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This report describes altered cell foci observed 9-14 days after treatment with 3-methylcholanthrene of mouse-embryo tissue cultures that had previously been Mouse-Embryo Cell Lines. Secondary cultures of embryo cells, after inoculation with AKR leukemia virus, became continuously infected carrier cultures that regularly produced complement-fixing antigens characteristic of the murine leukemia-sarcoma virus complex, and yielded infectious virus (3.0-4.0 logs of virus/ml) when tested in the in vitro COMul test (5). Cell-pack preparations of antigen from infected and uninfected cells were made as described (5). Complement fixation was tested by the microtiter technique described for tumor and viral-antigen studies (6). Titers were recorded as reciprocals of the highest dilution giving 3+ to 4+ fixation of 1.8 units of complement.Toxicity Tests. Toxicity tests were done as follows: 1 day after 5 X 104 cells/ml from the 4th subculture were planted in Falcon plastic Petri dishes, the medium was removed and fresh medium containing various concentrations of MCA in 0.5% dimethyl sulfoxide (Me2SO) was added. The control medium contained 0.5% MeSO. The media with MCA were changed twice in 7 days, and then the attached cells were removed with trypsin and counted.Oncogenicity of Transformed Cells. The transformed and untransformed cell lines were tested for their ability to produce tumors in newborn NIH Swiss mice; mice were inoculated subcutaneously with different amounts of cells. RESULTSEffect of MCA on the multiplication of AKR-infected and uninfected mouse-embryo cells 1 day after embryo cells at 4th subculture were plated, at a density of 5 X 104 cells/ml, the medium was removed and replaced with medium containing MCA (0.5 or 1.0 ,g/ml in 0.5% Me2SO). The medium for the control cells contained 0.5% Me2SO. After 7 days of treatment with the chemical carcinogen, two dishes from each group were trypsinized and the cells were counted. The results in Table 1 show that these cells were susceptible to the toxicity of MCA, as indicated by the fact that they grew more slowly than the Me2SO controls. It was also evident that these cells grew well in the presence of of 0.5% Me2SO. In addition, it appeared that AKR-infected embryo cells may be somewhat more susceptible than uninfected embryo cells to the toxicity of MCA; however, this observation requires confirmation.Altered cell foci in MCA-treated, virus-infected cells In the above experiment, after incubation of the virus-infected and uninfected embryo cells with MCA for 7 days, replicate dishes were washed and fed with carcinogen-free medium and then observed daily for morphological alterations. Every 2-3 2212 Abbreviation: MCA, 3-methylcholanthrene.

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