Monitoring of epstein-barr virus DNA load in peripheral blood by quantitative competitive PCR.
(33/1897)
A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin's lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt's lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters. (+info)
A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells.
(34/1897)
Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system. (+info)
Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji.
(35/1897)
Human lymphoid cells (Raji) were exposed to water-soluble compounds from cigarette smoke (CS) generated in a smoking machine. DNA damage, as detected by alkaline single-cell microelectrophoresis (COMET assay), was induced in a time- and concentration-dependent manner in the cells. Most of the rapidly induced DNA damage was attributable to direct-acting compounds since cytochrome P450-related metabolic activities (ethoxy- and pentoxyresorufin-O-deethylases and coumarin-7-hydroxylase) were absent or very low. In addition, induction of DNA damage could be inhibited only slightly by beta-naphthoflavone and coumarin. Vitamin C enhanced DNA damage in Raji cells probably by redox cycling of catechol and hydroquinone present in CS implicating reactive oxygen intermediates as another source of DNA damage. N-acetylcysteine, a radical scavenger and glutathione precursor, reduced DNA damage in Raji cells when exposure to CS was followed by 2 h post-incubation in culture medium. Unrepaired DNA damage caused by CS persisted longer than gamma-irradiation-induced DNA damage. Among the CS constituents, acrolein, but not formaldehyde and acetaldehyde, induced DNA damage although less intensely than CS itself. At 50 and 100 microM concentrations, acrolein also inhibited repair of gamma- irradiation-induced DNA damage in the COMET assay. Inhibition of DNA synthesis by acrolein at 50 microM was demonstrated by an immunochemical assay for bromo-deoxyuridine incorporation; however, inhibition of a representative repair enzyme, 8-oxoguanosine hydrolase, by either CS or acrolein was not observed. The present results further confirm the presence of direct-acting genotoxic components and inhibitors of DNA repair in the gas phase of tobacco smoke, that may contribute to DNA damage and smoking-associated cancers of the upper aerodigestive tract. (+info)
The cytotoxicity of DNA carboxymethylation and methylation by the model carboxymethylating agent azaserine in human cells.
(36/1897)
Carboxymethylating agents are potential sources of endogenous DNA damage that have been proposed as possible contributors to gastrointestinal carcinogenesis. The cytotoxicity of the model DNA carboxymethylating agent azaserine was investigated in human cells. Expression of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) did not affect sensitivity to the drug in two related Raji Burkitt's lymphoma cell lines. DNA mismatch repair-defective variants of Raji cells which display increased tolerance to DNA methylation damage were not selectively resistant to azaserine. Complementary results were obtained with a second carboxymethylating agent, potassium diazoacetate. In contrast, lymphoblastoid cell lines representative of each of the xeroderma pigmentosum complementation groups, including the variant, were all significantly more sensitive to azaserine than nucleotide excision repair-proficient cells. The hypersensitivity of XP cells was not due to systematic differences in the concentrations of intracellular thiol compounds or related thiol metabolizing enzymes. The data indicate that of the two types of potentially lethal DNA damage which azaserine introduces, carboxymethylated bases and O(6)-methylguanine, the former are repaired by nucleotide excision repair and are a more significant contributor to azaserine lethality in human cells. (+info)
Mitotic separation of daughter cells in the human lymphoma B cell line Daudi involves L-selectin engagement and shedding.
(37/1897)
BACKGROUND AND OBJECTIVE: A novel role for shedding of the surface molecule L-selectin has been proposed as an adjunctive phenomenon during cell detachment from marrow stroma or vessel endothelium. We wished to examine whether variations in expression of L-selectin on a lymphoma B cell line were linked to shedding. DESIGN AND METHODS: Mapping of L-selectin expression on the surface of Daudi lymphoma cells was performed by flow cytometry, fluorescence microscopy, and electron microscopy. Levels of shed L-selectin were evaluated by Western blotting of culture supernatants. Evaluation of cell cycle and proliferative activity was performed by flow cytometry. RESULTS: Large Daudi cells in S+G(2)/M phases were L-selectin positive, whereas small Daudi cells in G(0)/G(1) phase were L-selectin negative. During mitosis, L-selectin was distributed along the cleavage furrow, and gradually lost. Electron microscopy revealed that separating Daudi cells were negative for L-selectin on the entire surface, except minute aggregates of L-selectin within the cleavage furrow. Addition of agents known to interfere with the ligand-binding portion of L-selectin (sulfatides, MoAbs: Lam1.3 and TQ1) results in loss of L-selectin. Removal of L-selectin by digestion with chymotrypsin inhibits Daudi proliferation. The MoAb FMC46 did not interfere with proliferation. Proliferating Daudi cells produced large quantities of shed L-selectin. Inhibition of Daudi proliferation resulted in levels of shed L-selectin below the limit of detection. INTERPRETATION AND CONCLUSIONS: L-selectin is re-distributed on the cell surface of Daudi cells during the last phase of mitosis, in which plasma membrane invagination occurs between newly formed daughter cells. Shedding of L-selectin is involved in the cytokinesis of Daudi cells. (+info)
Inheritance of chromosomally integrated human herpesvirus 6 DNA.
