Control Lymphoblastoid Cell Lines Research Articles

REVERSAL OF MALIGNANT CELL GROWTH PHENOTYPE BY HYDROXYUREA-INDUCED LOSS OF EPSTEIN-BARR VIRUS EPISOMES

Background: Little consideration has been given to virus-directed therapy of EBV-associated AIDS malignancies, first, because latent herpesviruses are intractable to available antiviral chemotherapy and, second, because of the uncertainty that EBV makes any contribution to the long-term maintenance of the malignant cell phenotype. Using cell lines that reflects the phenotypic heterogeneity of AIDS-related lymphomas, we examined the effect of the ribonucleotide reductase inhibitor hydroxyurea (HU), known to accelerate loss of extrachromosomal DNA, on EBV episomal copy number and subsequent cell proliferation. Methods: EBV-positive lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cells were treated with low concentrations (50µM) of HU. EBV DNA content in treated versus untreated cells was measured serially by Southern blot and fluorescent in situ hybridization (FISH). Single cell clones from HU-treated BL cells, obtained via the cell sorter, were examined for growth characteristics in tumorigenicity assays. Results: Primary B cells immortalized by EBV (LCL) ceased to proliferate in HU and could not be maintained as long-term cultures. On FISH analysis of treated LCLs, individual cells were detected that did not contain hybridization signals implying complete climination of EBV episomes. The median episome count in HU-treated versus untreated LCLs was 6 versus 21(p<0.0001, Wilcoxon 2-sample test). A control LCL, IB4, which contains only integrated (not episomal) EBV DNA, was not growth inhibited in the presence of HU, implicating episomal loss and not drug toxicity in the altered growth phenotype. In the case of the BL cell line Akata, there was a 99.5% reduction of EBV DNA by Southern blot analysis after 15 cell population doublings on HU. By contrast, there was no EBV DNA loss from untreated controls. At an individual cell level, approximately half the treated cells were EBV-negative by FISH. Of 131 single cell clones obtained from the treated population, 57 were EBV-negative. Unlike EBV-positive counterparts, Akata clones "cured" of EBV episomes were no longer able to grow in 0.1% serum, form colonies in soft agar or produce tumors in SCID mice despite retaining the characteristic (8,14) rearrangement. Conclusion: These findings provide the first evidence for successful eradication of a latent herpesvirus from any cell population. They challenge the view that EBV is important only in the initiation of tumorigenesis, but plays no role in the long-term maintenance of the malignant cell phenotype. Together with the apparent reversal of EBV immortalization observed in primary B cells, these data suggest latent EBV is an appropriate and accessible therapeutic target and have prompted initiation of clinical trials of HU in EBV-related lymphoproliferative diseases of AIDS

Cytoplasmic transfer of the mtDNA nt 8993 T--&gt;G (ATP6) point mutation associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation with a decrease in state III respiration and ADP/O ratio.

A point mutation in the mtDNA-encoded ATP6 gene (T-->G at nt 8993) associated with Leigh syndrome in two pedigrees was found to decrease ADP-stimulated (state III) respiration and the ratio of ADP molecules phosphorylated to oxygen atoms reduced (ADP/O ratio) but did not affect 2,4-dinitrophenol (DNP)-uncoupled respiration, suggesting a defective mitochondrial H(+)-translocating ATP synthase. Intact mitochondria isolated from patient and control lymphoblastoid cell lines were tested for state III, ADP-limited (state IV), and DNP-uncoupled respiration with various substrates. Mitochondria isolated from patient lymphoblasts harboring 95-100% of mtDNAs carrying the nt 8993 T-->G mutation showed state III respiration rates 26-50% lower than controls while having normal DNP-uncoupled rates. This resulted in state III/DNP ratios of 0.52-0.70 in patient mitochondria versus 0.88-0.97 in controls. The ADP/O ratio was also decreased 30-40% in patient mitochondria. Patient lymphoblasts heteroplasmic for the nt 8993 mutation were enucleated by using Percoll gradients and the cytoplasts were fused to mtDNA-deficient (rho 0) cells by electric shock. Cybrid clones homoplasmic for the wild-type nucleotide (T) at nt 8993 gave state III/DNP and ADP/O ratios similar to those of control cybrids, whereas cybrid clones homoplasmic for the mutant nucleotide (G) showed a 24-53% reduction in state III respiration, a state III/DNP ratio of 0.53-0.64, and a 30% decrease in the ADP/O ratio. Thus, the reduced state III respiration rates and ADP/O ratios are linked to the T-->G mutation at nt 8993.

