothelial-specific Factor VIII. The toxic and mutagenic effects of N -methyl- N ′-nitro- N -nitrosoguanidine on RuBa 7E cells were studied and are similar to those reported for diploid human fibroblasts. Mutant cells lacking hypoxanthine-guanine phosphoribosyltransferase were selected by their resistance to 6-thioguanine. The spontaneous incidence of mutants was ≤6 × 10 -6 , and the induced incidence was 4.4 × 10 -4 at a survival frequency of 0.05. All 17 mutants that were tested lacked detectable hypoxanthine-guanine phosphoribosyltransferase activity, and none grew in medium containing azaserine and hypoxanthine. Autoradiography showed that mutant cells incorporated radioactive adenine but did not incorporate radioactive hypoxanthine. Unlike human fibroblasts, in which the recovery of 6-thioguanine-resistant mutants is reduced by contact feeding when the inoculum size during selection is increased above 10 4 cells per P60 dish, 5 to 10 × 10 4 RuBa 7E cells can be plated per P60 dish without reducing mutant recovery. This apparent lack of plating density suppression of mutant recovery makes RuBa 7E cells a comparatively compact and economical system for quantifying mutagenesis in diploid human cells. In order to determine whether RuBa 7E cells would undergo a distinct morphological transformation toward cancer in vitro , we infected them with SV40. As early as 14 days postinfection, discrete foci of morphologically transformed, mitotically active cells were seen against a monolayer background of normal cells when cultures were maintained in medium with low serum. Seven of the 33 foci which were obtained were studied for SV40-specific viral T-antigen, and all were positive. The facility with which RuBa 7E cells can be mutagenized and the ease with which morphological transformants can be identified make these cells potentially useful for studies comparing the mutagenic and transforming effects of chemicals and other agents on diploid human cells.