Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells.

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers.

Epstein-Barr virus transcription activator R upregulates BARF1 expression by direct binding to its promoter, independent of methylation.

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides -554 and -327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.

Variable EBV DNA load distributions and heterogeneous EBV mRNA expression patterns in the circulation of solid organ versus stem cell transplant recipients.

UNLABELLED: Epstein-Barr virus (EBV) driven post-transplant lymphoproliferative disease (PTLD) is a heterogeneous and potentially life-threatening condition. Early identification of aberrant EBV activity may prevent progression to B-cell lymphoma. We measured EBV DNA load and RNA profiles in plasma and cellular blood compartments of stem cell transplant (SCT; n = 5), solid organ transplant recipients (SOT; n = 15), and SOT having chronic elevated EBV-DNA load (n = 12). In SCT, EBV DNA was heterogeneously distributed, either in plasma or leukocytes or both. In SOT, EBV DNA load was always cell associated, predominantly in B cells, but occasionally in T cells (CD4 and CD8) or monocytes. All SCT with cell-associated EBV DNA showed BARTs and EBNA1 expression, while LMP1 and LMP2 mRNA was found in 1 and 3 cases, respectively. In SOT, expression of BARTs was detected in all leukocyte samples. LMP2 and EBNA1 mRNA was found in 5/15 and 2/15, respectively, but LMP1 mRNA in only 1, coinciding with severe PTLD and high EBV DNA. CONCLUSION: EBV DNA is differently distributed between white cells and plasma in SOT versus SCT. EBV RNA profiling in blood is feasible and may have added value for understanding pathogenic virus activity in patients with elevated EBV-DNA.

Purified hexameric Epstein-Barr virus-encoded BARF1 protein for measuring anti-BARF1 antibody responses in nasopharyngeal carcinoma patients.

WHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1 and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric BARF1 in sera of Indonesian NPC patients (n = 155) compared to healthy EBV-positive (n = 56) and EBV-negative (n = 16) individuals. BARF1 (B95-8) expressed in Escherichia coli and baculovirus, as well as BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1 protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV proteins. Higher IgG reactivity was found in NPC patients (P = 0.015) than in regional Indonesian controls or EBV-negative individuals (P < 0.001). IgA responses to native BARF1 were marginal. NPC sera with the highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.

Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1).

The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.

Hypoxia-induced acidification causes mitoxantrone resistance not mediated by drug transporters in human breast cancer cells.

Hypoxia has clinically been associated with resistance to chemotherapy. The aim of this study was to investigate whether hypoxia induces resistance to doxorubicin and mitoxantrone, two common drugs in cancer treatment, in MCF-7 breast cancer cells, and SW1573 non-small lung cancer cells. In addition, the role of drug transporters P-gp, BCRP and MRP1 was analysed. Hypoxia induced resistance in MCF-7 cells to mitoxantrone shifted the IC(50) value from 0.09 microM (+/-0.01) to 0.54 microM (+/-0.06) under hypoxia, whereas survival of MCF-7 and SW1573 cells in the presence of doxorubicin was not altered. Accumulation of mitoxantrone and daunorubicin, a doxorubicin fluorescent homologue, appeared to be 5.3 and 3.2 times lower in MCF-7 cells, respectively. Cytotoxicity assays showed no increased functionality of the drug transporters P-gp, BCRP and MRP1 under hypoxia. In addition, protein levels of these drug transporters were not changed. Medium of the MCF-7 cells became more acidic under hypoxia thereby causing a decreased uptake of mitoxantrone. Hypoxia induces mitoxantrone resistance in MCF-7 cells not mediated by the three major MDR transporters. Hypoxia-induced acidification may cause this resistance by decreased cellular uptake together with a lowered cytotoxicity due to pH-dependent topoisomerase type II activity.

Differential prognostic impact of hypoxia induced and diffuse HIF-1alpha expression in invasive breast cancer.

BACKGROUND: Intratumorous hypoxia triggers a broad cellular response mediated by the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1alpha concentrations increase during breast carcinogenesis, and are associated with poor prognosis. An earlier study noted two HIF-1alpha overexpression patterns: diffuse scattered throughout the tissue and confined to perinecrotic cells. AIMS: To investigate the prognostic impact of these different HIF-1alpha overexpression patterns in relation to its downstream effectors carbonic anhydrase (CA) IX and glucose transporter 1 (GLUT-1). METHODS: HIF-1alpha, CA IX, and GLUT-1 expression was studied by immunohistochemistry, including double staining for CA IX and HIF-1alpha. Clinical data included disease free survival, lymph node status, and tumour size. RESULTS: HIF-1alpha overexpression (44% of cases) had a perinecrotic (13.5%) or diffuse staining pattern (30.5%). CA IX expression was detectable in 12.5% of breast cancers, whereas GLUT-1 expression was seen in 29%, with both showing perinecrotic membrane staining. Perinecrotic HIF-1alpha overexpression was highly associated with CA IX and GLUT-1 overexpression, and double staining for HIF-1alpha and CA IX showed strong expression in the same cells. Diffusely overexpressed HIF-1alpha was not associated with CA IX or GLUT-1 expression. Patients with diffuse HIF-1alpha staining had a significantly better prognosis than patients with perinecrotically overexpressed HIF-1alpha. CONCLUSIONS: Different regulation pathways of HIF-1alpha overexpression exist in breast cancer: (1) hypoxia induced, perinecrotic HIF-1alpha overexpression with strong expression of hypoxia associated genes (CA IX and GLUT-1), which is associated with a poor prognosis; and (2) diffuse HIF-1alpha overexpression lacking major hypoxia associated downstream effects, resulting in a more favourable prognosis.

The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis.

Apoptosis can be induced in response to hypoxia. The severity of hypoxia determines whether cells become apoptotic or adapt to hypoxia and survive. A hypoxic environment devoid of nutrients prevents the cell undergoing energy dependent apoptosis and cells become necrotic. Apoptosis regulatory proteins are delicately balanced. In solid tumours, hypoxia is a common phenomenon. Cells adapt to this environmental stress, so that after repeated periods of hypoxia, selection for resistance to hypoxia induced apoptosis occurs. These resistant tumours probably have a more aggressive phenotype and may have decreased responsiveness to treatment. The key regulator of this process, hypoxia inducible factor 1 (HIF-1), can initiate apoptosis by inducing high concentrations of proapoptotic proteins, such as BNIP3, and can cause stabilisation of p53. However, during hypoxia, antiapoptotic proteins, such as IAP-2, can be induced, whereas the proapoptotic protein Bax can be downregulated. During hypoxia, an intricate balance exists between factors that induce or counteract apoptosis, or even stimulate proliferation. Understanding the regulation of apoptosis during hypoxia and the mechanisms of resistance to apoptosis might lead to more specific treatments for solid tumours.

Lack of lymphangiogenesis during breast carcinogenesis.

BACKGROUND: Recent evidence suggests that functional intratumorous lymph vessels may be absent from some human cancers. This could result from either the failure of tumours to induce lymphangiogenesis, or the collapse of lymph vessels, caused by high interstitial tumour pressure. METHODS: To differentiate between these two hypotheses, paraffin wax embedded clinical specimens from normal breast (n = 13), usual ductal hyperplasia (n = 11), ductal carcinoma in situ (n = 21), and invasive breast cancer (n = 40) were compared for lymphatic and blood vessel density by immunohistochemistry with antibodies to the lymphatic endothelial hyaluronan receptor (LYVE-1) and CD31, respectively. RESULTS: Lymph vessel density was lower than blood vessel density in normal breast tissue. Within breast lobuli, lymph vessels were absent. In premalignant lesions blood microvessel density increased, whereas no increase in lymph vessels could be seen intralesionally. In invasive cancers, lymph vessels were absent in all but a few cases, where probably some pre-existing lymph vessels remained, although blood microvessel density was once again increased. CONCLUSION: Unlike angiogenesis, lymphangiogenesis is absent during breast carcinogenesis. This, and not rising interstitial pressure caused by an increase in the size of lesions, explains the absence of intratumorous lymph vessels in invasive breast cancer.

Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.

Expression dynamics of human cytomegalovirus immune evasion genes US3, US6, and US11 in the blood of lung transplant recipients.

