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.     

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.     

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.     

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.     

Effects of fractionated X-irradiation on subsequent response to acute X-irradiation in two human tumour cell lines in vitro

The exposure of two human tumour cell lines, one derived from a squamous cell carcinoma of the tongue (HN-1) and the other from an adenocarcinoma of the breast (MCF-7), to fractionated X-irradiation in vitro, resulted in altered sensitivity to subsequent acute X-irradiation exposure in the former but not the latter tumour cell type. The X-ray-pretreated HN-1 cells, designated HN-1/DXR11 cells, showed a significantly increased sensitivity to X-irradiation with a D0 of 0.97 Gy, compared with a figure of 1.39 Gy for the parental cells. No significant changes were noted in a number of basic cell kinetic or biological parameters in the X-ray-pretreated cells. However, this enhanced X-ray sensitivity in the HN-1/DXR11 cells was associated with decreased cellular levels of total intracellular glutathione. These findings are consistent with the theory that intracellular thiols are involved in protection from radiation damage. This is one of the first observations that prior exposure to X-irradiation can modify subsequent responses to acute X-irradiation treatment in human tumour cells.     

Different behavioural responses to anthropogenic noise by two closely related passerine birds

Anthropogenic noise, now common to many landscapes, can impair acoustic communication for many species, yet some birds compensate for masking by noise by altering their songs. The phylogenetic distribution of these noise-dependent signal adjustments is uncertain, and it is not known whether closely related species respond similarly to noise. Here, we investigated the influence of noise on habitat occupancy rates and vocal frequency in two congeneric vireos with similar song features. Noise exposure did not influence occupancy rates for either species, yet song features of both changed, albeit in different ways. With increases in noise levels, plumbeous vireos (Vireo plumbeus) sang shorter songs with higher minimum frequencies. By contrast, grey vireos (Vireo vicinior) sang longer songs with higher maximum frequencies. These findings support the notion that vocal plasticity may help some species occupy noisy areas, but because there were no commonalities among the signal changes exhibited by these closely related birds, it may be difficult to predict how diverse species may modify their signals in an increasingly noisy world.     

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.     

Isolation and characterization of mitochondria from human B lymphoblastoid cell lines.

Mitochondria were isolated from detergent-treated Epstein-Barr virus-transformed human lymphocytes to examine their potential use in the study of the functional expression of genetic disorders of the respiratory chain. The increase of cytochrome c oxidase activity in the mitochondrial fraction indicated a 6-fold purification of intact mitochondria. Polarographic and spectrophotometric studies revealed that the isolated mitochondria were functionally well preserved. Furthermore, the isolated mitochondria supported an active in organello protein synthesis, which was dependent on the presence of a respiratory substrate generating ATP and was essentially abolished by chloramphenicol or by a specific respiratory chain inhibitor, such as antimycin. Thus, B lymphoblastoid cell lines constitute a valuable source of mitochondria to investigate mitochondrial functions in patients affected by respiratory chain disorders.     

Search for inhibitors against herpes simplex virus type-I in cell extracts derived from human lymphoblastoid cell lines.

Cell extracts obtained from KB cells and 5 human lymphoblastoid cell lines including 2 from Burkitt's lymphoma (P3HR-1 and Raji), one each from nasopharyngeal carcinoma (no.223), acute lymphatic leukemia (MOLT-4) and a healthy person (NC-37) were tested for their inhibitory effects on the growth of herpes simplex virus type-1 (HSV-1) in green monkey kidney (GMK) cells by the plaque titration method. The relationship between the production of HSV-1 inhibitors and the degree of Epstein-Barr virus (EBV) genome repression in lymphoblastoid cells were also examined. Among the cell lines used P3HR-1 and no.223 cells produced a few EBV particles, Raji and NC-37 cells contained EBV genomes only, and MOLT-4 as well as KB cells were EBV genome-negative. The results revealed that P3HR-1 cell extract showed a tendency to inhibit HSV-1 growth in GMK cells but the other 4 lymphoblastoid cell lines and KB cells did not produce HSV-1 inhibitors, indicating that EBV genomes governing the formation of EBV structural antigens were not related to the production of HSV-1 growth inhibitors. The extracts from MOLT-4 cells, which are only a T lymphocyte cell line used in this study, stimulated HSV-1 growth in GMK cells significantly.     

Stimulation with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines generates HLA class I-specific CTLs with different target cell avidity.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.     

Immunoglobulin genes of the kappa light chain type from two human lymphoid cell lines are closely related.

As a first step in our studies of functionally rearranged K genes of man we cloned the germline JK-CK region from placenta DNA employing a mouse JK clone as hybridization probe. Subclones of the human JK-CK region were then used to characterize and clone the rearranged K genes of the lymphoid cell lines Walker and Daudi. The Walker cell line contains one rearranged and one germline K allele (K+,KO; ref. 1). Only one K gene was found in Daudi cells (K+). Restriction mapping and DNA sequencing showed, that the rearranged K genes from both cell lines are closely related. These features make the two cell lines particularly suitable for studies on the chromatin structure of K light chain genes. The 5' flanks of the two genes (388 bp) are identical while there is a 12% divergence between the VK gene segments themselves. This situation may reflect somatic mutation processes and/or gene conversion like events.     

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.     

Stimulation of peripheral human lymphocytes by autologous EBV genome-carrying lymphoblastoid cell lines.

EBV-carrying lymphoblastoid cell lines (LCL) can stimulate lymphocytes of the autologous donor [autologous stimulation (AS) assay] to blast transformation and generation of killer cells of broad-range cytotoxicity. We have tested the possibility of developing an EBV-specific AS assay for use in the demonstration of EBV-specific memory cells in the peripheral blood of normal donors. For this purpose, the stimulating lines were treated by heat and protein synthesis inhibitors to prevent the release of possible nonspecifically mitogeneic factors. Moreover, an extensive purification of the effector cells was achieved in the hope of removing a possible cellular contributor to the observed nonspecific cytotoxicity. None of these approaches was able to narrow down this nonspecific cytotoxicity to an EBV-specific response. We have shown that AS reaction (1) is not related to the release of lymphocyte mitogeneic factors by stimulating LCL, (2) is mediated by Fc receptor-negative T cells, and (3) does not require macrophage nor any other non-T helper cells.