Biosynthesis of the third component of complement in rat liver epithelial cell lines and its stimulation by effector molecules from cultured human mononuclear cells.

Rat liver epithelial cell lines, growing in a serum-supplemented medium, synthesize and secrete into the culture medium the third component of complement (C3). We studied the regulation of C3 production in this system. We found that human peripheral blood mononuclear leukocytes in culture released one or more soluble factors which stimulated rat liver epithelial cells to produce increased quantities of C3. This stimulating effect was strongly enhanced when the mononuclear cell cultures were treated with phytohemagglutinin, a T-lymphocyte mitogen. The factor(s) failed to enhance C3 biosynthesis by rat dermal fibroblasts, which are known to produce this protein. This reveals a tissue-specific differential response between the fibroblasts and the liver epithelial cells. The physical and chemical characteristics, such as heat sensitivity, 2.8 M ammonium sulphate precipitation, and lower activity after digestion by proteases unambiguously indicate that the effector molecules are proteins. When the crude supernatant of mononuclear leukocytes was fractionated by gel filtration, the stimulating factor(s) eluted as two peaks with apparent molecular weight of 25 to 60 and 15 to 20 kdalton, respectively. As to the cellular origin of the C3-stimulating factor(s), several observations were made: (a) in separate cultures containing either T-cells or monocyte-enriched populations from the same sample of blood mononuclear cells, no activity was detected in the presence or absence of phytohemagglutinin, (b) conditioned media from each of these cultures could not substitute for the corresponding intact cell populations, and (c) the addition of purified T-cells to the monocyte-enriched population in the presence of phytohemagglutinin restored the production of the stimulating activity by the mixed culture. Finally, experiments were carried out to verify whether monokine interleukin 1 affects the hepatic C3 biosynthesis. It was demonstrated that interleukin 1 enhanced this biosynthesis, but could not completely substitute for conditioned medium from stimulated mononuclear cells.

Third component of rat complement. Purification from plasma and radioimmunoassay in culture media from cell lines.

A double antibody radioimmunoassay for rat C3 has been developed. The assay required the preparation of C3 from plasma. A new purification procedure using ion exchange high performance liquid chromatography is described. The final product was homogeneous on SDS-PAGE analysis. Rat C3 has an apparent molecular weight of 187,000 and is composed of two polypeptide chains with molecular weights of 125,000 and 73,000, respectively. The purified C3 antigen with high hemolytic reactivity, as assessed by its specific functional activity, was used in preparing anti-C3 sera to perform a specific radioimmunoassay for quantifying C3 in the presence of heterologous sera contained in the cell culture media. All the validating criteria, such as precision, recovery and dilution studies, were investigated. The high sensitivity of the method allowed replicate determination of C3 in small aliquots of the cell culture medium.

The cytotoxicity of bleomycin A2 on adult rat liver epithelial cell lines at different stages of spontaneous cell transformation.

Cytotoxic effects of Bleomycin A2 on adult rat liver epithelial cell lines were evaluated by three methods: the incorporation rate of (3H) thymidine for DNA biosynthesis, the incorporation rate of L-(3H) leucine for protein biosynthesis and Giemsa dye staining of surviving cells. Chromosome investigations at successive passages of cell lines have shown that spontaneous chromosome abnormalities in distribution and structure after 15-20 passages, i.e. 50 to 60 cell generations, were the earliest morphological sign of spontaneous transformation. In this study a highly spontaneously transformed cell line was very sensitive to the drug. Another cell line at the beginning of spontaneous transformation appeared to be insensitive although on further passage it became more sensitive. The use of microtitration plates made it easier for us to undertake a comparative study of the different parameters. Following Bleomycin A2 exposure, Giemsa staining gave the best evaluation of cell killing whereas thymidine incorporation allowed the estimation of cell recovery. The antineoplastic effect of Bleomycin A2 can probably be used to evaluate the malignant potential of different rat liver epithelial cell lines.

Interactions of caffeine and phenobarbital on sister chromatid exchanges in rat liver epithelial cell lines treated with 2-acetyl-aminofluorene and N-acetoxy-2-acetylaminofluorene.

Using "Fluorescence plus Giemsa" technique, sister chromatid exchanges (SCE) were determined on second-division metaphases in rat liver epithelial cell lines treated with 2-acetylaminofluorene (2-AAF), the procarcinogen, and N-acetoxy-2-AAF (N-OAc-2AAF), an ultimate carcinogen analog. The SCE frequency was found decreased after some conditions of 2-AAF treatment and increased with N-OAc-2-AAF. Phenobarbital (PB) decreased also the SCE frequency but cancelled the 2-AAF action when incubated after the procarcinogen. The addition of caffeine (Caf) cancelled the action of 2-AAF but not of phenobarbital. It is suggested that the mechanism of SCE may have several origins.