The appearance of γ-glutamyl transpeptidase (GGT) in focal areas of hepatocytes is a widely used histochemical marker for the identification of preneoplastic cell populations. The characterization of these GGT-positive preneoplastic cells in relation to possible alterations in protooncogene expression may help define cellular changes occurring during the early stages of hepatocarcinogenesis. Female Sprague-Dawley rats were subjected to a two-thirds partial hepatectomy, followed 18 h later by a single intragastric administration of 30 mg of diethylnitrosamine per kg and subsequent feeding of a diet containing 0.05% phenobarbital for 6 or 11 mo. Primary cell suspensions were obtained after the perfusion of liver with collagenase. Cell debris and nonviable cells were removed with multiple washes and a Percoll gradient step. GGT-positive hepatocytes were enriched from the cell suspension by adherence to an affinity-purified GGT antibody affixed to Petri dishes. These dishes allowed the selective adherence and collection of up to 2.28 × 106 GGT-positive cells per liver. The starting cell population and the isolated GGT-positive and -negative cells were then used for subsequent analysis. RNA was prepared from the cell isolates and from hepatocellular carcinomas induced with the same diethylnitrosamine and phenobarbital regimen as that used to induce GGT-positive foci; 10 µg of total cellular RNA were used for Northern blot hybridizations. The blots were probed with 32P-labeled c-myc, H-ras, and albumin DNAs. The results indicate that GGT-positive hepatocytes do not differ from the other hepatocyte populations in either the size or amount of mRNA transcripts for the c-myc and H-ras protooncogenes. Increased expression of c-myc and H-ras was observed in some malignant lesions and may represent a secondary alteration occurring during the multistage process of hepatocarcinogenesis.
This work was supported by National Cancer Institute Grants CA-07652, CA-07175, and CA-22484 and by Contract N01-ES-3-5024 from the National Institute of Environmental Health Sciences.