(38/1897)
Human herpesvirus 6 (HHV-6) genome has been detected in several human lymphoproliferative disorders with no signs of active viral infection, and found to be integrated into chromosomes in some cases. We previously reported a woman with HHV-6-infected Burkitt's lymphoma. Fluorescence in situ hybridization showed that the viral genome was integrated into the long arm of chromosome 22 (22q13). The patient's asymptomatic husband also carried HHV-6 DNA integrated at chromosome locus 1q44. To assess the possibility of chromosomal transmission of HHV-6 DNA, we looked for HHV-6 DNA in the peripheral blood of their daughter. She had HHV-6 DNA on both chromosomes 22q13 and 1q44, identical to the site of viral integration of her mother and father, respectively. The findings suggested that her viral genomes were inherited chromosomally from both parents. The 3 family members were all seropositive for HHV-6, but showed no serological signs of active infection. To confirm the presence of HHV-6 DNA sequences, we performed polymerase chain reaction (PCR) with 7 distinct primer pairs that target different regions of HHV-6. The viral sequences were consistently detected by single-step PCR in all 3 family members. We propose a novel latent form for HHV-6, in which integrated viral genome can be chromosomally transmitted. The possible role of the chromosomally integrated HHV-6 in the pathogenesis of lymphoproliferative diseases remains to be explained. (+info)
Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt's lymphoma.
(39/1897)
Ligation of the Fas receptor induces death-inducing signaling complex (DISC) formation, caspase activation, and subsequent apoptotic death of several cell types. Epstein-Barr virus (EBV)-positive group III Burkitt's lymphoma (BL) cell lines have a marked resistance to Fas-mediated apoptosis, although expressing each of the DISC components, Fas/ APO-1-associated death domain protein (FADD), and caspase-8 (FLICE/MACH/Mch5). The apoptotic pathway distal to the DISC is intact because ceramide analogs, staurosporine, and granzyme B activate caspase-3 and induce apoptosis. Fas resistance was not explained by the putative death-attenuating caspase-8 isoforms. However, while Fas-activated cytosolic extracts from sensitive cells were capable of processing both procaspase-8 and procaspase-3 into active subunit forms, resistant cell extracts did not possess either of these activities. Accordingly, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed higher transcript levels for the FLICE-inhibitory protein (FLIP(L)) in resistant cells and the ratio of caspase-8 to FLIP(L) measured by competition RT-PCR analysis directly correlated with susceptibility to Fas-mediated apoptosis of all cell lines. In addition, modification of the caspase-8/FLIP(L) ratio by caspase-8 or FLIP(L) overexpression was able to alter the susceptibility status of the cell lines tested. Our results imply that the relative levels of caspase-8 and FLIP(L) are an important determinant of susceptibility to Fas-mediated apoptosis. (+info)
Replication of GB virus C (hepatitis G virus) in interferon-resistant Daudi cells.
(40/1897)
We previously reported that Daudi cells, a Burkitt's lymphoma cell line, were capable of supporting productive infection of hepatitis C virus (HCV). During continual cultivation after HCV infection, the culture became resistant to interferons (IFNs). This resistant cell line, coded as H-903, was used as host cells for replication of GB virus C (GBV-C), also known as hepatitis G virus. GBV-C RNA was detected in the culture by reverse transcription-PCR for more than 130 days after inoculation, while it was detected for 44 days but not later in the parental IFN-sensitive Daudi cells. Productive infection of GBV-C in the H-903 system was confirmed by serially inoculating supernatants from infected cultures into uninfected cells. The viral E2 antigen was detected by immunofluorescence in the cells inoculated with the fifth passage of GBV-C. The presumed capsid-coding region of the viral genome in the inoculum, in the serially passaged virus, or in the virus produced by a long-term culture was only 16 amino acids long, suggesting that the GBV-C with a short core sequence was replication competent. (+info)