Primary rat embryo fibroblasts were transformed by a p53 mutant (alanine to valine change at amino acid 135) plus ras. This p53^^'*^^ mutant is temperature sensitive for a conformational change detected by the binding of a monoclonal antibody, PAb246, which recognizes the wild-type protein or the great majority of p53 vail 35 at 32.5°C. At 37°C, both mutant and wild-type p53 conformational forms co-exist in the cells, while at 39.5°C, the majority of the p53^"'^^^ in the cell is in a mutant conformation not recognized by PAb246 antibody. At 39.5°C, the mutant p53 is localized in the cytoplasm of the cell. At 32.5°C, the p53 protein enters the nucleus and stops the growth of these cells. At 37°C where there is a mixture of mutant and wild-type p53, the wild-type p53 protein is in a complex with hsc70 and mutant p53 protein in the cytoplasm of the cell during Gj. This wild-type protein enters the nucleus as the cells enter the S-phase of the cell cycle. At 32.5°C, the cells stop replication and arrest at the G^/S border. After 48 hr at 32.5°C, 91% of the cells are in the Gj fraction of the cell cycle. The S-phase cells appear to be immune to the p53 negative regulation of growth until they enter the next G^ period. These data strongly suggest that mutant p53 proteins in transformed cells act to sequester the wild-type p53 protein in an hsc70-p53 complex, which resides in the cytoplasm during the stage of the cell cycle, G^, when nuclear wild-type p53 would normally act to regulate cell growth and progression through the cycle. In this way, mutant p53 proteins can act in a trans-dominant fashion to overcome growth regulation by the wild-type p53 allele and protein in a cell.

Normal adult rat hepatocytes remained viable and functional for at least 43 days when plated on collagen-coated dishes and fed chemically defined medium supplemented with dimethyl sulfoxide (Me2SO). Hepatocytes isolated by collagenase perfusion and cultured in the presence or absence of Me2SO were (i) examined by light and electron microscopy for morphological changes; (it) analyzed for the production of albumin and other plasma proteins; and (iiM) tested by autoradiography for DNA synthesis. Me2SO-treated cells continued to produce specific plasma proteins during the entire culture period; albumin production was consistently high (11)(12)(13)(14)(15)(16)(17)(18)(19) ,.g/ml of culture medium per 24 hr) from day 2 to at least day 43 after plating. Ultrastructural analyses demonstrated that Me2SO-treated hepatocytes resembled those from intact liver in organization of cytoplasmic organelles and cellular junctions. The optimal concentration for observing the morphological and biochemical effects of Me2SO was 2% (vol/vol). We conclude that supplementation of chemically dermed medium with Me2SO enables maintenance ofdifferentiated hepatocytes in culture for extended periods of time.Dimethyl sulfoxide (Me2SO) is a dipolar aprotic solvent that is active in biological systems (1). Addition of 1-2% (vol/vol) Me2SO to the culture medium of Friend virus-induced murine erythroleukemia (MEL) cells for 4-5 days causes 90% of the cells to express characteristics associated with normal erythroid differentiation, including alterations in morphology (2), induction of a-and P-globin synthesis (3, 4), and loss of the capacity for cell division (5). Me2SO-induced differentiation has also been observed in a human leukemia cell line (6) and in cultured fibrosarcoma (7), neuroblastoma (8), human colon carcinoma (9), human lung cancer (10), and murine embryonal carcinoma (11, 12) cell lines.Past efforts to achieve long-term culture of differentiated normal adult hepatocytes have not been successful. Limited proliferation and maintenance of adult hepatocytes can be achieved by supplementing culture medium with serum from partially hepatectomized animals (13) or by plating hepatocytes on liver extracellular matrix and maintaining them in serum-free hormonally defined medium (14). Proliferation also can be achieved by culturing hepatocytes at low cell density in the presence of insulin and epidermal growth factor (EGF), but maintenance of hepatocyte-specific characteristics requires high density or supplementation with hepatic plasma membrane material (15,16).In the present study, we employed a collagen-coated surface and supplemented the culture medium with Me2SO in an attempt to extend the time in vitro that hepatocytes remain biochemically and morphologically differentiated. The addition of Me2SO had a dramatic effect; hepatocytes retaining morphological and biochemical characteristics of normal liver could be maintained in culture for as long as 43 days. Note that Me2SO, used previously to induce differentiation in tumor cells ...

Abstract. Normal adult rat hepatocytes plated on rat tail collagen-coated dishes and fed a chemically defined medium supplemented with epidermal growth factor and dimethylsulfoxide (DMSO) were examined over a 40-d culture period for (a) the amount of albumin secreted; (b) steady-state albumin mRNA levels; (c) steady-state mRNA levels for six other liver-specific genes and three common genes; and (d) transcription of several liver-specific and common genes using isolated nuclei. DMSO-treated hepatocytes in culture for 40 d expressed albumin mRNA at 45 % the level of normal liver and five other liver-specific genes at levels ranging from 21% to 72% of those in normal liver. The rate of synthesis of ligandin RNA using nuclei from 40-d hepatocytes in a nascent chain extension assay was 130% of the value obtained for normal liver, indicating that liverlike transcriptional activity for ligandin was maintained in this in vitro culture system. In contrast, the rates of synthesis of albumin and phosphoenolpyruvate carboxykinase (PepCK) mRNAs using nuclei from 40-d hepatocytes were 8% and <1%, respectively, and, therefore, were at levels that were much lower than was expected given the steadystate mRNA levels for these two genes. The discrepancy between the steady-state mRNA levels and rates of synthesis of RNA was analyzed, and the results suggest that the albumin and PepCK mRNAs from hepatocytes in culture may be more stable than those from liver. A plateau period for secretion of albumin, expression of albumin, a~-antitrypsin, ligandin, phenylalanine hydroxylase, and PepCK mRNAs, and synthesis of albumin RNA using isolated nuclei was observed from days 6 to 40. The usefulness at a biological and molecular level of a hepatocyte culture system in which liver-specific genes are expressed over a long plateau period is discussed.

Transformation of rat hepatocytes by simian virus 40 in chemically defined medium was examined. When hepatocytes plated on collagen-coated plates were infected with simian virus 40, colonies of replicating cells appeared as early as 40 days after infection, whereas no colonies appeared in control cultures. Medium from 85% of the transformed cultures contained albumin. When collagen was eliminated and hepatocytes were plated on Primaria plastic cell culture dishes, transformation occurred; medium from 86% of the transformed cultures contained albumin but the maximum albumin level secreted per culture was only 62% of that produced by cultures on collagen-coated plates. Quantitative assays for transformation were established. Transformation was linear after infection with 2-50 plaque-forming units of virus per hepatocyte, and the transformation frequency was the same on the two plating surfaces. An immuno-overlay technique made it possible to identify, purify, and determine the morphology of the albumin-producing cells. When ornithine was substituted for arginine in the medium, the transformation frequency decreased markedly while the percentage of colonies producing albumin increased from 30% to 100%. We conclude that we have defined an assay for quantifying transformation of a normal hepatocyte population and for identifying and enumerating epithelial liver cell transformants that produce albumin.