A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3

A31–714, a subclone isolated from clone A31 of the BALB/3T3 line showed a high degree of contact inhibition and an extremely low incidence of spontaneous transformation. Treatment of this subclone with 4‐nitroquinoline‐1‐oxide, 3‐methylcholanthrene, benzo(a)pyrene or N‐methyl‐N'‐nitro‐N‐nitrosoguanidine produced foci of multilayered growth on the background of contact‐inhibited monolayer within 2 to 4 weeks. In this system the transformation frequency can be determined quantitatively on either an inoculated cell basis or a surviving cell basis. The transformation frequency increased with the concentration of carcinogens within a certain range, and was affected by the duration of treatment and the cell density at the time of treatment. Treatment with DMSO alone or with the non‐carcinogenic substances, 4‐nitroquinoline, 4‐aminoquinoline‐1‐oxide, phenanthrene and pyrene, did not cause any transformation. The cytotoxic effects of carcinogens were not directly correlated with the transformation frequencies. All transformed cell lines derived from each focus showed characteristics known as the indices of malignant transformation, such as a criss‐cross pattern or piling up of cells, a high saturation density and the ability to grow progressively in soft agar. When the transformed cells were injected into the skin of the back of newborn or adult mice at a dose of 106 cells, fibrosarcomas were produced at the site of injection after 3–6 weeks. Neither untreated A31–714 cells nor morphologically untransformed cells in cultures with foci produced tumors on injection at a dose of 107 cells. In general, transformed cells had a reduced cloning efficiency, and almost the same susceptibility to the cytotoxic effects of carcinogens as untransformed cells, though these properties varied in different lines.