Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: identification of tumor origin by idiotypic analysis.

Hurley, John N.; Fu, Shu Man; Kunkel, Henry G.; McKenna, Gillies; Scharff, Matthew D.

doi:10.1073/pnas.75.11.5706

Cited by 47 publications

(19 citation statements)

References 21 publications

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“…RESULTS Two Primary Translation Products of the 6 Chain. The human B lymphoblastoid cell line SeD expresses two immunoglobulin classes, IgM and IgD, which share idiotypic determinants (22). In previous work with this cell line (24), structural differences were delineated between Am and A, (the heavy chains ofmembrane-bound and secreted IgM, respectively).…”

Section: Methodsmentioning

confidence: 99%

“…1, lane 4). With an anti-idiotypic antiserum specific for IgM (SeD) (22), the pL chains as well as the four 8 forms were immunoprecipitated (Fig. 1, lane 5).…”

Section: Methodsmentioning

confidence: 99%

“…5127 Cell Culture and in Vivo Pulse-Labeling. Cell lines were maintained in suspension culture as described (22). Conditions for pulse-labeling with [3S]methionine were essentially as described (23).…”

Section: Methodsmentioning

confidence: 99%

“…Rabbit antibodies to human IgD and K light chains, sheep antibodies to rabbit IgG, and rabbit antibodies specific for the IgM (SeD) idiotype were prepared as described (3,22). Samples containing radiolabeled polypeptides, synthesized in vitro or in vivo, were adjusted in the 1% Triton X-100 buffer described above, incubated with antibody for 2 hr at room temperature or at 0C (as indicated), and then incubated for 2 hr at 40C with Sepharose CL-4B beads (Pharmacia) coated with sheep anti-rabbit IgG.…”

Section: Methodsmentioning

confidence: 99%

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Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

McCune¹,

Fu²,

Kunkel³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Structural differences between the heavy chain of membrane IgD (Bin) and the heavy chain of secreted IgD (8s) were investigated by using a human lymphoblastoid cell line that expresses idiotypically identical IgM and IgD. In a wheat germ cell-free system, mRNA from this cell line was shown to encode two distinct 8 Further analysis of these functional systems must eventually be accompanied by a detailed structural examination ofthe IgD heavy chain (8). Until recently, the low incidence of IgD myeloma and the extreme lability of IgD to proteolysis hampered the determination of the Fc amino acid sequence of the heavy chain of secretory IgD (8,) (7). The study of the heavy chain of membrane IgD (Sm) has been even more difficult. It is extremely susceptible to proteolysis and is present in low amounts, with a slow turnover rate on B cell membranes. Early structural studies on membrane IgD demonstrated that reduced 8 chains migrate as a broad band on NaDodSO4/polyacrylamide gel electrophoresis (8, 9). Subsequent analyses by two-dimensional gel electrophoresis indicated that the biosynthetic forms of 8 exist as a heterogeneous group, reflective of modifications of N-linked oligosaccharides (10). As shown by charge-shift electrophoresis, these forms probably have a hydrophobic COOH terminus (11, 12). Studies using one-dimensional NaDodSO4 gel electrophoresis systems have demonstrated that am exists as two molecular entities (8im and 8m2), distinguished from one another by a difference in molecular weight (ranging from 2000 to 7000) (13-16). Likewise, 8, has been resolved into two species (5s1 and 8s2) that differ in molecular weight (17)(18)(19). The relationship between these various forms ofthe 8 chain is obscure at present. As a result, the structural relationships between 5im and 8, as well as those between am and ILm have yet to be resolved.In this study, IgD biosynthesis was analyzed in a human B lymphoblastoid cell line (SeD) that coexpresses IgM. All Ig molecules synthesized by this cell line express the same idiotype. The 8 chains of this line were found, by cell-free translation ofextracted mRNA, to exist as two distinct primary translation products, most likely corresponding to 8m and 8s. When followed by pulse-chase experiments in vivo, these chains were found to be processed to four forms, differing in relative mobility as a result of differential N-glycosylation. 8m appears to be a higher molecular weight than 8, and does not have a large cytoplasmically exposed domain. MATERIALS AND METHODSPreparation of RNA and Cell-Free Protein Synthesis. Total cellular RNA was extracted with NaDodSOjphenol/chloroform/isoamyl alcohol and proteinase K as described (20) and translated in a staphylococcal nuclease-treated wheat germ system (21).[35S]Methionine (New England Nuclear; 700 Ci/ mmol; 1 Ci = 3.7 x 10'°becquerels) was used at a final concentration of 1 mCi/ml, and salts were adjusted to final concentrations of 150 mM KOAc and 3.8 mM Mg(OAc)2. In indicated experiments, dog pancreatic microsomal membran...

