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Monkey kidney cells tested in their first culture passage, 24 hours after their isolation from the animal host, required the same 13 amino acids for survival and growth as cell lines serially propagated in culture for years. Under the conditions of the present experiments, arginine, cystine, glutamine, histidine, and tyrosine proved necessary, over and above the 8 amino acids required for nitrogen balance in man. With the serially propagated lines, glutamic acid substituted for glutamine only at extremely high and non-physiological levels. In the monkey kidney cell cultures, however, glutamic acid and glutamine were interchangeable, mole for mole; and aspartic acid and asparagine were also effective as glutamine substitutes. Glycine was growth-stimulatory for monkey kidney cells in primary culture, and the cells grown in a glycine-deficient medium usually failed to survive subculture.

Fischer rat embryo cells were treated with 3-methylcholanthrene before or after inoculation with Rauscher murine leukemia virus. Transformation was not observed in untreated control cultures, cultures given virus or 3-methyl-cholanthrene alone, or cultures treated first with 3-methylcholanthrene followed by inoculation with the virus after removal of the chemical. Transformation was dependent upon the presence of Rauscher murine leukemia virus at the time of chemical treatment.

Extracts of particulate matter from condensates of city air were tested for their ability to transform rat or hamster cell cultures. Uninfected rat embryo cultures were not transformed, but cultures chronically infected with Rauscher leukemia virus were transformed by benzpyrene or by extracts of city smog. The smog extracts were 600 times more active than pure benzpyrene as transforming agents. Hamster embryo cultures infected with hamster leukemia virus were equally as sensitive as leukemia-infected rat cultures to the transforming effects of smog; uninfected hamster cultures were also transformed, although tenfold higher doses of smog extract were required.

Cell cultures of Syrian hamster embryo were treated for 7 days with selected chemicals. Certain cultures were morphologically transformed by three different chemical preparations and yielded cell lines that subsequently produced malignant tumors in hamsters. Although the cell lines were negative for infectious virus before inoculation into animals, hamster-specific C-type RNA virus was isolated from tumors or from cell lines derived from the tumors. Since infectious C-type viruses are usually not demonstrable in hamster tissues of normal or tumor origin, we conclude that the chemical treatment and activation of the viruses are related events.

Low-passage rat-embryo cells were not transformed by 3-methylcholanthrene or by 5-bromo-2' deoxyuridine. However, prior treatment with bromodeoxyuridine, followed by treatment with methylcholanthrene, resulted in cell transformation about three subpassages after removal of the carcinogen. RNA-directed DNA polymerase activity could not be detected in either normal or transformed cells. However, gs-1 antigen specific for rat C-type RNA virus was detected in cultures derived from bromodeoxyuridine-treated cells. No gs-1 antigen for the C-type RNA virus was detected in cultures that had not been treated with bromodeoxyuridine during the 25 subpassages of these experiments.High-passage rat-embryo cells, derived from a different cell pool, were transformed by either methylcholanthrene or dimethylbenzanthracene without prior infection with an exogenous virus, and without prior treatment with bromodeoxyuridine, gs-1 antigen for C-type RNA virus was also detected in 4 of 4 randomly selected transformed cell lines; the gs-1 antigen was not detected in any of 4 nontransformed control cultures. Considering these and previously published findings, we conclude that the lowpassage cells cannot be transformed by methylcholanthrene because of powerful cellular controls over the endogenous virus. Bromodeoxyuridine triggers some expressions of the endogenous virus; thus, the bromodeoxyuridine-treated cells are more susceptible to the transforming effects of methylcholanthrene. High-passage rat cells do not maintain perfect control over expression of their endogenous virus; the cell cultures are susceptible to the transforming effects of chemical carcinogens.

Adenovirus type 12 (Huie) inoculated into cultures of primary whole rat embryo produced foci of morphologically altered cells. The number and identification of these transformed areas was dependent upon the calcium concentration of the medium; more foci appeared in 0.1 mm than in 1.8 mm calcium. Cell lines derived from these inoculated cultures did not yield infectious virus, and also were similar to cell lines derived from adenovirus type 12-induced tumors with respect to morphology, presence of virus-specific tumor antigen, and oncogenicity. Dose-response curves revealed that transformation of rat embryo cells by adenovirus type 12 followed one-hit kinetics, and that approximately 7 x 10(5) infectious virus particles were required for one transformation event. Our results indicate that the transformation system described for adenovirus type 12 is reproducible, and that previous difficulties experienced in developing such a system may well be explained by the higher calcium concentration of the tissue culture media used.