Ataxia Telangiectasia Mutated Kinase Controls Chronic Gammaherpesvirus Infection

Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.     

Epstein-Barr virus and oral squamous cell carcinoma in patients without HIV infection: viral detection by polymerase chain reaction.

In order to test the hypothesis that Epstein-Barr virus (EBV) may be a cofactor for oral squamous cell carcinoma (OSCC) the authors evaluated tumour cells from OSCC of 108 patients without HIV infection, for the presence of EBV DNA by polymerase chain reaction. The sequences of oligonucleotides used in the amplification and hybridization included a set for the DNA polymerase region. The amplification was detected using an ELISA assay with peroxidase. EBV DNA was detected in 17.59% of the tumours. Inhibition studies showed that the ability to detect EBV DNA was not affected by the pathological material, suggesting that the negative PCR results in these samples were not caused by PCR inhibitors in the biopsy. Results revealed that 63.1% of the tumours (12 cases) were DNA positive affecting the lateral margin of the tongue, and were statistically significant (p < 0.001; chi 2). In the pool of tumours with EBV DNA only 26.3% (5 of 19 cases) were well differentiated OSCCs whereas the remaining 73.7% (14 of 19 cases) were moderately and poorly differentiated OSCCs, with a statistical significance of p = 0.08; chi 2. This study suggests a relationship between OSCC and EBV.     

Instability of extrachromosomal cosmid DNA in SV40-transformed human (ataxia-telangiectasia) cells

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.     

Coupling of histone mRNA levels to radioresistant DNA synthesis in ataxia-telangiectasia cells

Cloned genomic DNA for human histone H1, H3 and H4 genes has been used to determine the effects of -radiation on histone mRNA levels and synthesis in ataxia-telangiectasia cells. Synthesis of histone mRNA was determined in cells synchronized with aphidicolin. Effects of irradiation on DNA synthesis and passage through S phase were also monitored. Irradiation was found to slow the passage of control cells through the cell cycle but had no effect on progression of ataxia-telangiectasia cells. H1 and core histone mRNA synthesis was inhibited by radiation in two control cell lines after release from aphidicolin block. No inhibition was observed in one ataxia-telangiectasia cell line and a small degree of inhibition in a second. An increased level of mRNA was observed in both irradiated control and ataxia-telangiectasia cells at 5–7 h post-irradiation compared to unirradiated cells. Similar results were obtained in log phase cells. These results demonstrate that histone mRNA synthesis is radioresistant in ataxia-telangiectasia cells and is coupled to radioresistant DNA synthesis in these cells.     

Defective DNA topoisomerase II in ataxia-telangiectasia cells

A number of characteristics in the human genetic disorder ataxia-telangiectasia are compatible with an alteration to chromatin structure or the recognition of that structure by an enzyme or DNA binding protein. We describe here reduce activity of DNA topoisomerase type II in a number of Epstein Barr Virus-transformed ataxia-telangiectasia lymphoblastoid cell lines. Enzyme activity was reduced 10-fold or greater in 4 out of 5 cell lines compared to controls. In the remaining cell line approximately a 2-3 fold reduction was evident in partially purified extracts. DNA topoisomerase type I activity was found to be the same as controls in all the cell lines. Northern blot analysis revealed that the same level of DNA topoisomerase II mRNA was expressed in ataxia-telangiectasia and control cell lines. The size and amount of the enzyme did not differ appreciably from that observed in control cells. The reduced activity of DNA topoisomerase II in ataxia-telangiectasis cells might be explained by amino acid substitutions, small deletions in DNA or by a defect in post-translational modification in these cells.     

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.     

ATM signaling and genomic stability in response to DNA damage

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed.     

Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments

Nasopharyngeal carcinoma (NPC) is a prevalent tumour in southern China and southeast Asia, particularly in the Cantonese population, where its incidence has remained high for decades. Recent studies have demonstrated that the aetiology of NPC is complex, involving multiple factors including genetic susceptibility, infection with the Epstein-Barr virus (EBV) and exposure to chemical carcinogens. During development of the disease, viral infection and multiple somatic genetic and epigenetic changes synergistically disrupt normal cell function, thus contributing to NPC pathogenesis. NPC is highly radiosensitive and chemosensitive, but treatment of patients with locoregionally advanced disease remains problematic. New biomarkers for NPC, including EBV DNA copy number or methylation of multiple tumour suppressor genes, which can be detected in serum and nasopharyngeal brushings, have been developed for the molecular diagnosis of this tumour. Meanwhile, new therapeutic strategies such as intensity-modulated radiation therapy and immuno- and epigenetic therapies might lead to more specific and effective treatments.     

Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells.

Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome.     

Replicative DNA synthesis in permeable fibroblasts from normal individuals and ataxia-telangiectasia patients

Replicative DNA synthesis in normal human fibroblasts was inhibited by 50% when they were X-irradiated (8 Gy) and made permeable 30 min later, whereas only a slight inhibition (20%) was observed in similarly treated ataxia-telangiectasia cells. Treatment of irradiated normal cells with caffeine (2 mM) before permeabilization reversed the inhibitory effects of X-rays, buf caffeine had no effect on DNA synthesis in permeable ataxia-telangiectasia cells. Diadenosine tetraphosphate (0.1 mM) did not affect DNA synthesis in permeable normal fibroblasts.     

Differential sensitivities to gamma radiation of human bladder and testicular tumour cell lines

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.     

DNA of Epstein-Barr virus. I. Comparative studies of the DNA of Epstein-Barr virus from HR-1 and B95-8 cells: size, structure, and relatedness.

We have compared the properties of the DNA of Epstein-Barr virus (EBV) purified from HR-1 (EBV HR-1 DNA) and B95-8 (EBV B95-8 DNA) continuous lymphoblast cultures. Our data indicate that (i) the S suc of native EBV DNA relative to T4D DNA is 55S. Using the modified Burgi-Hershey relationship (5), we estimate the molecular weight of native EBV DNA is 101 (plus or minus the molecular weight of native FBV DNA by measurement of the length of 3) times 106. Estimation of the molecule relative to form II PM2 DNA yields a value of 105 (plus or minus 3) times 106. (ii) After alkali denaturation, less than 50% of EBV DNA sediments as a single band in alkaline sucrose gradients in the region expected for DNA of 50 times 406 daltons. (iii) Intact EBV HR-1 and EBV B 95-8 DNAs band at 1.718 g/cm3 and a smaller band (approximately 25% of the DNA) AT 1.720 G/CM3. (IV) EBV HR-1 DNA possesses greater than 97% of the sequences of EBV B95-8 DNA. Hybrid DNA molecules formed between (3H)EBV HR-1 DNA and EBV HR-1 DNA or EBV B95-8 DNA had identical thermal stability. EBV B95-8 DNA lacks approximately 15% of the DNA sequences of EBV HR-1 DNA. We interpret these data to mean that EBV B95-8 is derived from a parental EBV through loss of genetic complexity. This defect may be linked to the ability of EBV B95-8 to "transform" lymphocytes invitro.     

Exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer

Several different viruses have been proposed to play a role in breast carcinogenesis. The aim of this study was to investigate the prevalence of a subset of viruses in breast cancer tissue. We investigated the prevalence of 12 DNA viruses: EBV and CMV from the Herpesviridae family and SV40, BKV, JCV, MCV, WUV, KIV, LPV, HPyV6, HPyV7, and TSV from the Polyomaviridae family in 54 fresh frozen breast tumour specimens. Relevant clinical data and basic lifestyle data were available for all patients. The tissue samples were DNA extracted and real-time PCR assays were used for viral detection.The highest prevalence, 10% (5/54), was found for EBV. MCV, HPyV6, and HPyV7 were detected in single patient samples (2% each), while WUV, KIV, JCV, BKV, LPV, SV40, TSV and CMV were not detected in the 54 breast cancer specimens analysed here. Further investigations are needed to elucidate the potential role of viruses, and particularly EBV, in breast carcinogenesis.     

实时定量PCR方法检测鼻咽癌病人全血EB病毒拷贝数的实验研究

EBV is detected in more and more tumors, and is relative to carcinogenesis. We studied the copies of EBV DNA in whole blood of NPC patients and healthy controls by real-time quantitative PCR. In the 73 NPC patients and 83 controls, the positive rate of EBV in blood of NPC is 46.6%, while 13.3% in control. The mean copy number is 3.9 x 10(4) copys/microgram DNA in controls, which is much higher than NPC patients (which is 1.7 x 10(5) copies/microgram DNA). EBV infection is relative with NPC, while the lytic form of EBV maybe more important than its latent form. These results suggest that whole blood EBV DNA may be a valuable tool for molecular diagnosis of NPC.     

Circular Epstein-Barr virus genomes of reduced size in a human lymphoid cell line of infectious mononucleosis origin.

