Hamster-Embryo-Cell Transformation in vitro

Cell culture provides a potentially useful system for studying carcinogenesis, but available systems of transformation in vitro have limitations. Permanent, non-tumorigenic mouse-embryo cell lines have been widely used, but were shown to form tumours when grown on solid supports and implanted into mice (Boone, 1976), and so may represent a pre-neoplastic rather than a normal state. The use of early-passage cultures circumvents doubts about normality of the parent cells, but with mouse cells the problem of spontaneous transformation arises (Aaronson & Todaro, 1968). SV40-Virus transformation of human fibroblasts has been accomplished (Potter & Potter, 1975), but X-rayinduced (M. M. Clynes & E. J. Duke, unpublished work) or chemical (Parshad et al., 1973; Rhim et al., 1975) transformation of normal human cells has not been observed, except with fibroblasts from von Recklinghausen’s-syndrome patients (Igel et al., 1975). An apparently satisfactory system was reported by Jones et al. (1972), namely the transformation of secondary hamsterembryo cells by cytosine arabinoside, hydroxyurea and 5-fluorodeoxyuridine, involving morphological transformation correlated with tumorigenicity in vivo within 1-3 weeks. The untreated control cultures died out within about 30 days or eight passages, providing an objective marker in vitro of malignancy. We decided to examine this system for possible use in experiments on inhibition of transformation in vitro (Clynes & Duke, 1976). Pilot experiments indicated the necessity, in the absence of a CO, incubator, of buffering the medium with 20m~-Hepes*, to prevent loss of the cultures during a crisis period of poor growth (usually in the first 3-5 weeks of culture). Provided that trypsin treatment was not attempted during this period, we experienced no difficulty in establishing permanent cell lines from both carcinogen-treated and control cultures. Two experiments with separate Syrian hamster-embryo-cell pools were performed. In the first experiment, six control cultures were maintained successfully for 90 days (12 subcultures), and in the second experiment four control cultures were maintained successfully for 120 days (22 passages for two of the cultures, 18 passages for the others). All lines were growing well and apparently established as permanent lines when the experiments were terminated. This represents a discrepancy between our results and those of Jones et al. (1972), Heidelberger et al. (1973) and Berwald & Sachs (1965). Other workers, however, including Sanford et al. (1974), Kuroki et al. (1975) and Levy et al. (1976), have readily established permanent cell lines from untreated hamster embryo cells. For chemical transformation, replicate cultures (at least three for each concentration) were treated with cytosine arabinoside hydrochloride ( 1 0 p ~ , 1 O O ~ M , 1 mM), hydroxyurea (1OmM) or 5-fluorodeoxyuridine ( 2 p ~ ) for 24h as described by Jones et al. (1972), or with benzo[a]pyrene (IOpg/ml) for 79h (Kouri et al., 1975). In the second experiment, different protocols of frequent or infrequent trypsin digestion after carcinogen treatment were compared, in view of the importance of cell division in fixation of the transformed state (Kakunaga, 1975). Initial cytotoxicity was observed, but no ‘criss-crossing’ colonies of transformed cells appeared. Both treated and control cultures were tested (79 days and ten passages after treatment in the first experiment, 60 days and eight or twelve passages in the second) for ability to grow in 0.33 % agar medium (MacPherson, 1973), a property which correlates well with tumorigenicity of cultured hamster cells (Kirkland, 1976). No colony formation was observed with any of the hamster cell lines or with Balb/3T3 mouse cells; colonies were formed by 3T6 (spontaneously transformed) mouse cells. Thus we did not find the apparently simple system of Jones et al. (1972) repeatable, in terms of lifespan of untreated cells in vitro, morphological transformation and tumorigenicity (judged by us in terms of growth in agar). There is also disagreement among