Spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells.
Journal: 1998/December - American Journal of Pathology
ISSN: 0002-9440
Abstract:
Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.
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Am J Pathol 153(6): 1913-1921

Spontaneous Neoplastic Transformation of WB-F344 Rat Liver Epithelial Cells

From the Curriculum in Toxicology and Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina
Accepted 1998 Sep 12.

Abstract

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.

Abstract

Rat liver epithelial cell lines have been used extensively for investigation of the cellular stages of neoplastic transformation in vitro. Such cell lines have been established in culture from both normal rat livers 1-3 and from the livers of carcinogen-treated rats. 4,5 Previous studies have shown that neoplastic transformation of these cells in vitro can be achieved spontaneously, 6-8 through carcinogen treatment, 2,9 and by transfection with activated oncogenes. 10-13 Neoplastic derivatives of rat liver epithelial cells produce a variety of undifferentiated and differentiated tumors after transplantation to subcutaneous or intraperitoneal sites of nude mice or syngeneic rats, including hepatocellular carcinomas, adenocarcinomas, epidermoid carcinomas, hepatoblastomas, and sarcomas. 14-16 The differentiated types of tumors reflect the spectrum of liver neoplasms that develop in mammalian liver, suggesting that an undifferentiated stem-like cell may represent the origin of various liver tumors in vivo.

The WB-F344 rat liver epithelial cell line has been used to investigate the process of neoplastic transformation in vitro and hepatocarcinogenesis in vivo. 14,17,18 We have shown previously that recovery of spontaneously transformed WB-F344 cells was selectively enhanced from a single parental population when cultures were maintained at confluence with infrequent passaging compared with cultures maintained in exponential growth. 6 In the present study, we initiated multiple independent lineages of low-passage WB-F344 cells and subjected them in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. We reasoned that if the ability to produce tumors resulted from the stochastic accumulation of several spontaneous alterations, a large number of independent cultures established from the WB-F344 parental culture and grown under selection growth conditions would acquire the ability to produce tumors at different times and express different paratumorigenic and tumorigenic phenotypes. However, if a pre-existing heritable alteration, which predisposed the cells to neoplastic transformation, was present in the parental WB-F344 cell line, tumorigenicity would arise in all of the lineages at similar times and/or the tumors would be phenotypically similar.

Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of appearance of tumorigenicity, frequency, and latency of tumor formation and histology of the tumors formed. Although spontaneously transformed WB-F344 cells produced diverse tumor types, individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro was responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, commitment to a specific differentiation program was stable with time in culture and with site of transplantation.

Lineages and selection cycles that were negative for tumor formation are not displayed in this table. There were no differences in tumor formation for individual lineages between male and female host rats (data not shown).

*Selection cycle following which cells were transplanted into syngeneic rats.

Fraction represents number of rats with tumor over total number of rats transplanted with cells.

Mean time to detection and harvest of 1 cm. diameter tumors.

Range of tumor latencies.

Refer to the text for a description of each morphological pattern. Positive growth in soft agar was indicated by the formation of 0.05-mm-diameter colonies by more than 0.1% of the 1.25 × 10 cells plated under standard conditions. The number of population doublings per selection cycle = 1n[(nf)/(ni)]/1n2 where nf is the final number of cells present on the dish at the end of a selection cycle and ni is the initial number of cells seeded at the beginning of the selection cycle. ND, not done.

*Statistically significant differences from the WB control (n = 2).

Positive growth in soft agar was indicated by the formation of colonies by more than 0.1% of the cells plated under standard conditions. The fraction of intraperitoneal tumors represents the number of rats with tumors over total number of rats transplanted with cells. Latency is the mean time to detection and harvest of tumors.

*Statistically significant differences from the WB control (n = 3).

Acknowledgments

We thank Dr. Bob Bagnell and Vicky Madden for the preparation of electron microscopic sections and photographic assistance.

Acknowledgments

Footnotes

Address reprint requests to Dr. Gary J. Smith, Department of Pathology and Laboratory Medicine CB 7525, 414 Brinkhous-Bullitt Building, University of North Carolina, Chapel Hill, North Carolina 27599-7525. E-mail: .ude.cnu.dem@trosllec

Supported by National Institutes of Health grant CA59486 and grants ES07126 (M.J. Hooth) and ES07017 (S.C. Presnell). S.C. Presnell is supported in part by a post-doctoral grant awarded by the American Liver Foundation.

Footnotes

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