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Single-stranded DNA transcripts of rat type-C viruses prepared in the presence of actinomycin D, hybridized specifically to DNA of several rat cell cultures with no obvious qualitative or quantitative differences. Similar products prepared from a pseudo-type sarcoma virus with contributions from rat and mouse type-C viruses hybridized to both rat and mouse cellular DNA, while mouse viral transcripts did not hybridize to rat cell DNA. Viral RNA was detected in all rat cells by means of the rat viral DNA transcripts, with some differences between untreated low-passage cells and sister cultures treated with bromodeoxyuridine or bromodeoxyuridine and methylchol-anthrene. Cells treated with both compounds were previously shown to be transformed and tumorigenic, and these were distinguishable by kinetic analysis from the control cells.

The customary benzene extraction of airborne particulate matter collected in Los Angeles has previously been shown to yield carcinogenic material. The residue from the benzene extraction contains substances soluble in methanol, some of which are organic. This methanol extract and a number of its component fractions have been tested in rodent cell culture systems developed as assay methods for screening carcinogens. In one system a high-passage Fischer rat embryo cell line was used. In the other a low-passage cell line derived from NIH Swiss albino mouse embryos and infected with AKR leukemia virus was used. In both systems the methanol extract showed cell transformation activity approaching that of 3-methylcholanthrene. In the mouse cell system the activities of several fractions were also examined. The major inorganic component, ammonium nitrate, showed no activity. Neither the neutrals alone nor the recombined non-neutral fractions separately showed any activity, although they were active together. The basic materials separated by ion exchange were also active. The methanol extract combined with the conventional benzene extract showed much lower activity than either extract alone.

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.

A short, simple test involving interferon-mediated protection of cells in vitro from cytopathogenic effects produced by vesicular stomatitis virus or Sindbis virus has been developed to help in determining the genus of origin of cells. By using the observed pattern of protection of five cell types by five interferons, cells could be grouped as of primate, rabbit, rat, and mouse origins. The primate grouping resulted from bilateral cross-reactions between the human and monkey systems. An unexpected observation was that African green monkey interferon preparations protect both monkey and rat cells, but not the converse. Chromosomal analysis of the cell cultures confirmed the genus determined by the interferon test.

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.

CELLS derived from several animal species, including mouse1,2, hamster3 and rat4, have been transformed in vitro from the normal to the malignant state by diverse chemical carcinogens. In similar conditions, however, attempts to transform human cells have usually been unsuccessful and as far as we know, such transformation has been reported only once5. This was a case of two cell lines treated with urethane, obtained from siblings with von Recklinghausen's disease, a familial disorder transmitted by an autosomal dominant gene, and characterised by multiple fibromas with a high predisposition to malignant transformation in vivo6. Although morphologically altered foci of transformed cells were reported, there was the possibility that a few tumour cells in the original population had been selected for by urethane. Therefore we characterised in detail the urethane-treated and untreated cultures. We have found that human cells can indeed be chemically transformed in vitro.

RAT embryo cells infected with either CF-1 or Rauscher C-type RNA murine leukaemia virus, when treated with diethylnitrosamine (DENA), undergo morphological transformation and become aneuploid1. Untreated cells and cells treated with either virus or chemical alone do not transform. We describe here a similar effect of 3-methylcholanthrene (3 MC) on rat cells infected with Rauscher leukaemia virus.

Low passage Fischer rat embryo cultures, which are normally very resistant to transformation by 3-methylcholanthrene but are highly susceptible when chronically infected with the Rauscher murine leukemia virus, were completely protected from transformation by methylcholanthrene when treated with neutralizing antibody specific for the leukemia virus prior to and during treatment with methylcholanthrene. Sister cultures were not protected by neutralizing antibody specific for the B-tropic radiation leukemia virus. This demonstrates clearly a definite type specific role for Rauscher murine leukemia virus in the 2-methylcholanthrene transformation system in rat cells.

Eight food dyes or commercial color mixtures certified for use in the United States were tested for their ability to transform in vitro a serial line of Fischer rat embryo cells previously reported to be a sensitive indicator of chemicals having carcinogenic potential. Malignant cell transformation was induced by a commercial mixture (G2024) of two of these dyes (Blue 1 and Yellow 5) and by Blue 2, Green 3 (one of two experiments) and Red 4. Food dyes Blue 1, Red 3, Yellow 5 and Yellow 6 did not induce cell transformation. One to 1.5 mg of each dye was injected into suckling LVG or Graffi hamsters which were monitored for tumor induction and/or death over a 330-day period. None of the non-transforming dyes (Blue 1, Red 3, Yellow 5, Yellow 6) or Green 3 induced a significant increase in tumor (mostly lymphoma) incidence or animal mortality. Three of the transforming dyes (Blue 2, Green 2024, Red 4) did increase tumor incidence and/or mortality in at least one strain of hamster. We conclude the the in vitro assay suggested that certain food dyes were carcinogens and that in vivo studies in hamsters supported this interpretation.

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 m m than in 1.8 m m 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 × 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.

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.

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.