Possible Role of Chromatin Alteration in the Radiosensitivity of Ataxia-telangiectasia

Cells derived from individuals with ataxia-telangiectasia (A-T) are known to exhibit increased sensitivity to ionizing radiation and certain radiomimetic chemical agents. Here we summarize our findings regarding the role of chromosome damage and repair in this radiosensitivity. Lymphoblastoid cells derived from A-T homozygotes were characterized for initial chromosome (premature chromosome condensation) and DNA (neutral filter elution) damage and repair kinetics in cells from G1 and G2 cell cycle phases. Despite initial levels of DNA damage being similar to normal controls, A-T cells exhibited nearly a two-fold higher initial amount of chromosome damage. Different A-T cell lines exhibited differing chromosome repair capacities compared with control lymphoblastoid cell lines. These results suggest that A-T cells have an altered chromatin structure whereby DNA double-strand breaks are apparently more efficiently converted into chromosome breaks. Four A-T heterozygote cell lines were examined for chromosome damage and repair in the same fashion and all exhibited increased levels of chromosome damage, although the degree of sensitivity was more prominent in G2 phase cells (two-fold higher) than in G1 phase cells (1.5-fold higher than normal controls). These results suggest that A-T heterozygotes also exhibit an altered chromatin structure which impacts on chromosome damage expression. Of interest, A-T cells also exhibited increased chromosome stickiness after irradiation, and telomere regions appeared to be frequently involved. While the molecular basis for preferential telomere involvement is not understood, these results again suggest that structural alterations in the chromatin of A-T cells may play an important role in A-T radiosensitivity.

Methylation of the 5' flanking sequences of the ribosomal DNA in human cell lines and in a human-hamster hybrid cell line.

In a human lymphoblastoid cell line (Z83) in which rDNA genes on chromosome 22 are amplified but transcribed at a low level, immunocytological studies with antibodies to 5 methylcytidine provided evidence for hypermethylation of the rDNA. The extent of methylation of the 5' flanking sequences of the ribosomal DNA was examined by comparing the size of restriction fragments obtained by digestion of genomic DNA with EcoRI and HpaII or EcoRI and MspI. Southern blots indicated hypermethylation of the 5' flanking sequences of many copies of rRNA genes in these cells, but not in a control lymphoblastoid cell line without rDNA amplification. Results obtained with a somatic hybrid human-hamster cell line, in which the rRNA genes on the single human chromosome 22 are inactive, showed that only a small fraction of the CCGG sites in the 5' flanking sequences of the transcriptionally silent rRNA genes in this hybrid were methylated. Since inactive rRNA genes can show such a minimal level of methylation, it is likely that the extreme hypermethylation of the amplified rRNA genes in Z83 occurred in association with their inactivation rather than following it.

Two Types of DNA Ligase I Activity in Lymphoblastoid Cells from Patients with Bloom's Syndrome

DNA ligases I and II were separated by hydroxylapatite (HA) column chromatography in cell‐free extracts of lymphoblastoid cell lines (LCLs) derived from two unrelated patients with Bloom's syndrome (BS) and two healthy individuals. The specific activity of ligase I from the crude extract was consistently lower in GM3403, a BS LCL from an Ashkenazi Jewish patient, than in normal control LCLs. By contrast, the level of ligase I activity in BSL‐2KA, another BS LCL derived from a Japanese patient, was equivalent to those in normal LCLs, although GM3403 and BSL‐2KA shared the feature of exceedingly high frequency of spontaneous sister‐chromatid exchange. The levels of total ligase activity in crude extracts without the separation into the two forms, however, were approximately two‐fold higher for the two BS LCLs than for the normal LCLs. Partial purification by chromatography on a DEAE‐cellulose 23 column and a phosphocellulose column did not affect the superiority of the two BS LCLs over the normal LCLs in the specific activity of the total ligases. Nonetheless, subsequent application to an HA column again resulted in much less elevation of the specific activity of ligase I for GM3403 than for BSL‐2KA and control LCLs. The levels of ligase II activity, accounting for 4‐13% of total ligase activity, were similar among the LCLs examined. Irrespective of the extent of purification, essentially no difference in the heat lability of DNA ligase I was detected among the four LCLs. These findings suggest that there may exist among BS LCLs at least two types of subtle abnormality of DNA ligase I itself and/or a putative substance modulating the enzyme function.

