Student Theses and Dissertations
Date of Award
1977
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Samuel Silverstein
Keywords
C3d receptor, lymphoblastoid cells, clonal variation, receptor expression, complement system, surface proteins
Abstract
A human lymphoblastoid cell line, 8866, was used to investigate a surface marker, the C3d receptor. A method for cloning these cells at a relatively high efficiency was developed and C3d receptor bearing (CR+) and receptor free (CR-) subclones isolated. Receptor bearing cells were identified by their capacity to bind to complement coated sheep erythrocytes. Of the many clones isolated, only a few contained uniformly receptor positive or negative cells. Most expressed an intermediate character, not only in the percentage of CR+ cells, but also in the degree of resettability of the individual CR+ cells. Clones initially wholly CR+ remained CR+ both on prolonged culture and on recloning. CR-populatons always developed some CR+ cells with continued culture. Upon recloning, CR- clones gave rise to largely CR- clones, but also to CR+ clones, and to clones of intermediate character. Recloning of a CR+ clone derived from a CR- clone yielded mainly intermediate clones, but also CR- clones: the CR+ trait was unstable in such derivatives. Having thus demonstrated the phenotypic stability of some clones, and the instability of others, attempts were made to induce or repress the expression of the receptor. A variety of compounds (BrdU, DMSO, 2-deoxyglucose and sodium butyrate) had no effect. Neither did mixing CR+ and CRcells, nor growing CR- cells in medium conditioned by CR+ cells. There was an apparent increase in the number of CR- clones produced by the picked single cell cloning method (in which cells grow for some time at suboptimal cell densities), and CR+ cells always appeared in cultures of cells kept at optimal density in the logarithmic phase of growth. These were the only conditions found to modify the expression of the receptor. Since 1) the growth rates of CR+ and CR- cell types were similar, and 2) continued culture of clones containing mixtures of the two cell types never became wholly CR+ or CR-, and 3) continued cultures varied the degree of receptor expression rapidly and significantly, it is considered unlikely that the conversion of CR- to a partially CR+ population could be explained simply by the emergence of a CR+ variant with a growth advantage. The ability of clonally derived cells to alter their degree of receptor expression indicates that the CR- cells are not mutants deficient in the structural gene necessary for the production of the receptor, nor are they bearers of a mutant structural gene which produces a receptor molecule with altered activity. Examination of karyotypes of the different clones has shown near diploid and near tetraploid clones of both CR+ and CR-types. Limited examination of individual karyotypes has not revealed any distinctive chromosomal abnormalities. The nature of the receptor with respect to complement components was investigated. Using purified complement components, and specific antisera, it has been established that 8866 cells bear a C3d receptor exclusively. A number of physico-chemical studies were carried out to define the ligand-receptor interaction. It was concluded that the receptor may be regarded as representative of that present on the plasma membrane of normal peripheral blood lymphocytes. The receptor is presumed to be an externally disposed protein, since its activity is destroyed by proteases, and its regeneration is inhibited by cycloheximide at concentrations which inhibit protein synthesis. In an effort to identify the C3d receptor molecule(s), the lactoperoxidase-mediated iodination procedure of Hubbard and Cohn (1975a) was slightly modified for 8866 cells, and the major iodinatable surface proteins of these cells defined. Three major species, with molecular weights of approximately 11,000, 34,000, and 50,000 daltons were found, and eight other species were reproducibly present. No significant differences were found between the iodine labelling patterns of membrane proteins of CR+ and CR- cell populations by this technique. Further studies with selective protease treatment, and affinity binding techniques were also unavailing in detecting the C3d receptor molecule(s). The proteolysis of iodinated cells did reveal a uniquely protease sensitive moiety of 35,000 daltons, which was cleaved by trypsin, chymotrypsin, papain and pronase, leading to the appearance of a new cell-associated moiety with a molecular weight of 22,000 daltons. These studies have established the clonal character of the C3d receptor, and provided homogeneous populations of cells bearing a receptor like that on peripheral blood lymphocytes. In addition, an unstable system has been defined. This may prove useful in the investigation of the regulation of the expression of the C3d receptor.
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Recommended Citation
Hoffman, Gary John, "Studies on the Complement Receptor of a Human Lymphoblastoid Cell Line" (1977). Student Theses and Dissertations. 535.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/535
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy