Cell lines with heterogeneous phenotypes result from a single isolation of albumin-sv40 T-antigen transgenic rat hepatocytes

Abstract

Transgenic animals and the cell lines derived from these Several immortalized cell lines were established from transgenic animals have become important tools in the study the livers of two transgenic rats expressing the simian of many diseases. [1][2][3][4][5][6] The construction of transgenic animals virus protein large T antigen under the control of the involves the introduction of a specific gene into the germ line albumin promoter. Hepatocytes from transgenic rats of an animal, resulting in one or more copies of the transgene were isolated by a two-step perfusion procedure from a in every cell of the adult animal. Expression of the gene may normal-appearing liver and from a liver that contained be regulated through the use of tissue-specific promoters, a single neoplasm. Cells were also isolated from the diswhich allow the gene to be expressed in specific tissues and sociated liver neoplasm. After 5 weeks in culture, cell not to be active in other tissues. colonies were isolated and subcloned as individual cellIn our laboratory, we have developed a line of transgenic lines. Electron microscopy revealed that all cell lines rats to study the process of hepatocellular carcinogenesis in had a high nuclear-to-cytoplasmic ratio compared with this species. 7 These animals were produced by microinjection normal hepatocytes. The cytoplasm contained numerinto fertilized rat eggs of the DNA coding sequence for the ous organelles, including smooth and rough endoplassimian virus large T antigen (SV40) under the control of the mic reticulum, Golgi, mitochondria, peroxisomes, and 5 regulatory region of the mouse albumin gene. 8 These aniannulate lamellae. However, the lines exhibited a varimals develop hepatocellular carcinomas, adenocarcinomas, ety of different cell morphologies. All cell lines, including and hepatoblastomas at about 3 to 5 months of age. Focal those derived from neoplastic cells, exhibited a similar lesions and nodules are observed as early as 2 months, with doubling time of 26 hours and the ability to grow in soft metastases observed in some animals later in life. By 6 to 8 agar. Northern blot analysis revealed that the cell lines months of age, the animals either die of the disease or from differentially expressed hepatocyte markers. Large T other pathological abnormalities that can be attributed to the antigen was expressed in the cultured cell lines at much expression of the transgene. 7 higher levels than was observed in transgenic hepatoThis report presents data characterizing a number of cell cytes in vivo. This suggests that the viral protein is relines derived from isolated alb-SV40 T-antigen transgenic rat quired to maintain cell viability in culture. In addition, hepatocytes. It is important to note that, although the cell the tumor suppressor proteins, p53 and Rbp105, were lines were derived from a single population of isolated hepatodetected in all cultured cell lines. In contrast, these same cytes, the established cell lines exhibit very different cellular proteins were not detected by West...

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…simian virus 40 large T antigen, c-myc, cH-ras) (Osanai et al 1997, reviewed by Castell et al 2006), hybrid cells obtained by the fusion of hepatocytes and immortalized cell lines (Widman et al 1979; Cassio et al 1991) or the generation of hepatic cell lines from transgenic animals (reviewed by Castell et al 2006). Immortalized hepatic cell lines have been established from livers of transgenic mouse and rat expressing the SV-40 large T antigen under the control of the hepatic L-pyruvate kinase (Courjault-Gautier et al 1997) or albumin (Bulera et al 1997) promoter, respectively; or from animals over-expressing growth factors (TGF-α or human growth hormone), early growth signals (constitutively active met-protooncogene) or truncated growth suppressor genes (p53-knockout mice) (reviewed by Castell et al 2006). Although using these strategies some hepatic functions were maintained following cell transformation, the cells show a very low expression of CYP enzymes, which, in general, makes them unsuitable for drug metabolism and hepatotoxicity studies (Castell et al 2006; Donato et al 2008b).…”

Selective loss of nucleoside carrier expression in rat hepatocarcinomas

“…Several hepatocyte-related cell lines isolated from albumin-SV40 T-antigen transgenic rats have recently been characterized. 21 L25 cells were isolated from tumor lesions, whereas the L37 cells were derived from normal-appearing liver tissue from the same rat. Both cell lines show similar growth characteristics but differ in their basal level of expression of selected markers of hepatocyte differentiation and transformation.…”

Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis

“…The albumin±SV40 T-Ag Sprague Dawley hepatocyte cell lines L-37, L-60, L-61, and L1-3 were derived from normal tissue, and L-25 was derived from a liver tumor [40]. The ®ve hepatocyte lines were maintained in Dulbecco's modi®ed Eagle's medium/high glucose medium (GIBCO BRC, Grand Island, NY) with 5% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, UT), 5 mg/mL insulin (Collaborative Research, Bedford, MA), 0.1 mM dexamethasone (Elkin Sinn Inc., Cherry Hill, NJ), and penicillin (100 units/ml)/streptomycin (100 mg/ml) (Sigma Chemical Co.).…”

“…Nevertheless, these mutations should be considered rare in our model. Because this cell line was derived from the normal liver of a rat carrying the SV40 T-Ag transgene [40], we attribute the identi®ed aberrations to a clonal expansion in the in vitro establishment of the cell line rather than to a mutation within the primary tumor. The mutations were detected in DNA samples from the original parental L-60 cells derived from T-Ag transgenic liver and persisted during cell passage.…”

“…The mutations were detected in DNA samples from the original parental L-60 cells derived from T-Ag transgenic liver and persisted during cell passage. Although L-60 expresses T-Ag molecules [40], which bind p53 protein, the mutation may produce a functional p53 protein with a selective growth advantage for the cells, as it occurs under certain cell culture conditions [52]. Most p53 mutations occur between residues 102 and 292 in the protein, and they result in a loss of DNA binding [1,52,53].…”