Interferon production in human T lymphoblastoid cells

Human T lymphoblastoid cell (RPMI 8402 cell) produced interferon (IFN) through the induction by Sendai virus. The priming effect on the interferon production in the RPMI 8402 cell could be found by the pretreatment of human leukocyte IFN (Hu IFN-alpha), but not by that of the IFN produced in the RPMI 8402 cell (T-IFN). The superinduction by the irradiation of ultraviolet rays or the treatment of antimetabolites (actinomycin D and cycloheximide) or 5-bromodeoxyuridine was not found. The T-IFN was completely neutralized by the anti-Hu IFN-beta serum, but not by the anti-Hu IFN-alpha serum at all. In conclusion, it was confirmed that the IFN produced in the RPMI 8402 cell through the induction by Sendai virus was antigenically identical to Hu IFN-beta.     

Instability of Mex- phenotype in human lymphoblastoid cell lines

Three lymphoblastoid cell lines (LCLs) had extremely low activities of O6-alkylguanine-DNA alkyltransferase (O6-AGT), and were classified as Mex-. They were highly sensitive to cell killing by 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a hydrochloride (ACNU), whereas NMO2, a Mex+ LCL with a high O6-AGT activity, was resistant to the agent. Small fractions of these Mex- LCLs survived the treatment with 10 micrograms/ml of ACNU for 24 h, and the surviving cells were found to be resistant to subsequent treatments with the agent. In addition, they contained O6-AGT activities comparable to that of NMO2 and were therefore regarded as Mex+. These results suggest that the Mex- phenotype in LCLs is unstable.     

Platelet-activating factor enhances Ig production in B lymphoblastoid cell lines

Platelet-activating factor (PAF) is a highly potent phospholipid mediator known to be active in many biologic systems. To date, little is known of the effect of PAF on B lymphocytes. Using two Ig-secreting B lymphoblastoid cell lines, we have demonstrated that PAF can enhance Ig production by these cells in a dose-dependent fashion. PAF also causes significant alteration of the kinetics of Ig secretion in these lymphoblastoid cell lines. The effect of PAF is rapid, with detection of 6- to 12-fold increases in Ig production in the first 24 h of cell culture, followed by a plateau during the next 24 to 48 h. The specificity of the PAF effect on Ig secretion is emphasized by lyso-PAF having no Ig-enhancing properties and by the inhibition of Ig enhancement in the presence of the structural analogue PAF antagonist CV3988 and the soluble nonstructural analogue PAF receptor antagonist Web 2086. PAF does not cause an increase in the kinetics of cell proliferation or an increase in cell numbers at any time during a 72-h culture period. In an attempt to explain the increase in Ig secretion in the absence of changes in growth parameters, an ELISA spot assay for enumeration of Ig-secreting cells was developed. This assay demonstrated that the increase in Ig production is likely due to enhancement of single cell Ig secretion rather than an increase in cell number. These data indicate that PAF may have an important immunomodulatory role in the production of Ig by B lymphocytes.     

Adaptive response induction and variation in human lymphoblastoid cell lines

Adaptive response is a term used to describe the ability of a low, priming dose of ionizing radiation to modify the effects of a subsequent higher, challenge dose, but it has been observed to be highly variable in both presence and magnitude. To examine this variability, 10 human lymphoblastoid cell lines were screened for adaptability to 137Cs radiation by determining the frequency of micronuclei in binucleated cells. Of these, six adapted, three did not adapt and one was synergistic. The assay was then repeated on each of the cell lines to test for reproducibility. Five cell lines showed the same result both times; four of these adapted and one did not.To determine whether fluctuations in the cell cycle distribution in the irradiated population of cells could alter the adaptive response, and therefore explain some of the observed variability, two of the cell lines were tested for adaptation after enriching the population, by synchronization, for a given cell cycle stage. In both cell lines, the direction of the response was altered when the distribution of cells within the cell cycle was changed, suggesting that the adaptive response can be affected by cell cycle stage at the time of irradiation.     

Cytogenetic analysis of lymphoblastoid cell lines

Cytogenetic abnormalities were discovered in more than half of 16 lymphoblastoid cell lines established from fragile X individuals and their relatives upon routine cytogenetic analysis of early passage cultures. Subsequently, a second series of lymphoblastoid lines was analyzed to determine if the aneuploidy was a characteristic of lymphoblastoid cell lines derived from fragile X families or a result of the use of cyclosporin A in the establishment of these lines. In the second series of 33 lymphoblastoid cultures, no aneuploid clones were found in the fragile X group, while two were detected in the control cultures, one in a line initiated with cyclosporin A and the other in a line established without cyclosporin A. We conclude that the abnormal clones in our preliminary series were not a characteristic of lines derived from fragile X families and probably not due to the use of cyclosporin A. However, the finding of chromosome abnormalities in a large proportion of lines during the first 3 mo of culture contrasts with previous reports of chromosome stability for the first 12-18 mo of cultivation and indicates that the chromosomes of lymphoblastoid lines should be monitored in any experiment in which a normal diploid complement is critical.     

Transferrin binding by human lymphoblastoid cell lines and other transformed cells

Viable cells of 18 human cell lines, including 15 transformed cell lines of malignant and lymphoblastoid origin, were examined by an indirect immunofluorescence method for their ability to bind purified transferrin and transferrin in normal human serum. The specificity of the reaction was investigated by study of the binding reactions of several other serum proteins, including albumin, α-1-antitrypsin, and α-2-macroglobulin. Membrane binding of human transferrin was demonstrated in less than 5% of normal peripheral blood mononuclear cells or cultured diploid fibroblasts, but in more than 80% of the cells from 13 of the transformed lines, and the data obtained indicated that this binding reaction reflected the presence of specific receptors for transferrin.     

EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines

Recent research suggests that epigenetic alterations involving DNA methylation can be causative for neurodevelopmental, growth and metabolic disorders. Although lymphoblastoid cell lines have been an invaluable resource for the study of both genetic and epigenetic disorders, the impact of EBV transformation, cell culturing and freezing on epigenetic patterns is unknown. We compared genome-wide DNA methylation patterns of four white blood cell samples, four low-passage lymphoblastoid cell lines pre and post freezing and four high-passage lymphobastoid cell lines, using two microarray platforms: Illumina HumanMethylation27 platform containing 27,578 CpG sites and Agilent Human CpG island Array containing 27,800 CpG islands. Comparison of genome-wide methylation profiles between white blood cells and lymphoblastoid cell lines demonstrated methylation alterations in lymphoblastoid cell lines occurring at random genomic locations. These changes were more profound in high-passage cells. Freezing at low-passages did not have a significant effect on DNA methylation. Methylation changes were observed in several imprinted differentially methylated regions, including DIRAS3, NNAT, H19, MEG3, NDN and MKRN3, but not in known imprinting centers. Our results suggest that lymphoblastoid cell lines should be used with caution for the identification of disease-associated DNA methylation changes or for discovery of new imprinted genes, as the methylation patterns seen in these cell lines may not always be representative of DNA methylation present in the original B-lymphocytes of the patient.     

DNA repair in lymphoblastoid cell lines established from human genetic disorders

Lymphoblastoid cell lines (LCLs) established from chromosomal breakage syndromes or related genetic disorders have been used to study the effects of mutagens on human lymphoid cells. The disorders studied include xeroderma pigmentosum, ataxia telangiectasia, Fanconi's anemia, Bloom's syndrome and Cockayne's syndrome. Three approaches were used to assess the cells' ability to cope with a particular mutagen: (1) assaying recovery of DNA snythetic capabilities as measured by [³H]thymidine (dT) incorporation; (2) measurements of classical excision DNA repair by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (3) determining cell survival by colony formation in microtiter plates. LCLs established from xeroderma pigmentosum showed increased sensitivities to ultraviolet (354 nm) light and N-acetoxy-2-acetylaminofluorene (AAAF) as determined by DNA synthesis or colony formation and had diminished levels of excision-repair. Cockayne's syndrome LCLs, on the other hand, had increased sensitivities to ultraviolet (UV) light, AAAF and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) while showing near normal levels of DNA-repair after treatment with each agent. An LCL established from ataxia telangiectasia had decreased DNA repair synthesis and defective colony-forming ability following treatment with MNNG. LCLs, in addition to ease of establishment, appear likely to provide useful material for the study of DNA repair replication and its relationship to carcinogenesis.     

Transferrin receptors on human B and T lymphoblastoid cell lines.

Experiments demonstrating the existence of receptors for iron-saturated transferrin on both B and T lymphoblastoid cell lines of human origin are described. Binding of 125I-labeled transferrin is rapid, saturable and reversible. It can be specifically inhibited by unlabeled transferrin but not by other proteins. The number of receptors on T cell lines determined by Scatchard analysis is almost double the number on B cell lines but the binding affinities are equal. The putative transferrin receptor can be removed from the cell by the proteolytic enzymes papain and trypsin, and is re-expressed during overnight incubation at 37 degrees C. Resynthesis is inhibited by puromycin. The receptor can be solubilized by deoxycholate, and retains transferrin binding capacity when non-covalently attached to an amphipathic matrix consisting of deoxycholate-coupled poly(L-lysyl) Agarose.     

Differences in the incorporation of bromodeoxyuridine by human lymphoblastoid cell lines.

Long term human lymphoblastoid lines differ in their ability to grow in medium containing bromodeoxyuridine (BrdU) and to incorporate analog into their DNA. Eight Burkitt's lymphoma cell lines divided at least twice in BrdU-containing medium and made DNA in which over 90% of the thymidine residues were substituted with analog in both strands. Three infectious mononucleosis-derived lines and 24 lines transformed in vitro were inhibited by BrdU after one cell division and made only hybrid DNA in which one strand was substituted with analog. One out of eight normal individuals from whom long term lines were prepared gave cell lines which divided at least twice in BrdU and gave DNA in which both strands were substituted with analog. It would appear that intrinsic cellular factors regulate the response to BrdU and that Burkitt's tumor lines are characterized by their ability to make stable doubly substituted DNA containing a high proportion of halogenated analog.     

Lysis of C1Q-coated chicken erythrocytes by human lymphoblastoid cell lines

Human lymphoblastoid cells lysed chicken erythrocytes (E) that carried cell surface bound human C1q. Antibody to E(A) was not required for the C1q-dependent reaction. The effect of C1q was inhibited by Fab'2 anti-C1q and by the serum C1q inhibitor. The action of the lymphoblastoid cells was inhibited by anti-metabolites and by pretreatment of the cells with trypsin which is known to destroy their C1q receptor. Lymphoblastoid cell lysate was inactive. The time course of the C1q-dependent lysis was comparable to that of the antibody-dependent cellular cytotoxic reaction of human K-cells. Lysis of EA by human peripheral lymphocytes was enhanced up to 50% by human C1q.     

Use of cyclosporin A in establishing Epstein-Barr virus-transformed human lymphoblastoid cell lines

We have investigated the potential for using cyclosporin A to increase the efficiency with which Epstein-Barr virus-transformed human lymphoblast lines can be prepared. Use of this immunosuppressive drug has permitted the development of a procedure with success rates exceeding 95% despite the processing of very large numbers of samples.