Efficient one-step selection of hepatoma cell variants of a variety of phenotypes by use of aflatoxin B1

We have developed a method to select for rat hepatoma cells that fail to express hepatocyte-specific functions. Well-differentiated cells descended from the H4IIEC3 hepatoma line express aldrin epoxidase (AE) activity, an indicator of the liver-specific forms of cytochromes P450 and, concurrently, are able to activate the procarcinogen aflatoxin B1 (AFB1) into highly toxic metabolites. Thus, differentiated hepatoma cells are highly sensitive to AFB1, while dedifferentiated derivatives, which fail to express AE activity, are resistant. Exposure of differentiated Fao cells to 10 microM AFB1 for 24 h permits the isolation, at a frequency of 5 x 10(-5), of resistant colonies that exhibit strongly reduced AE activity. Strikingly, various morphological types can be observed. In more than 90% of the colonies, cells are morphologically similar to the original differentiated cells and accumulate all liver-specific mRNAs examined in amounts comparable to Fao cells. Moreover, they are able to carry out gluconeogenesis, as judged by their capacity to grow in glucose-free medium. For a minor fraction of colonies, the cells exhibit nonhepatic morphology. These cells fail to express three or more of the liver functions and are not able to proliferate in glucose-free medium. Our results demonstrate that the use of AFB1 constitutes a simple and efficient single-step selective method for obtaining variant hepatoma cells of a wide variety of phenotypes.