Mouse L cells express a molecular complex carrying the human epitopes recognized by monoclonal antibodies 44D7 and 44H7 after DNA-mediated gene transfer.
In a previous study, we isolated from the non-T, non-B acute lymphoblastic leukemia cell line, HOON, a molecular complex comprised of a minimum of two polypeptide chains linked by disulfide bonds and expressing two distinct epitopes, 44D7 and 44H7. The DNA from the HOON cell lines has been co-transfected with the herpes simplex thymidine kinase gene into mouse L cells. By using a flow cytometer, a stable thymidine kinase-positive cell population expressing both 44H7 and 44D7 antigens of the human leukemic cells has been generated by repeated sorting of cells reactive with monoclonal antibody 44H7. After three sequential sorting experiments, cloned cell lines were established, and a subclone designated 3D5 has been additionally characterized. Greater than 90% of the 3D5 cells stained positively with monoclonal antibodies 44H7 and 44D7 and with 4F2, which reacts with an epitope identical or spatially related to that seen by 44D7. The ratio of antigenic density 4F2:44D7 calculated from the relative fluorescence indices was similar on the HOON leukemic cells and on the 3D5 transfectant cells. However, 3D5, which was selected with antibody 44H7, expressed a higher ratio of 44H7:44D7 than did the HOON cells. The molecular complex carrying these epitopes was isolated from a 3D5 cell extract on a column of 44D7-IgG-Sepharose and was additionally purified by immunoprecipitation. Although several polypeptide chains were present in the immunoprecipitates, the major polypeptide band had an apparent m.w. of 127,000 under nonreducing conditions. After reduction, three bands of apparent m.w. 91,000, 38,000, and 33,000 appeared. The presence of the 91,000 and 38,000 subunits linked by disulfide bonds was also observed for the 44D7 antigen isolated from HOON cells, whereas the polypeptide of apparent m.w. 33,000 was only seen in immunoprecipitates of the transfectant cell extracts. Because the antigen expressed in the transfectants is associated with a multimeric complex containing disulfide-linked subunits, it is possible that only the gene encoding one of the polypeptide chains, namely that carrying the epitopes, was in fact transfected. This HOON gene product could be one subunit associating with murine subunits encoded by genes of the L cell. Alternatively, the antigenic complex may be the product of closely linked genes transfected together, or of a single human gene that is modified post-translationally to create a disulfide-linked complex.
E J
EJ
Quackenbush
P
P
Linsley
M
M
Letarte
D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000032:analysis / Q000302:isolation & purification; D000954:Antigens, Surface / Q000032:analysis / Q000302:isolation & purification; D002457:Cell Extracts / Q000276:immunology; D002469:Cell Separation; D004247:DNA / Q000235:genetics*; D000939:Epitopes / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D005434:Flow Cytometry; D006801:Humans; D007739:L Cells / Q000276:immunology*; D007945:Leukemia, Lymphoid / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D014162:Transfection
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D002457:Cell Extracts; D000939:Epitopes; D004247:DNA