Ellen Borenfreund

Differences of expression of cytoskeletal proteins in cultured rat hepatocytes and hepatoma cells

Abstract Cytoskeletal elements, enriched in intermediate-sized filaments and insoluble in buffers of high salt concentrations and Triton X-100, were isolated from various cultures of rat hepatocytes and hepatoma cells, and their proteins were studied by one- and two-dimensional gel electrophoresis and immunofluorescence microscopy. The cells examined included several permanent cell lines (MH1C1, HTC, hepatoma 72 22 , clone 12 from Gunn rat hepatocytes, and cell clones from normal rat hepatocytes), as well as freshly dissociated hepatocytes that were cultured and allowed to attach to substratum for increasing periods of time, beginning at 24 h after removal of the liver from the animal. Filaments containing vimentin, which were not found in hepatocytes grown in liver tissue, were detected in most of the cultured hepatocytes and hepatoma cells, except in MH1C1 cells, and were shown to be newly synthesized during the first days of primary culture. Maintenance of expression of filaments containing proteins immunologically related to epidermal prekeratin (‘cytokeratins’) was observed in all cells examined but HTC cells. Detailed comparison of the cytokeratin polypeptides present in various hepatocyte and hepatoma cell cultures showed that, in some of the cultured epithelial liver cells, cytokeratins are expressed which are identical with, or similar to, those of normal hepatocytes grown in the liver. On the other hand, differences in cytokeratin polypeptides were also found among different hepatocyte-derived cell cultures. Changes of expression of cytoskeletal proteins were found to occur even in cloned cell populations, and cells positive for certain cytokeratins could be seen next to other cells that were negative. The results demonstrate that profound changes of cytoskeletal composition, especially concerning intermediate filament protein patterns, can occur during culturing in vitro. Moreover, we show that different intermediate filament proteins can be expressed in different hepatocyte-derived cell cultures and that changes of cytoskeletal composition can occur in a given cell population, without obvious effects on cell growth rate and cell morphology. During culturing of hepatocytes and hepatoma cells, there seems to be a general tendency to induce the production of vimentin filaments as well as to maintain the production of cytokeratins similar to the hepatocyte-specific cytokeratins in liver tissue. However, the demonstrated exceptions speak against a role of these filament proteins as prerequisites for the growth of an epithelial cell in vitro. Rather, the presence of filaments containing certain cytokeratins and of desmosomes in epithelial cells growing in vitro seems to reflect the synthesis of specific differentiation markers which may be lost, independently, in some cells during culturing.

Constitutive aggregates of intermediate-sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate.

Abstract Cloned hepatoma cells ( 72 22 ) derived from the liver of a rat treated with the carcinogen, diethylnitrosamine, exhibit a genetically stable, large, acentric, juxtanuclear, hyaline aggregate of loosely packed intermediate-sized (7–11 nm) filaments, interspersed with variable but minor amounts of microtubules, polyribosomes and membranous structures. Immunofluorescence microscopy shows that the these filaments react specifically with antibodies to bovine prekeratin and to murine vimentin. The aggregates contain aster-like foci common to the arrangement of both tonofilament-like and vimentin-containing intermediate-sized filaments, although both filament systems show different fibrillar patterns in other cytoplasmic regions. While the cytokeratin filament system is not significantly altered during exposure to colcemid, the vimentin in the abnormal aggregate is rearranged during such treatment into extensive and complex perinuclear ‘whorls’ of filaments. Treatment of the cells with butyrate results in a markedly flattened, hepatocyte-like morphology, a reappearance of typical actin-containing ‘cables’, and a progressive disintegration of the filament aggregate concomitant with a normal display of filaments of both the cytokeratin and vimentin type. The results show that ( i ) some cells contain aggregates consisting of two different types of intermediate-sized filaments oriented onto a common focal center; ( ii ) such an abnormal filament arrangement is clonally stable; ( iii ) the vimentin-type filaments contained in such aggregates are still susceptible to the action of antimitotic drugs and can be rearranged into characteristic perinuclear whorls; and ( iv ) this abnormal aggregate of intermediate filaments can be reverted to normal patterns upon treatment of the cells with butyrate.

Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dimethylsulfoxide

The murine BW 1 and rat 32III 6/d tumor cell lines, derived from a spontaneous mouse hepatoma and a carcinogen-induced rat hepatocellular carcinoma, were used to investigate the effect of dimethylsulfoxide (DMSO) on liver cells in vitro. After a 4-day exposure to DMSO in final concentrations of 0.5 and 1.0%, BW 1 cell-associated albumin increased by 41.6 and 94.2%; extracellular albumin levels in these same cultures rose by 131.4 and 214.2%. Exposure of 32III 6/d cells to 2% DMSO produced increases in cell-associated and extra-cellular albumin concentrations of 67.8 and 188.7%, respectively. The lack of inducible gamma-glutamyl transpeptidase in BW 1 cells and its decrease in 32III 6/d cultures following DMSO treatment suggests that the DMSO-mediated enhancement of albumin production is not reflective of a random increase in the expression of cellular genes.

Differential association of fetal antigen with hepatoma tissue grown in vivo and in vitro.

Abstract Production of two distinct fetal antigens, alpha-fetoprotein (AFP) and γ-fetal antigen (γ-FA) was associated with growth of the BW7756 mouse hepatoma in vivo . Synthesis of AFP, but not γ-FA, continued during in vitro propagation of the tumor cells. After re-inoculation of these cultured hepatoma cells into inbred mice, both AFP and γ-FA could be detected again in the growing tumor tissue and in the sera of tumor-bearing mice. It is evident that different growth conditions effect synthesis of these two tumor-associated antigens.

In vivo initiated rat liver carcinogenesis studied in vitro; formation of alcoholic hyaline-type bodies

Epithelial liver cells isolated from rats after oral diethylnitrosamine administration were established in culture. When propagated in vitro for 2--3 months, over half the cells contained acentric nuclei and large juxtanuclear inclusions resembling the hyaline deposits (Mallory bodies) in cirrhotic livers of alcoholics. The morphological changes and disarrayed filaments in these bodies were retained on serial passages for many months. Cells inoculated into nude mice or newborn rats produced carcinomas from which cells with these abnormalities were reestablished in continuous culture.

The attachment and penetration of T7 DNAphage in Syrian hamster embryonic cells

Bacteriophage T7 DNA can penetrate Syrian hamster embryonic cells after a mandatory initial pretreatment with DEAE-dextran. In 3 h an extracellular complex between T7DNA and the cell monolayer is formed which is equivalent to 105 T7 genomes per cell. During the ensuing 24-48 h of cell growth, an average of 102-103 T7 genomes are transported to the nucleus in 90% of the cells of the culture.

In Vitro consequences of sperm-somatic cell interactions

Abstract Following interaction with rat spermatozoa and subsequent proliferation in vitro , Chinese hamster lung fibroblasts synthesized fetal antigen and grew in semisolid agar suspension culture but were not tumorigenic in nude mice. Control hamster cells were consistently negative for these properties even after long-term cultivation in liquid medium. Admixture with spermatozoa was also found to induce marked multinucleation and to enhance the cloning efficiency of human tumor (HeLa) cells in soft agar culture. Sperm-mediated induction of fetal antigen synthesis and anchorage-independent growth by cultured mammalian somatic cells may thus represent an early stage in the multi-step sequence leading to malignant transformation. Sperm-somatic cell culture systems may provide a convenient model system for the study of cell-cell interactions similar to those which may normally occur in vivo .

Alpha-Fetoprotein and Albumin Synthesis by Heterotransplanted Rat Liver Tumor Cells

Epithelial cells, isolated from the hepatic tissue of rats bearing carcinogen-induced neoplastic liver nodules, were analyzed by immunological methods for the production of specific liver proteins during alternate in vitro-in vivo passage. Heterotransplantation of several long-term rat liver cell lines into nude mice and subsequent re-establishment in vitro resulted in the derivation of hepatocyte cultures expressing both alpha-fetoprotein and albumin. Prior to passage in vivo these cell lines synthesized by one or neither of these proteins in vitro. Alternate in vitro-in vivo passage thus appears to potentiate tissue-specific protein expression by established liver cell lines.