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Ras-transduced Diethylnitrosamine-treated Hepatocytes Develop into Cancers of Mixed Phenotype in Vivo¹

Departments of Internal Medicine, and Biochemistry and Molecular Biophysics [Y-Z. L., S. C. K., K. P. P.], and Surgery [W. B., D. G. H., M. W. F.], Washington University School of Medicine, St. Louis, Missouri 63110, and Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63103 [E. M. B.]

The cell of origin of hepatocellular carcinoma (HCC) is controversial. A method for marking cells of different lineages in vivo and then determining their carcinogenic potential should resolve this issue. A retroviral vector expressing activated ras and ß-gal genes (Ras-gal) was transferred into adult rat hepatocytes in vivo, and some animals were treated with diethylnitrosamine (DEN). Bile ductule cells and the putative stem cells of the liver (the oval cells) did not appear to be transduced by this method. At 1 month after transfer, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining was performed on transduced rat livers to determine the blue cluster size. Eight % of the clusters in Ras-gal-transduced, DEN-treated livers contained at least twice as many cells as the largest cluster in Ras-gal-transduced, DEN-untreated rats, demonstrating that they had acquired markedly abnormal growth properties. When the retroviral vector containing ß-gal without ras (Gal-509) was transferred into DEN-treated rats, 2.5% of the cells were present in clusters containing at least twice as many cells as the largest cluster in Gal-509-transduced, DEN-untreated animals. Thus, p21-ras may increase the percentage of cells that acquire mutations in response to DEN, or it may behave synergistically with other mutations to increase the replication rate of cells. Occasional foci in Ras-gal-transduced, DEN-treated rats had extramedullary hematopoiesis. Forty % of the Ras-gal-transduced, DEN-treated rats developed unifocal HCC, mixed HCC/cholangiocarcinoma (CC), or CC at 3–6 months after transduction, suggesting that hepatocytes can develop into HCC or CC if sufficient genetic alterations occur.

¹ This work was supported by NIH Grant R29 DK44593, a Liver Scholar Award from the American Liver Foundation awarded to K. P. P., and a postdoctoral research fellowship from the American Liver Foundation awarded to Y-Z. L.

² To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, 660 South Euclid Avenue, St. Louis, MO 63110. Phone: (314) 362-5188; Fax: (314) 362-7183.

Received 7/27/95. Accepted 10/ 4/95.

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