Delayed elimination of human cytomegalovirus (HCMV)-infected cells by the host immune system may contribute to viral dissemination and pathogenesis of HCMV infection. The mRNA expression dynamics of HCMV-encoded immune evasion genes US3, US6, and US11 expressed after active HCMV infection were analyzed in blood samples of lung transplant recipients by means of quantitative nucleic acid sequence-based amplification. The results were compared with the expression dynamics of IE1 mRNA and pp67 late mRNA, levels of pp65 antigenemia, and antiviral treatment. During acute infection, high levels of US3 and US6 RNA were detected before antigenemia, which were detected simultaneously with IE1 RNA. US11 RNA was detected simultaneously with antigenemia but before late pp67 RNA. These data suggest an active role of viral immune evasion during HCMV infection in vivo. Interestingly, immune evasion RNA remained detectable after clinical recovery, often independently of IE1 RNA expression, indicating persistent viral activity, which may have implications for long-term control of HCMV.

Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification.

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10(4) copies per 100 microl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.

Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process.

While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

Molecular fine-specificity analysis of antibody responses to human cytomegalovirus and design of novel synthetic-peptide-based serodiagnostic assays.

To identify single immunodominant marker proteins which can replace complex virion antigen in serodiagnostic assays, we investigated in detail the molecular fine specificity of antibody responses in different individuals with latent or active human cytomegalovirus (HCMV) infection. An overview of the HCMV proteins recognized by human antibodies was obtained by immunoblotting. For selected immunodominant proteins the epitope fine specificity of the antibody response was determined by a peptide-scanning enzyme-linked immunosorbent assay (ELISA). Epitope clusters were synthesized as combination peptides and were used for further serologic analysis of immunoglobulin M (IgM) and IgG reactivities with panels of sera from different groups of patients in comparison to those with cytomegalovirus (CMV) virion antigen. Several serum samples had significantly higher reactivities with peptides than with the CMV virion antigen. However, individual serum samples occasionally recognized diverse peptide epitopes, stressing the importance of using combinations of peptides in serologic assays. From these studies we were able to define a specific combination of peptides derived from pp52 (UL44) and pp150 (UL32) for the specific and highly sensitive early detection of HCMV IgM, whereas a combination of peptides from pp150 (UL32), gB (UL55), and pp28 (UL99) was selected to give optimal and specific reactivity with HCMV IgG. On the basis of the results obtained with these peptide combinations, new, highly specific serodiagnostic assays were constructed. These assays had sensitivities of 98.9 and 96.4% for IgG and IgM, respectively, in comparison with the results obtained with the "gold standard," the virion antigen-based ELISA. From the results of this study we conclude that specific combinations of highly defined synthetic peptides can replace complex HCMV virion extracts used in current serodiagnostics and may add to further standardization of HCMV serology.

Optimized conditions for the production of recombinant amphotropic retroviral vector preparations.

The production and stability of recombinant retroviral vectors was examined at various temperatures. The two studied recombinant retroviral vectors, based on different packaging cell lines, exhibited a four-fold increased half-life at 32 degrees C as compared to 37 degrees C. Surprisingly, this increased stability at 32 degrees C was only observed within a very narrow temperature window. At 30 degrees C and 34 degrees C, retroviral vector half-lives were quite similar to that at 37 degrees C. Regardless of the vector half-life, retroviral vectors accumulated in the culture medium for a period of 48 h before an equilibrium was reached between retroviral vector production and decay. Maximal accumulated recombinant retroviral titers were five- to ten-fold increased after a medium incubation at 32 degrees C as compared to 37 degrees C. Furthermore, multiple cycles of freezing and thawing of retroviral vector supernatants hardly affected the recombinant retroviral vector titer, independent of the presence of serum. This knowledge on characteristics of recombinant retroviral vectors has practical implications for the manufacturing of these viruses for clinical gene therapy protocols.

Intracellular analysis of in vitro modified HIV Tat protein.

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that specifically activates transcription from the viral long terminal repeat. To characterize the properties of the Tat proteins, we have expressed them in Escherichia coli. The purified Tat protein was biochemically analyzed and tested for activity upon electroporation into human cell lines. This protein electroporation was used for the intracellular analysis of in vitro modified Tat protein. Our results indicate that the transcriptionally active form of the Tat protein is a monomer. Furthermore, we found that Tat activity is dramatically inhibited by preincubation of the protein with strongly reducing agents. In contrast, no inhibitory effect was measured upon incubation with metal-chelating reagents. These results suggest that the cysteine residues of Tat are involved in the formation of intramolecular disulfide bonds.