Infection of Epstein‐Barr virus‐transformed lymphoblastoid B cells by the human immunodeficiency virus: evidence for a persistent and productive infection leading to B cell phenotypic changes

Four Epstein-Barr virus-positive lymphoblastoid cell lines (LCL) were successfully infected in vitro with immunodeficiency virus type 1 (HIV-1) as demonstrated by reverse transcriptase activity and p24 HIV antigen in culture supernatants, positive cell staining for gag-encoded HIV proteins, presence of viral HIV genome by Southern blot analysis and ulstrastructural observations. In addition, both HIV-1-infected B cells and their supernatants efficiently transactivated the chloramphenicol acetyl transferase reporter gene which is under the control of the HIV-1 long terminal repeat. The LCL cells displayed long-term HIV-1 infection and production, but no cytopathic effects were observed. Cytofluorimetric analysis did not detect membrane CD4 presence in the LCL cells before and after HIV-1 infection; moreover, a minute amount of CD4 mRNA was observed only in one of the LCL. A monoclonal antibody specific for the viral binding site of the CD4 molecule delayed, but did not block, HIV-1 infection of the LCL cells. Following HIV-1 infection, changes in LCL phenotype were observed, consisting of a decrease in CD23- and CD39-positive cells, and a concomitant increase of cells with surface CD10 and Bac-1. Furthermore, HIV-1-infected LCL cells did not grow in tight clumps, as usually observed in uninfected LCL, but as disperse suspensions, and formed more agar colonies than control LCL. However, despite this apparent acquisition of a malignant-like phenotype, c-myc proto-oncogene rearrangement was not detected. The appearance of cells with new characteristics did not seem due to clone selection by HIV-1 infection, since all the LCL conserved their clonotypic pattern of IgH chain rearrangement. The acquisition of malignant-like features by HIV-infected B cells might be clinically significant in terms of the pathogenesis of non-Hodgkin's B cell lymphomas, which occur frequently in AIDS patients.

TaqI polymorphism within the c-Ha-ras-1 VTR is associated with melanoma

N. K. Hayward, D. J. Nancarrow, P. G. Parsons, C. Kidson, and K. A. O. Ellem Queensland Institute of Medical Research, Bramston Terrace, Herston, 4006, Australia Rare alleles of the c-Ha-ras-1 locus detected by cleavage of DNA with MspI/HpaII have been reported to occur signifi- cantly more frequently in patients with various types of cancer than in healthy controls (Krontiris et al. 1985; Lidereau et al. 1986; Hayward et al. 1988). However, others have failed to find an association (Heighway et al. 1986; Thein et al. 1986; Ceccherinni-Nelli et al. 1987; Ishikawa et al. 1987; Gerhard et at. 1987; Radice et al. 1987; Wyllie et al. 1988). TaqI cleavage results in two major types of polymorphism at the human c-Ha-ras-1 locus (Thein et al. 1986; Pierotti et al. 1986; Ceccerinni-Nelli et al. 1987). One of these overlaps with the same alleles as MspI/HpaII (depending on variations in VTR length), whereas the other occurs because of the pres- ence of TaqI sites within the VTR itself, resulting in patterns designated Tp alleles (Pierotti et al. 1986). The Tp alleles appear closely linked to VTR length, occurring always within the largest (a4) common allele and in rare alleles of similar size (Thein et al. 1986; Pierotti et al. 1986). However, in one report, the Tp alleles appeared to occur in a proportion of samples with the second largest (a3) common allele, but not in the single case studied with an a4 allele (Ceccherinni-Nelli et al. 1987). Recently, melanoma patient lymphocyte DNAs have been reported to have a significantly higher frequency of Tp alleles than lymphocyte DNAs from unaffected controls (Radice et al. 1987). To investigate this association further and to clarify in which alleles the Tp fragments occur, we have undertaken a study of melanoma (MM) cell line and melanoma tumour DNA samples, together with a series of DNAs from control lymphoblastoid cell lines (LCLs) for comparison. All DNA samples were derived from Caucasian (pred- ominantly Anglo-Saxon) individuals. These comprised 66 un- affected control LCLs, 34 MM cell lines and 22 fresh MM biopsies. After overnight digestion with 20 units of TaqI, DNA fragments were electrophoresed in 1.4% agarose gels. DNA was Southern blotted onto nylon membranes and hybridised with the 6.6-kb BamHI c-Ha-ras-1 fragment, contained within the plasmid pEJ, labelled with S2p-dCTP. Hybridisation was carried out for 22h at 65°C in 6 x SSC, 5raM EDTA, 0.1% SDS, 5 × Denhardt's solution and 500gg/ml denatured sheared salmon sperm DNA. Washes were performed at 65°C Offprint requests to: N. K. Hayward for lh in 2 x SSC, 0.1% SDS followed by lh in 0.3 x SSC, 0.1% SDS at the same temperature. By studying MspI/HpaII data previously obtained from our samples (Hayward et al. 1988), we found that Tp alleles occurred without exception in all control and melanoma DNAs with MspI/HpaII VTR-containing fragments larger than or equal to 2.32kb. In no instance did a sample possessing an a3 common allele, or rare allele of similar size, give rise to Tp Fig. 1. DNAs from 7 different MM cell lines digested with TaqI and hybridised to the plasmid pEJ containing the c-Ha-ras-i genomic se- quence. All major allelic forms (Pierotti et al. 1986) are shown, e.g. lanes A, G contain bl and Tpl alleles; lanes B, F have Tml and Tpl alleles; lane C is homozygous for Tpl; lane D has bl and b2; and lane E a b3 and a Tpl allele. There is little or no microvariation of the smallest (300 bp) Tpl fragment although considerable microvariation is seen with the two Tp fragments around 690 and 830 bp (lanes A-C). The major 2200-bp Tpl allele fragment also shows a degree of micro- variation and co-migrates with a faint invariant fragment from the 5' end to the gene observed in DNAs lacking Tp alleles (lane D). Sizes of the major allelic fragments are indicated in base pairs (bp). The 300-bp fragments have run off the end of the gel in lanes E, F, G. Note: bl, b2 and b3 alleles in TaqI digested DNA are equivalent to al, a2 and a3 alleles respectively in MspIIHpaII digested DNA (Kron- tiris et al. 1985; Pierotti et al. 1986)

