Quantitative analysis of tumor initiation in rat liver: role of cell replication and cell death (apoptosis)

The formation and development of initiated cells has been studied at the be ginning of hepatocarcinogenesis. Rats received the genotoxic carcinogen N-n itrosomorpholine (NNM); placental glutathione S-transferase was used as a m arker of initiated cells (G + cells). Single G + cells appeared within 24 h after NNM; their frequency increased steeply for similar to 2 weeks, then decreased and finally remained constant. G + foci consisting of greater tha n or equal to 2 G + cells appeared successively after the single cells. His tological determination of DNA replication and apoptosis revealed that: the formation of single G + cells may not depend on DNA replication of precurs or cells; single G + cells showed considerably lower DNA replication than G - normal hepatocytes; from the 2-cell stage onwards GS foci displayed enhan ced DNA replication and apoptosis, Data from histological sections were tra nsformed into the third dimension by a new stereological method which consi ders the non-spherical shape of many G + lesions. Rates of division and dea th of G + cells and of formation and growth of G + foci were estimated by a stochastic model: initially G + clones appeared at a rate of 12 000 per da y and liver until a maximal number of 176 000 (phase I) was reached; therea fter they declined to 134 000 (phase II); they then remained constant (phas e III). Estimated division rates of G + cells decreased from phase I to pha se III, while the death rate increased in phase II, when every third G + cl one disappeared. As a result, at day 50 after NNM only 0.3% of G + single c ells had formed a clone containing greater than or equal to 5 cells. In con clusion, experimental and computed parameters provide direct evidence that hepatocarcinogenesis evolves clonally and that initiated hepatocytes have a selective proliferation advantage, associated with an enhanced potential t o undergo apoptosis, Thereby, depending on the conditions, initiated clones expand or become extinct, Extinction may lead to reversion of the biologic al effects of initiation.