Preneoplastic rat liver cells in vitro: slow progression without promoters, hormones, or growth factors

In order to determine early changes in liver cells during carcinogenesis and to compare them with normal or neoplastic hepatocytes, an experimental model was established which allowed enrichment of this population at early stages of carcinogenesis and provided sufficient viable material for biochemical and cytogenetic analysis. This paper describes a method that allows in vitro selection and propagation of hepatocytes after in vivo initiation by alkylating agents, without the use of hormones, growth factors, or promoters which might affect their progression. From 6 different rat livers (5 initiated by continuous diethylnitrosamine feeding, 1 by a single exposure to N-methyl-N-nitrosourea) we established slow-growing lines, each of which had its own typical characteristics of growth behavior, morphology, and chromosome number. One of these lines (CL 38) transformed spontaneously after 8 weeks in primary culture, with an abrupt change to typical tumor cell behavior such as focal growth, anchorage independence, cloning ability in soft agar, and tumorigenicity in nude mice and newborn rats. In none of the other lines (now in culture for 11-15 months) has a similar abrupt change yet been observed, but all of them show a steady, albeit slow progression towards the properties of neoplastic liver cells, together with a reduction in chromosome number.