Studies on the oncogenic potential of epstein-barr-virus (EBV)-infected B cells in aids-related disorders

Spontaneous lymphoblastoid cell lines (LCLs) were established from the peripheral blood of 10 human immunodeficiency virus (HIV)-seropositive patients in order to investigate whether or not progression of the cells towards a malignant state could be traced. The LCLs studied displayed no differences in their surface phenotype, karyotype, and tumorigenicity in nude mice as compared with a wide panel of control LCLs. Furthermore, no c-myc rearrangement could be detected in any of the LCLs. However, 4 of the 10 LCLs derived from HIV-seropositive patients formed colonies in agar with a cloning efficiency of 0.1-0.9%. This percentage was much lower than that of a control neoplastic B cell line (50%), but consistently higher than that observed for a battery of spontaneous LCLs. The cells of a number of sublines that were derived from the agar colonies expressed new activation markers (CD10 and Bac-1) but did not induce tumors in nude mice or display chromosomal abnormalities. These sublines might comprise cells that have progressed towards a more markedly transformed state.

Heterogeneity of B cell abnormality in 13 patients with primary a/hypo-gammaglobulinemia.

B cell abnormality in 13 patients with primary a/hypo-gammaglobulinemia were studied. Number of peripheral B cells was normal in 7 of 13 patients but very small in the remaining six patients.In the first step, we established lymphoblastoid cell lines (LCL) by EB virus and evaluated immunoglobulin production by LCL. LCLs were established in 7 patients, whose number of peripheral B cells was normal. IgM dominant pattern of immunoglobulin production was seen in 6 of the 7 LCLs as LCLs from cord blood cells. These results suggested that the B cells of these patients were immature, like the B cells of cord blood. Only three LCLs could be established from the remaining 6 patients, whose number of peripheral B cells was very small. These three LCLs produced dominantly IgM, dominantly IgG and no immunoglobulin. Various B cell abnormalities were sugested, even when the number of peripheral B cells was very small.Next, we purified B cells from 5 patients and evaluated B cell functions, such as activation, proliferation and differentiation by using SAC and IL 2. In this assay system, B cell abnormality was devided into three groups. We concluded that the abnormality was in the step of activation/proliferation by SAC in 1 patient, in the step of proliferation response by IL 2 in 1 patient and in the step of differentiation to Ig secreting cell by IL 2 in 2 patients. B cells from 1 patient, whose LCL secreted IgG and IgM equally, showed no abnormality even in this assay.Finally, we examined DNA and mRNA of immunoglobulin of the LCL, which produced no immunoglobulin. It was confirmed that the LCL was pre B cell line, and suspected that μm-RNA of the LCL had an abnormality because the number of M. W. was small compared to that of control LCLs secreting IgM.