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Section: Methodsmentioning

confidence: 99%

“…1, lane 4). With an anti-idiotypic antiserum specific for IgM (SeD) (22), the pL chains as well as the four 8 forms were immunoprecipitated (Fig. 1, lane 5).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

McCune¹,

Fu²,

Kunkel³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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“…), supplemented with 10% fetal calf serum. 17 B-lymphoblastoid cell lines derived from one tonsil and other cell lines from normal donors were initiated and maintained as previously described (11).…”

mentioning

confidence: 99%

Isolation and immunological characterization of a major surface glycoprotein (gp54) preferentially expressed on certain human B cells.

Wang¹,

Fu²,

Kunkel³

1979

The Journal of Experimental Medicine

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A major membrane glycoprotein with mol wt of approximately 54,000 has been isolated from membrane preparations of B-type lymphoid cell lines. Antiserum prepared against the isolated material specifically precipitated this glycoprotein from membranes labeled by surface radioiodination or by metabolic labeling. This antiserum was shown by complement-mediated cytotoxicity assay, membrane immunofluorescent staining, and by quantitative absorption analysis to react preferentially with certain B-lymphoblastoid cell lines, with a minor population of peripheral blood B lymphocytes, and a major population of tonsillar B lymphocytes. Certain B-cell leukemias also expressed the antigen, whereas others did not. Considerable variability was observed among positive B cells in the intensity of fluorescent staining even among the leukemic cells from a single individual. Although T cells, including T cells, were negative by direct immunofluorescent and cytotoxicity assay, evidence for low levels of the antigen on the cells of T cell lines was obtained. The whole specific antiserum and its F(ab')2 fragments stimulated B lymphocytes to proliferate. This proliferation did not produce differentiation to plasma cells and was T-cell independent. The monovalent Fab fragments had no effect. None of these preparations timulated T cells. The possibility that this antigen, termed gp54, may play some role in B-cell activation is discussed.

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Analysis of transformation with epstein‐barr virus and phenotypic characteristics of lymphoblastoid cell lines established from patients with hairy cell leukemia

et al. 1983

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In order to assess the role of Epstein-Barr virus (EBV) in patients with hairy cell leukemia (HCL), we have sought to characterize 1) the ability of EBV to infect and transform hairy leukemic cells in vitro and 2) the phenotypes of cell lines putatively derived from those leukemic cells. Analysis of EBV-induced transformation and the kinetics of Epstein-Barr nuclear antigen (EBNA) induction in leukemic preparations indicated that most leukemic cells were not susceptible to EBV infection but that at least a small subpopulation of leukemic cells could be infected with EBV. Lymphoblastoid cells lines were established after exposure of peripheral blood or splenic cells from HCL patients to B95-8 or QIMR-WIL EBV. Splenic leukemic cell preparations were more sensitive targets for EBV transformation than were peripheral blood cell samples. The newly established cell lines, but not long-established B lines such as Raji, demonstrated high levels of synthesis of p35, (a protein complex expressed abundantly by cells of a subset of HCL patients) and high levels of tartrate-resistant acid phosphatase (an enzyme relatively diagnostic for HCL). Lymphoblastoid lines from one patient with HCL expressed lambda light chains and no kappa chains as did the patient's leukemic cells. Virus expression in these lines showed that HCL-derived lines had spontaneous early antigen (EA) and viral capsid antigen (VCA) expression. Transforming EBV could be rescued from HCL-derived cell lines but not from cord blood-derived lines.

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Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: identification of tumor origin by idiotypic analysis.

Cited by 47 publications

References 21 publications

Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

Biogenesis of membrane-bound and secreted immunoglobulins: two primary translation products of the human delta chain, differentially N-glycosylated to four discrete forms in vivo and in vitro.

Isolation and immunological characterization of a major surface glycoprotein (gp54) preferentially expressed on certain human B cells.

Analysis of transformation with epstein‐barr virus and phenotypic characteristics of lymphoblastoid cell lines established from patients with hairy cell leukemia

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