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.     

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.     

In vitro establishment of tumorigenic human B-lymphoblastoid cell lines transformed by Epstein-Barr virus

We studied tumorigenic and phenotypic characteristics of pre- and postimmortal human B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV): preimmortal LCLs showed low telomerase activity and a normal diploid karyotype while postimmortal LCLs showed much higher telomerase activity and maintained a clonal aneuploidic state. Among five postimmortal LCLs tested, LCLs N0005 and N6803 formed colonies in agar medium and showed marked aneuploidy, and N6803 was transplantable into nude mice indicating that it had a complete malignant phenotype, but all preimmortal LCLs and the remaining three postimmortal LCLs lacked these characteristics. The products of tumor suppresser genes, p16(INK4A) and pRb, were downregulated in these two LCLs, and the p53 gene was mutated in N0005 LCL. We believe these results showed for the first time that some postimmortal EBV-transformed LCLs can become tumorigenic, contrary to previous reports, and that these LCLs provide an in vitro model of tumorigenesis induced by EBV.     

Studies on host-virus genome relationship in Epstein-Barr virus immortalized lymphoblastoid cell lines

Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.     

Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines.

Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.     

Use of cyclosporin a in establishing epstein-barr virus-transformed human lymphoblastoid cell lines

Summary 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. This work was supported by National Institute of Neurological and Communicative Disorders and Stroke Grants NS16367 (Huntington's Disease Center Without Walls) and NS 20012.     

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.     

Lysosomal acid hydrolases in established lymphoblastoid cell lines,transformed by Epstein-Barr virus,from patients with genetic lysosomal storage diseases

Summary Lysosomal acid hydrolases were determined in established lymphoblastoid cell lines, transformed in vitro by Epstein-Barr virus (EBV) from lymphocyte-rich cell populations isolated from the peripheral blood of patients with genetic lysosomal storage diseases—Hurler syndrome, Scheie syndrome, G_M1-gangliosidosis type 1 and type 2, Tay-Sachs disease, and I-cell disease—and from obligate heterozygotes for these diseases.The respective enzyme activity was undectectable in lymphoblastoid cells from the patients, but not from controls. Obligate heterozygotes could not always be distinguished from controls in lymphoblastoid cells as well as in leukocytes. These results suggest that established lymphoblastoid cell lines are useful material for the enzymatic study of genetic lysosomal storage diseases.     

Functional CD4(+) and CD8(+) T-cell responses induced by autologous mitomycin C treated Epstein-Barr virus transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4(+) and CD8(+) T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4(+) and (granzyme B positive) CD8(+) T-cells rosetting with McLCL. Granzymes A and B, IFN-gamma, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4(+) and CD8(+) T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.     

Acute and chronic infection of human lymphoblastoid cell lines with measles virus.

Several human continuous lymphoblastoid cell lines (LCL) having T or B characteristics were infected with low and high passage strains of measles virus. All of the cell lines were susceptible to one or the other or to both strains of measles virus with the production of typical syncytial giant cells and released cell-free infectious virus into the supernatant medium. There was no consistent pattern of susceptibility of LCL with either T or B characteristics to infection by measles virus. Viral induced cytolysis of the lymphoblastoid cells in many of the lines was marked, but in the LCL that could be maintained over longer periods of time, a state of chronic, less cytolytic and persistent infection could be established. The infection was characterized by the production of moderate amounts of cell-free infectious virus for up to 4 1/2 months after initial infection with little change in the number of viable cells in culture. Long-term low multiplicity of infection (MOI) experiments demonstrated that the cell-free infectious virus was being produced only by a small number of cells, but the majority of cells in culture contained measles antigen that was in a cell-restricted, noninfectious, or defective form. Electron microscopic examination of the chronically infected cells demonstrated that many of them contained aggregates of hollow tubular intranuclear nucleocapsids whose "stripped" appearance was in marked contrast to the larger granular intracytoplasmic nucleocapsids found during earlier stages of infection. It is theorized that the persistent infection of LCL may serve as a model in understanding the immune mechanisms which permit latent and chronic measles infection in man.     

Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse cells failed to relase TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.     

Epstein-Barr virus-transformed lymphoblastoid cell lines as antigen-presenting cells and "augmenting" cells for human CMV-specific Th clones

The ability of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) to present human cytomegalovirus (HCMV) antigen to a panel of HCMV-specific T helper (Th) clones was evaluated. Among the seven Th clones studied, only one clone (SP-CN/T3-16) proliferated well to HCMV presented by both autologous mononuclear cells (MNC) and LCL, and one clone (SP-CN/T3-9) proliferated significantly better to HCMV presented by autologous LCL than by autologous MNC. The majority of the HCMV-specific Th clones tested (five out of seven) responded much better to HCMV presented by MNC than to HCMV presented by LCL. The mechanism(s) responsible for the inefficiency of LCL to present HCMV to certain clones was studied. Our results suggested that the defect of LCL is not due to insufficient interleukin 1 production, insufficient MHC class II molecule expression, nor an inhibitory mechanism or factor. In this report, we also demonstrate that by adding a minimum amount of LCL along with MNC as antigen-presenting cells (APC), one can restimulate and expand Th clones much more efficiently than by using MNC alone as APC.     

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines.

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.     

Functional analysis and molecular characterization of spontaneously outgrown human lymphoblastoid cell lines

In vitro, the infection of human B-cells with the lymphotropic gammaherpesvirus Epstein-Barr virus (EBV) induces formation of permanently growing lymphoblastoid cell lines (LCL). In a spontaneously outgrown LCL (cell line CSIII), we detected nucleotide sequence variations of the EBV nuclear antigen 1 (EBNA1) RNA that was different from the reference sequence of EBNA1 in the prototypic EBV strain B95-8. In the present study, we molecularly and functionally characterized this virus isolate in comparison to LCL with the prototypic nucleotide sequence. Although we detected high functional similarity between CSIII and the other LCL, our data suggest that the lytic cycle might be ineffective in the CSIII LCL. DNA microarray analysis indicated that RNA binding motif, single stranded interacting protein 1 (RBMS1), which is typically expressed in latency III of EBV to prevent the lytic cycle, was the most overexpressed gene in CSIII LCL.     

Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines

DNA methylation is involved in development and in human diseases. Genomic DNA derived from lymphoblastoid cell lines (LCLs) is commonly used to study DNA methylation. There are potential confounding factors regarding the use of LCL-derived DNA, however, such as Epstein-Barr (EB) viral infection and artifacts induced during cell culture. Recently, several groups compared the DNA methylation status of peripheral blood leukocytes (PBLs) and LCLs and concluded that the DNA methylation profiles between them might be consistent. To confirm and extend theses results, we performed a comprehensive DNA methylation analysis using both PBLs and LCLs derived from the same individuals. Using the luminometric methylation assay, we revealed that the global DNA methylation level was different between PBLs and LCLs. Furthermore, the direction of change was not consistent. Comparisons of genome-wide DNA methylation patterns of promoter regions revealed that methylation profiles were largely conserved between PBLs and LCLs. A preliminary analysis in a small number of samples suggested that the methylation status of an LCL may be better correlated with PBLs from the same individual than with LCLs from other individuals. Expectedly, DNA methylation in promoter regions overlapping with CpG islands was associated with gene silencing in both PBLs and LCLs. With regard to methylation differences, we found that hypermethylation was more predominant than hypomethylation in LCLs compared with PBLs. These findings suggest that LCLs should be used for DNA methylation studies with caution as the methylation patterns of promoter regions in LCLs are not always the same as those in PBLs.