Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells

A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-methyl-pyrazolate)]²⁺ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na⁺/K⁺-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.     

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin.

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wild type H4-II-E cells, pCXN2 vector-transfected cells (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 microM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.     

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Involvement of intracellular signaling factors in the serum-enhanced Ca2+-binding protein regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E).

The involvement of signaling factors, which are related to serum component, on the regucalcin mRNA expression in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). H4-II-E cells were cultured for 2 or 6 h in a medium containing various reagents in the presence of serum (10% fetal bovine serum) after the subconfluent with 3-day-culture. The regucalcin mRNA expression was significantly increased by serum addition. This increase was clearly inhibited by the presence of EGTA (10(-3) M), A23187 (10(-6) M), trifluoperazine (10(-5) M), staurosporine (10(-7) M), or genistein (10(-5) M) with 6-h-culture, although the beta-actin mRNA expression was not altered by the reagents. Meanwhile, the regucalcin mRNA expression was significantly stimulated by the addition of Bay K 8644 (2.5 x 10(-6) M) in the presence of serum. This effect was also seen in the presence of genistein (10(-5) M). The present study suggests that the regucalcin mRNA expression is mediated through signaling pathways which are partly involved in Ca2+-dependent protein kinases and tyrosine kinase in H4-II-E hepatoma cells.     

Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.     

Properties of the H-4-II-E tumor cell system. I. Growth and cell proliferation kinetics of an experimental hepatoma.

Growth and cell proliferation kinetics of hepatoma H-4-II-E and its tissue culture derivative have been studied to establish the characteristics of an in vivo--in vitro solid tumor model. The H-4-II-E line, originating from the Reuber H-35 hepatoma, can be maintained and studied either in cell culture or as a transplantable solid tumor in ACI male rats. In addition it allows for the in vitro assay of cell survival following treatment of animal tumors in situ. In vivo, hepatoma H-4-II-E is rapidly growing tumor with a mean doubling time of 49-2 hr. The cell cyle time is 39-1 hr with a cell loss factor of 0-32. Retrospective examination of tumor specimens obtained during the establishment of the H-4-II-E tumor system demonstrates that both structural as well as cell population changes have occurred. The biological characteristics of the primary tumor (H-35) and an early intermediate stage (H-35tc2) are compared with H-4-II-E and the histopathological, growth and cell kinetic changes are discussed.     

PROPERTIES OF THE H-4-II-E TUMOR CELL SYSTEM

Growth and cell proliferation kinetics of hepatoma H-4-II-E and its tissue culture derivative have been studied to establish the characteristics of an in vivo—in vitro solid tumor model. The H-4-II-E line, originating from the Reuber H-35 hepatoma, can be maintained and studied either in cell culture or as a transplantable solid tumor in ACI male rats. In addition it allows for the in vitro assay of cell survival following treatment of animal tumors in situ. In vivo, hepatoma H-4-II-E is a rapidly growing tumor with a mean doubling time of 49.2 hr. The cell cycle time is 39.1 hr with a cell loss factor of 0.32. Retrospective examination of tumor specimens obtained during the establishment of the H-4-II-E tumor system demonstrates that both structural as well as cell population changes have occurred. The biological characteristics of the primary tumor (H-35) and an early intermediate stage (H-35_tc2) are compared with H-4-II-E and the histopathological, growth and cell kinetic changes are discussed.     

Activation of polycyclic hydrocarbons in Reuber H4-II-E hepatoma cells an in vitro system for the induction of SCEs

Activation of polycyclic aromatic hydrocarbons (PAHs) was examined in the Reuber H4-II-E established cell line without the use of exogenous enzyme preparations. Metabolism of PAHs to genotoxic products was determined by the induction of sister-chromatid exchanges (SCEs). The induction of SCEs followed a dose-response pattern with plateaus at high doses of PAH. The effects of metabolic enzyme inducers (3-methylcholanthrene, phenobarbital, Aroclor 1254) and the epoxide hydrase inhibitor 1,1,1-trichloropropylene oxide were assessed as changes in SCE induction and enhanced production of water-soluble metabolites. Results indicate that Reuber H4-II-E cells can be employed in the testing of carcinogens activated by the P₁-450 monooxygenase system and would be a useful in vitro system for the study of mechanisms of metabolic induction and their effect on genetic toxicity.     

Cytosolic LC3 Ratio as a Sensitive Index of Macroautophagy in Isolated Rat Hepatocytes and H4-II-E Cells

Macroautophagy, an intracellular bulk degradation process in eukaryotes, is sensitive to nutrient supply and deprivation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an indispensable role in macroautophagy formation and is a suitable marker for this process. Through analysis of the subcellular distribution of LC3, we determined that the cytosolic fraction contained not only a precursor form (LC3-I), but also an apparent active form (LC3-IIs). Both cytosolic LC3-I and LC3-IIs were more responsive to amino acids than those of total homogenate. Moreover, changes in the LC3-IIs/I ratio reflected those in the total proteolytic flux remarkably in both fresh rat hepatocytes and H4-II-E cell lines. Thus, in addition to a sensitive index of macroautophagy, calculating the cytosolic LC3 ratio became an easy and quick quantitative method for monitoring its regulation in hepatocytes and H4-II-E cells.     

Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells

Macroautophagy, an intracellular bulk degradation process in eukaryotes, is sensitive to nutrient supply and deprivation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an indispensable role in macroautophagy formation and is a suitable marker for this process. Through analysis of the subcellular distribution of LC3, we determined that the cytosolic fraction contained not only a precursor form (LC3-I), but also an apparent active form (LC3-IIs). Both cytosolic LC3-I and LC3-IIs were more responsive to amino acids than those of total homogenate. Moreover, changes in the LC3-IIs/I ratio reflected those in the total proteolytic flux remarkably in both fresh rat hepatocytes and H4-II-E cell lines. Thus, in addition to a sensitive index of macroautophagy, calculating the cytosolic LC3 ratio became an easy and quick quantitative method for monitoring its regulation in hepatocytes and H4-II-E cells.     

Tumour-cord parameters in two rat hepatomas that differ in their radiobiological oxygenation status

Summary Tumour cords have been examined quantitatively in two rat hepatomas, 3924A and H-4-II-E, that differ in their radiobiological oxygenation status (oxygen enhancement ratio for growth delay [tumour clamped: tumor in air] was 1.35 for 3924A and only 1.08 for H-4-II-E). The average thickness of tumour cords in 3924A was 118 µm and only 69 µm in H-4-II-E. The migration rates across the cords of the two tumours were approximately the same (1.7 and 1.4 µm h^–1) but for any given distance from the subtending blood vessel, the proportion of histologically-dead cells within the cord was always higher for H-4-II-E. Volume for volume, H-4-II-E contained four times as much vascular space as 3924A but it is suggested that the poorquality of this vasculature in H-4-II-E contributed to its relative radioresistance.