Circular Epstein-Barr virus (EBV) DNA molecules have been purified and characterized from a human lymphoid cell line derived from a case of heterophile antibody-positive, blood transfusion-induced infectious mononucleosis, 883L. The circular EBV DNA in three cell lines obtained by transformation of human umbilical cord blood leukocytes with a strain of EBV originally derived from 883L was also studied. As estimated from sedimentation velocity data and electron microscopy, the circular EBV DNA molecules are 10 to 15% smaller than either the circular EBV DNA previously found intracellularly in several other types of EBV-transformed cells or the linear EBV DNA present extracellularly in virus particles. In addition, the EBV-transformed cord blood cell lines studied here differed from other EBV-transformed cells in that integrated virus DNA sequences could not be detected.     

Use of plasma Epstein-Barr virus DNA monitoring as a tumor marker in follow-up of patients with nasopharyngeal carcinoma: preliminary results and report of two cases

Recent studies suggest that plasma Epstein-Barr virus (EBV) DNA may reflect tumor burden in patients with nasopharyngeal cancer. A prospective study was initiated to investigate this correlation in 125 patients (34 pretreatment [Group A], 78 in remission [Group B] and 13 relapsed [Group C]) and 19 healthy controls. In group A, EBV DNA was detected in plasma samples of 24 (70%) patients. In Group B, EBV DNA was detected in 7 patients (range 77-13,731 copies/mL) and further imaging in all but one of these patients revealed active disease confirmed by ultrasound-guided fine-needle biopsy. There was only one false-positive case; this patient is currently under follow-up. Here we describe 2 of the 7 patients with detectable plasma EBV DNA in whom recurrence was documented by PET scan during follow-up. Our results showed that in group B the positive predictive value of quantitative analysis of plasma EBV DNA was 85%. Quantitative analysis of EBV DNA in plasma seems to become an integral part of screening, staging, monitoring, and prediction of relapse in patients with nasopharyngeal carcinoma. However, previous studies cannot be considered definitive and more reports on the use of this technique are urgently needed from both endemic and non-endemic regions.     

Quantitative PCR in EBV-infected renal transplant patients

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.     

DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains

Raji cells, collected at various times from a synchronized culture, were gently lysed, and the high-molecular-weight DNA was enriched ca. 10-fold for latent Epstein-Barr virus (EBV) genomes by equilibrium density gradient centrifugation in neutral CsCl. The heavy-density DNA pool, which included more than 90% of the total intracellular EBV DNA sequences, was further fractionated by velocity sedimentation on neutral glycerol gradients, and material from fractions containing potential EBV DNA replicative forms was examined in the electron microscope. Early in the cellular S phase, when the EBV DNA content was found to be doubling in parallel with host chromosome replication, half of the 50- to 55-micron circular EBV genomes were observed to have two or more DNA branch points or forks. Most molecules were in a relaxed theta configuration, indicative of the Cairns mode of DNA replication. In the supercoiled state, the two daughter strands of the partially replicated molecules were seen to be wrapped around each other. Two theta structures had more than two DNA forks, indicating that DNA replication can initiate more than once on the same DNA molecule. Late in the S phase, the EBV DNA sedimenting at positions where theta structures were found with early S phase samples was composed of catenated dimers rather than partially replicated genomes. It is concluded that the circular EBV genomes, which are the major intracellular form in latently infected cells, are maintained as independent replicons and are not synthesized from an integrated template.     

Visualization of single copies of the Epstein-Barr virus genome by in situ hybridization

Conditions have been established for demonstrating small numbers of genes of the Epstein-Barr virus (EBV) in B-lymphoid cells by in situ hybridization using biotinylated EBV-specific DNA from cloned BamHI fragments of the viral genome. Single copies of EBV genomes were successfully visualized with minimal background when the probe concentration was 0.2 micrograms/ml, the DNA denaturation step was performed at 100 degrees C, and the immunochemical detection system employed a three-layer peroxidase protocol with gold-silver amplification of the diaminobenzidine substrate. The minimal target DNA detectable was about 10 kilobase pairs. In the case of sectioned cells fixed overnight with formalin, simulating conditions used in routine tissue fixation, this approach failed to demonstrate EBV DNA present at less than 100 copies per cell, that is, at the level found in Raji cells. However, when denaturation was performed using microwave irradiation with the other optimized conditions maintained, EBV DNA could be visualized in 10-20% of such cells, although not in cells known to contain fewer than 10 copies per cell. Thus, microwave irradiation partially overcomes the limit of DNA target detection imposed by formalin.