Comparison of soluble RNA methylase capacity in paired neoplastic and nonneoplastic cell lines in vitro.

Cell CUltUreS. Pooled 11to 13-day mouse embryo minces in NCTC 135 medium supplemented with 10% fetal calf serum (Flow Laboratories) were used to initiate the cell cultures. The cultures were maintained in T-15 flasks and grown as mono layers. After the cell line was 90 days old, a substrain was established by supplementing the NCTC 135 medium with 10% horse serum; this resulted in conversion of the normal cells into neoplastic cells (5). After 162 days on horse serum supplement, the cell line began producing tumors from intra ocular implants. After a total of 22 months on horse serum, the line was frozen in liquid nitrogen for four months. It was then thawed and switched back to 10% fetal calf serum supple ment and grown for four months before experiments were done. The original nonneoplastic line was continued on fetal calf serum supplement and served as the nonneoplastic control in the experiments described. The growth rates of the two lines were essentially the same, and the cultures were always split at the same time. Neoplastic conversion in these cells was determined by the production of transplantable tumors in syngeneic animals (14). Cell lines were injected both before and after the experiments reported were carried out to assure their neoplastic and nonneoplastic state. The tumor tissue originated as a single tumor which arose among many animals that had cells injected into the anterior chamber of the eye to monitor for neoplastic conversion. This single tumor was then carried in vivo by intramuscular injec tion in the thigh of irradiated syngeneic animals. The tumor tissue used in the experiments described was rapidly growing, and sections showed many mitotic figures. Enzyme Extraction and Assay. Cell extracts and incubation solutions were prepared by methods similar to those of Srinivasan and Borek (17). Six to ten T-60 flasks were grown for four to five days, and, while still in logarithmic phase, they were harvested at 37.5°C ±0.1°C by mopping with cellophane strips, and the cells were pelleted by centrifugation at 50 X g at 37°C. The pellet was suspended in one to three ml of ice cold solution (0.25 M sucrose, 0.01 M magnesium chloride) and homogenized for 30 seconds in a motor-driven 10-nil