Analysis of the Biology of Initiation Using the Resistant Hepatocyte Model

Abstract

Critical to the design of models for the analysis of the hepatocellular lineage of carcinogenesis was the demonstration in vivo that key populations of hepatocytes arising during hepatocarcinogenesis showed a relative resistance to a variety of hepatotoxins^1–3. It was then possible to show that in a cytotoxic environment, these resistant (R⁺) hepatocytes could be very selectively stimulated to grow while the surrounding hepatocytes did not^4–11. A more direct visualization of R⁺ hepatocytes prior to such clonal expansion was not possible until a marker for R⁺ hepatocytes in nodules was identified, namely, the glutathione S-transferase P-form or GST-P^12–15. GST-P positive (GST-P) single hepatocytes were first shown by Moore et al.¹⁴ in livers with nodules. We found singles, doublets and groups of GST-P⁺ hepatocytes in vivo within days to weeks after initiation with diethylnitrosamine (DEN) alone, prior to selection or promotion¹⁵. Using the resistant hepatocyte model^4–11, we found GST-P⁺ nodules of significant size within several days after the start of resistance-selection suggesting that the nodules did not arise de novo from single cells and that GST-P⁺ groups of hepatocytes are precursors of nodules¹⁵. A design for more direct visualization of hepatocellular lineage was based on studies which quite conclusively showed that for hepatocytes to become initiated they must undergo cell proliferation^16–20. We then demonstrated that hepatocytes proliferating in response to DEN-induced necrosis could incorporate bromodeoxyuridine (BUDR) and be seen in tissue sections using antibodies to BUDR and immunohistochemistry¹⁵. This paper reveals results of studies to look for putative initiated hepatocytes, namely, resistant (R⁺), GST-P⁺ and proliferating (BUDR⁺).