David P. Aden

Complement Biosynthesis by the Human Hepatoma-derived Cell Line HepG2

The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.

Cell-mediated cytotoxicity to sv40-specific tumour- -associated antigens.

CYTOTOXIC thymus-derived lymphoctyes (T cells) from mice infected with lymphocytic choriomeningitis virus, ectromelia virus, vaccinia virus, or parainfluenza virus interact only with H–2 compatible virus-infected target cells1–6. When congenic H–2 recombinant mice7 and mice containing mutations within genes mapped at H–2K end specificities6,8 are used, recognition of virus-infected target cells by the effector lymphocytes requires compatibility at either the K or D locus of the H–2 gene complex. These observations led to the hypothesis that cytotoxic T cells recognise either a complex of viral and histocompatibility antigens or virus-induced alterations of the histocompatibility antigens. The same restriction has been described for the cytotoxic T cell response to lymphocytes modified by trinitrophenyl (TNP)9, to minor histocompatibility antigens10,11 and to the male Y antigen12. To investigate the possibility of a similarly restricted specificity of the effector cells of cell-mediated immunity to tumours, cytotoxic cells specific for SV40 tumour-associated specific antigens (SV40-TASA) were generated in two inbred strains of mice, C57BL/6J and BALB/cAn. The SV40-transformed lines C57SV (SV40-transformed C57BL/6 mouse embryo fibroblasts), MKS/Bu100 (SV40-transformed BALB/c kidney cells13) and LN-SV (SV40-transformed human skin fibroblasts14) were used as immunising and target cells (Table 1). In addition, clones of human–mouse somatic cell hybrids, obtained by fusion of LN-SV and mouse peritoneal macrophages from inbred strains15 were used. These hybrid clones contain the complete mouse genome and, in addition, one to several copies of human chromosome 7, in which the SV40 genome is presumably integrated14,15. C121, N8 and N9 cells are derived from LN-SV fused with C57BL/6 macrophages16 and C136 cells from LN-SV fused with BALB/c macrophages17. These clones express the SV40 tumour (T) antigen (a nuclear antigen)16,17, and the histocompatibility antigens of the murine parental cells (our unpublished results). Neither LN-SV nor any hybrid clones derived from LN-SV make infectious SV4018. Only after fusion with permissive monkey kidney cells can some SV40 particles be rescued. This defective virus exhibits no biological activity. Both LN-SV and human–mouse somatic cell hybrids containing the human chromosome 7 derived from LN-SV exhibit tumour-specific transplantation antigens (TSTA), as demonstrated by their ability to protect SV40-inoculated newborn hamsters from developing SV40 tumours19. Both the SV40-transformed mouse cell lines (our unpublished results) and the hybrid clones16,17 are tumorigenic in immunodeficient “nude” mice, but these cells do not grow routinely in immunocompetent syngeneic mice, probably because of strong TSTA20.

Cell surface antigens coded for by the human chromosome 7.

Human-mouse somatic cell hybrids, containing chromosome 7 from an SV40-transformed human cell line as the only human chromosome, were injected into the same inbred strain of mouse as the mouse parental cell, and the humoral immune response assayed. A cell-surface antigen(s) coded for by the chromosome 7 common to all human fibroblastic cell lines tested and also found on African green monkey kidney cell lines was demonstrated. No reactivity to SV40-induced TSTA was detected.

Histone complements of human tissues, carcinomas, and carcinoma-derived cell lines

SummaryThe pattern of subtypes of the nucleosomal histories and of historic Hl was investigated in human cells from adult and fetal lung and liver, from carcinoma tissues and from carcinoma-derived cell lines, with the object of comparing these patterns, and their relationship to cell growth rate, with those in cells of other species. The subtype pattern of the nucleosomal histories H2A and H3 shows a correlation with replication rate. In adult tissues, subtype H3-3 predominates over H3-2 and H3-1, and the subtype H2A-1 and H2A-2 are approximately equally abundant. In fetal tissues, lung carcinoma and cultured carcinoma-derived cell lines, the subtype H3-1 is predominant and H2A-1 is more abundant than H2A-2. The subtype pattern of H 1 also differs between normal and carcinoma cells, among different tissues, and in different cell lines derived from the same type of carcinoma. In particular, the relative level of H1° differs in several cell lines showing relatively high rates of replication, and in some cases represents more than 25% of the total H1, similar to the level in slowly replicating normal adult liver and lung tissues. The relative level of H1° does not therefore appear to be correlated in a simple manner with cell growth rate in these human cells.

Partial Characterization of Cell-Surface Protein Coded for by Human Chromosome 7

An antigen reactive with antisera to a human chromosome 7-coded cell-surface antigen can be extracted from human cells with the nonionic detergent NP-40. Immune complexes of radiolabeled cell extracts analyzed on SDS polyacrylamide gels showed a single protein with an apparent M.W. of approximately 165,000. This protein was immunoprecipitated from extracts of hybrid cells containing chromosome 7 and human cell extracts, but not from mouse cell extracts or from extracts of a hybrid clone without human chromosome 7. The data presented here indicate that the protein identified on the gels is a cell-surface glycoprotein coded for by human chromosome 7.

Production of HLA antibody in mice immunised with syngeneic mouse-human hybrid cells containing human chromosome 6.

XENOIMMUNISATION with human cells or isolated molecules found on the surface of human cells results in the production of antibodies to a variety of human alloantigens (refs 1–3 and unpublished results). The genetic loci which specify the antigens being recognised can then be determined by using rodent–human hybrids containing various human chromosomes as target cells. In this manner, chromosome 11 has been found to include genes coding for a series of antigens designated AL (refs 1, 4) or HSA-1 (ref. 2) and chromosome 15 to code for β2 microglobulin3. Murine–human somatic cell hybrids with one or a limited number of human chromosomes have been used by us and by others to obtain antisera in mice syngeneic to the mouse parental cells reactive with antigens coded for by specific human chromosomes2,5–7. The advantage of this method is that antisera generated are specific for the antigen(s) coded for by a particular human chromosome and the reactivity is to a limited number of human cell surface antigens. This allows for both systematic genetic mapping of these antigens and detection of previously unknown cell surface molecules. Chromosome 6 has been shown both by pedigree analysis8 and by the use of interspecific somatic cell hybrid target cells9 to be the location of the genes coding for the serologically defined antigens (HLA) of the major histocompatibility complex (MHC). We have used a human–mouse somatic cell hybrid containing several human chromosomes, including chromosome 6, to immunise mice syngeneic to the mouse parental cell. The reactivity of the absorbed antiserum has been analysed both serologically and by sodium dodecyl sulphate polyacrylamide gel electrophoresis after immunoprecipitation. Evidence is presented here to show that mice immunised with somatic cell hybrids containing human chromosome 6 produce antibody which reacts with HLA as well as with other human lymphocyte cell surface antigens.

Heterotopic chondrogenesis and osteogenesis induced by transformed cells: use of nude mice as a model system.

A subline of HeLa cells was shown to induce cartilage and bone formation in congenitally athymic (nude) mice when injected intramuscularly. The chondrogenesis and osteogenesis observed was similar to that found when other inducing epithelia are injected intramuscularly into cortisonetreated mice. A tumorigenic SV40 T antigen-positive human-mouse somatic cell hybrid with human chromosomes C7 and C6 did not induce cartilage or bone formation under similar conditions. The nude mouse may thus provide a system in which to investigate experimental host-cell differentiation without the complications of immunosuppression.