Phenotypic and Karyotypic Changes Induced in Cultured Rat Hepatic Epithelial Cells That Express the "Oval" Cell Phenotype by Exposure to N-Methyl-N'-nitro-N-nitrosoguanidine MING-SOUND TSAO, MD, JOE W. GRISHAM, MD, KAREN G. NELSON, PhD, and JUDITH D. SMITH, BSc A diploid population of cultured rat hepatic epithelial cells that expresses the "oval" cell phenotype was exposed briefly and repetitively to N-methyl-N'-nitro-N-nitroso- guanidine (MNNG), and the effect on more than 20 phenotypic properties was evaluated during the neoplas- tic transformation of the population. MNNG treatments of this hepatic epithelial cell population resulted in a pro- gressively increasing phenotypic alteration and hetero- geneity including changes in specific activities of several cellular enzymes and expression of isozymes, synthetic functions, and various in vitro growth properties. Changes in phenotypic expression were clustered episod- PRODUCTION of hepatocellular carcinoma in rats is a prolonged process characterized by the reproducible occurrence of discrete populations of liver cells that ex- press altered phenotypic properties. 1-3 Included among the populations of phenotypically altered cells that ap- pear in the liver during chemically induced carcinogen- esis are "oval" cells4 and foci and nodules of altered hepatocytes. It has been hypothesized that one or more of these new populations is the precursor to hepatocel- lular carcinoma"5 and represents preneoplastically al- tered cells progressing to cancer. Although this provoc- ative hypothesis has stimulated many studies, it has not been possible to establish unequivocally that any of these altered populations are precursors of hepatocel- lular cancer. A major handicap to the tracing of cell lineages leading to hepatocellular carcinoma has been the inability to analyze clonally the in vivo process. Al- though various types of cells can be isolated from livers of chemically treated rats,6-8 and some of these isolated cells can be transplanted into recipient animals, where From the Department of Pathology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina ically and were associated with major karyotypic changes. The development of increasing phenotypic het- erogeneity preceding and accompanying tumorigenic- ity in cultured liver epithelial cells in vitro and the specific phenotypes that occur resemble superficially the pattern of phenotypic changes that occur in hepatocytes during chemical hepatocarcinogenesis in vivo. The results of this study provide the basis for future investigations to fur- ther elucidate the mechanistic and linkage relationship between specific pretumorigenic and paratumorigenic phenotypes and tumorigenicity. (Am J Pathol 1985, 118:306-315) they grow to form lesions,"` a lesion cannot be identified as a clone arising from a single transplanted cell. Several hundred thousand to several million genet- ically heterogeneous cells must be transplanted to yield a graft of liver tissue, and this situation prevents a clonally based tracing of the process of neoplastic progression in the chemically treated rat liver in vivo. It appears possible that the clonal analysis of the de- velopment of liver cancer may be accomplished with the use of cultured liver cells in vitro. Although mature hepatocytes cannot yet be continuously propagated in Supported by NIH Grant CA29323. Dr. Tsao is Centen- nial Fellow of the Medical Research Council of Canada. Dr. Nelson was supported by Postdoctoral Research Service Award, ES07017. Accepted for publication September 19, 1984. Address reprint requests to J. W. Grisham, MD, Profes- sor and Chair, Department of Pathology 228H, University of North Carolina, Chapel Hill, NC 27514. 306