VARIABLE EXPRESSION OF B CELL FUNCTION IN FAMILIES WITH X-LINKED AGAMMAGLOBULINEMIA (XLA)

XLA patients have a defect of B cell maturation involving the pre B to B cell stages. This block is not absolute since circulating immunoglobulin (Ig) B cells may be found in XLA. To evaluate B cell function in XLA, we studied 37 patients, including 31 from 13 families with multiple affected members. Sera from 33 untreated patients contained measurable IgG, 26 exceeding 200 mg%; IgG subclasses were proportionally diminished. IgM, at low concentrations, was detected in 27; IgA1 and IgA2 in 7, and IgA1 only in 1 patient. Following immunization with bacteriophage øX 174, 13 failed to clear antigen and to produce antibody (Ab), 2 cleared phage without making detectable Ab. Of the 22 with low Ab titers, 9 switched from IgM to IgG (IgG1-3) after repeated immunization. Six of the 13 families with multiple affected members showed discordance in their response: some did, others did not clear phage and make Ab, correlating with the number of circulating B cells. EBV induced lymphoblastoid cell lines (LCL) obtained, with difficulty, from 14/19 patients were analyzed for Ig synthesis: 7 LCL synthesized IgM, and 1 IgA1. Six LCL produced both IgM and IgG; 4 IgG subclasses were present in 1 LCL, IgG1-3 in 3 LCL, IgG1 and 3 in 2 LCL, IgG1 and 2 in 1 LCL. The amount of Ig produced was significant, although 6/14 LCL produced only 5-10% of control LCL, the quantity being highest for IgM, followed by IgG3>IgG1 >IgG2>IgG4. In contrast, mitogen stimulated peripheral blood mononuclear cells produced predominantly IgG (80% of cultures); IgM was found in only 10% and IgA in none. These studies demonstrate a broad spectrum of B cell defects in XLA, often variably expressed in members of the same family. B cells, although markedly restricted in number and function, were demonstrated in every XLA patient suggesting a broad defect of B cell maturation and differentiation into Ig synthesizing cells.

Activation and somatic mutation of the translocated c-myc gene in burkitt lymphoma cells.

In contrast to other human tumors in which the c-myc gene and its transcript are greatly amplified, careful analysis of t(8;14) Burkitt cell lines indicates that the c-myc transcript is marginally, and in some cases not at all, increased by comparison to control lymphoblastoid cell lines. Instead, there is a more subtle alteration in the expression of the translocated c-myc gene characterized by a shift in promoter utilization and an apparent insensitivity to the regulation that inactivates the normal c-myc allele within these same cells. In some Burkitt cell lines, such deregulation might be because of the loss of a putative control region through removal of the large dual promoter/leader segment of the c-myc gene. In other cell lines, however, this deregulation may be explained by somatic mutations that occur within the putative control region even though it is located many hundreds of bases from the translocation breakpoint.

Transformation and repair replication in lymphocytes from ataxia telangiectasia.

Peripheral blood leukocytes (PBL) isolated from five patients with ataxia telangiectasia (AT) proved more difficult to transform following addition of exogenous Epstein-Barr virus than PBL isolated from AT heterozygotes or normal adults. PBL isolated from one AT patient transformed within the range expected for normal PBL. Once established in culture, the resulting lymphoblastoid cell lines (LCLs) were immortal and, though they grew slower than normal control LCLs, provided useful material for studying cellular phenotypes associated with AT lymphoid cell lines. All the resulting LCLs established from ataxia were more sensitive to X-irradiation than were LCLs established from controls as measured by colony formation in microtiter plates. Furthermore, X-ray-induced inhibition of semiconservative DNA synthesis in ataxia LCLs was less than that seen in normal LCLs. These results are in agreement with those obtained using cultured AT fibroblasts, indicating that in vitro transformation by exogenously added Epstein-Barr virus does not alter the phenotype of the ataxia cell as measured by these two parameters. However, no deficiency in X-ray-induced excision repair of DNA was demonstrable in LCLs established from four AT patients. Nor was there a deficiency in AT LCL host cell reactivation of herpes simplex virus X-irradiated under anoxic conditions. Taken together, these data point toward a defect in ataxia lymphoblasts other than repair enzyme(s) per se, one possibly associated with chromosomal structure, function, or modification.

Ataxia telangiectasia: an anomaly in DNA replication after irradiation

The effect of increasing dose of gamma-radiation on DNA synthesis in an ataxia telangiectasia lymphoblastoid cell line and a number of control lymphoblastoid cell lines was investigated. No significant inhibition of low molecular weight DNA synthesis was observed in the AT cell line at doses which resulted in considerable inhibition in the control cell lines. At higher doses, 600 to 800 rad, low molecular weight DNA synthesis and chain elongation were enhanced in the AT cell line. At time course study of DNA synthesis after 200 rads of gamma-radiation, revealed no appreciable inhibition of low and high molecular weight DNA synthesis up to 60 minutes postirradiation. However, in control cell lines, overall DNA synthesis was depressed to a level 50% of that shown by the unirradiated cells.