Three parameters were evaluated as diagnostic of the malignant potential of cultured rat liver epithelial cells: cytology, growth in soft agar, and production of extracellular plasminogen activator. A total of 22 tumorigenic and nontumorigenic cultures from 15 cell lines were sent coded from their originators to two different laboratories for the evaluation of these three parameters. Cytologic diagnosis and growth in soft agar were reliable means of determining the malignant potential of the cultured cells. However, the production of extracellular plasminogen activator showed little correlation with tumorigenicity. Of cytologic properties evaluated, the two that correlated best with malignant potential were increased cytoplasmic basophilia and and increased nuclear:cytoplasmic ratio.

Epithelial-like cells originating from the livers of 10-day and 8-week-old BD rats were established in culture. The cells were treated in vitro for 1 or 4 weeks with dimethylnitrosamine (DMN) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although some structural changes were observed in treated cells, it was not possible to score for morphological transformation in vitro. Newborn syngeneic rats were injected with 1.5-2 times 10(6) treated or 1.5-5 times 10(6) control cells at various times up to 38 weeks from the beginning of treatment with the carcinogen. Following the injection of DMN-treated cells, a total of 32 of the 42 injected rat developed tumours, of which 17 were epithelial, 10 carcinosarcomas and 5 fibrosarcomas. Following the injection of the MNNG-treated cells into 61 rats, a total of 30 tumours were observed, including 8 carcinomas, 9 carcinosarcomas and 13 fibroscarcomas. Tumours, mainly of the mesenchymal type, were also observed in rats inoculated with control cells but at a lower frequency. The observation observed in rats inoculated with control cells but at a lower frequency. The observation of mesenchymal tumours is attributed to the presence of a mixed population of epithelial and mesenchymal cells in the orginal culture.

Summary.-Epithelial-like cells originating from 10-day and 8-week old BD rats were treated in vitro with dimethylnitrosamine (DMN) and N-methyl-N'-nitronitrosoguanidine (MNNG). Morphologically, no differences were seen between treated and untreated cells up to the time when the cells were transplanted into syngeneic hosts. However, the treated cells grew in soft agar and once injected subcutaneously or intraperitoneally into newborn rats, developed tumours after a latent period of 9-12 weeks. The tumours obtained with DMN-treated cells were well differentiated adenocarcinomata, whereas carcinosarcomata were observed with the MNNG-treated cells.

Summary.-The pattern of surface proteins of different types of normal and transformed rat liver cells have been studied in culture by means of lactoperoxidasecatalysed iodination procedures, followed by SDS-gel electrophoresis. The cells examined were primary cultures of epithelial liver cells, long-term cultures of epithelial liver cells, in vitro transformed epithelial liver cell lines and liver tumourcell lines; mesenchymal cells from liver and skin were also examined. The principal surface proteins of primary cultures of epithelial cells from adult or neonatal rats had components with mol. wts of 140,000-160,000, 100,000 and 40,000-70,000. A band that had the same position as fibronectin from mesenchymal cells was also present and this band, as well as other iodinated components, were less sensitive to trypsin than fibroblastic fibronectin. A similar pattern of iodinated proteins was seen in long-term cultures of epithelial liver cells, with a great reduction in the number and intensity of the bands in the mol. wt region below 100,000. Almost all the in vitro transformed and tumour epithelial cell lines contain a protein with a mol. wt 135,000 as one of the major iodinated bands, and in contrast to the observation in transformed fibroblasts, the fibronectin was retained by most of these transformed cell lines.

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