Epstein–Barr Virus (EBV) BamHI-A rightward frame 1 (BARF1) protein is considered a viral oncogene in epithelial cells and has immune-modulating properties. During viral lytic replication BARF1 is expressed as an early gene, regulated by the immediate early EBV protein R. However, in viral latency BARF1 is exclusively expressed in epithelial tumors such as nasopharyngeal (NPC) and gastric carcinoma (GC) but not in lymphomas, indicating that activation of the BARF1 promoter is cell type specific. Undifferentiated NPC is characterized by high expression of ΔNp63 isoforms of the epithelial differentiation marker p63, a member of the p53 family of transcription factors. Transcription factor binding site analysis indicated potential p53 family binding sites within the BARF1 promoter region. This study investigated ability of various p53 family members to transactivate the BARF1 promoter. Using BARF1 promoter luciferase reporter constructs we demonstrate that only p63 isoform ΔNp63α is capable of transactivating the BARF1 promoter, but not the TAp63 isoforms, p53 or p73. Direct promoter binding of ΔNp63α was confirmed by Chromatin Immune Precipitation (ChIP) analysis. Deletion mutants of the BARF1 promoter revealed multiple ΔNp63 response elements to be responsible for BARF1 promoter transactivation. However, ΔNp63α alone was not sufficient to induce BARF1 in tumor cells harboring full EBV genomes, indicating that additional cofactors might be required for full BARF1 regulation. In conclusion, in EBV positive NPC and GC, BARF1 expression might be induced by the epithelial differentiation marker ΔNp63α, explaining BARF1 expression in the absence of lytic reactivation.

UNLABELLED:The active metabolite (JM118) of the oral platinum analog satraplatin (JM216) was investigated for potential synergism with erlotinib, an epidermal growth factor receptor (EGFR) inhibitor. JM118 sensitivity of 7 cancer cell lines (ovarian: 2008, A2780; colon: Lovo92, WiDr; lung: A549, SW1573; epidermoid: A431), was enhanced most pronounced when JM118 preceded erlotinib, which was associated with increased formation of DNA-platinum adducts. The combination increased G2/M phase accumulation and enhanced apoptosis. JM118 increased the phosphorylation of the cell cycle proteins CDK2 and CHK1 after 24 hr exposure. JM118/erlotinib enhanced Erk and Akt phosphorylation after 2 hr. JM118 significantly decreased the phosphorylation of PTEN, VEGFR, EPHA1, ERBB4, FGF-R, andSTAT3 by 20 (PTEN) to >90% (STAT3).CONCLUSION:Erlotinib enhanced the effects of JM118, even in cells with mutations in Ras. The mechanism of synergy involved a combination of effects on platinum-DNA adduct formation, cell cycle distribution and signaling.

Epstein-Barr virus (EBV) BARF1 (BamHI-A rightward frame 1) protein is a viral oncogen and immunomodulator. BARF1 mRNA is highly and selectively expressed in nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC), however, evidence for presence of BARF1 protein in situ is lacking. In vitro, the hexameric form of BARF1 is rapidly secreted (sBARF1). Secretion of BARF1 from NPC tumor cells may result in the presence of sBARF1 in sera of NPC patients. This study describes the development of new monoclonal and polyclonal antibodies that strongly and selectively bind to ‘native’ NPC-derived sBARF1 protein in its natural hexameric conformation. Using these antibodies, a capture enzyme linked immunosorbent assay (ELISA) was developed to detect sBARF1 protein in sera of NPC patients. Although a detection limit of 10 ng/ml sBARF1 in serum was reached for the ELISA, only 3 of 71 NPC patients at diagnosis showed elevated levels of sBARF1 protein. Possibly sBARF1 is complexed with serum proteins shielding antibody recognition sites. However, sample denaturation prior to ELISA testing did not affect the levels observed. A MALDI-MS/MS approach was developed to overcome this problem, yielding a specific BARF1 identifier peptide. However, the sensitivity of the current setup was less than BARF1-capture ELISA. The BARF1 identifier peptide can be used to further develop a quantitative targeted proteomics approach. Using immunohistochemical staining with various anti-BARF1 antibody reagents, BARF1 could be detected in paraffin-embedded BARF1 expressing cell lines revealing a cytoplasmic localisation. Unfortunately, in patient material, no BARF1 protein expression could be demonstrated. In conclusion, the sensitive sBARF1 capture ELISA (10 ng/mL serum) demonstrated only low levels of sBARF1 in NPC patient sera, conflicting with previously published data. A higher sensitivity of both ELISA and proteomic approach is needed for a diagnostic tool. sBARF1 remains a potential serum biomarker for NPC and GC and its diagnostic value using a targeted proteomics approach should be further evaluated. Chapter 8 EBV BARF1 detection

Epstein-Barr virus (EBV) causes several benign and malignant disorders of lymphoid and epithelial origin. EBV-related tumors display distinct patterns of viral latent gene expression, of which the BamHI-A rightward frame 1 (BARF1) is selectively expressed in carcinomas, regulated by cellular differentiation factors including ΔNp63α. BARF1 functions as a viral oncogene, immortalizing and transforming epithelial cells of different origin by acting as a mitogenic growth factor, inducing cyclin-D expression, and up-regulating antiapoptotic Bcl-2, stimulating host cell growth and survival. In addition, secreted hexameric BARF1 has immune evasive properties, functionally corrupting macrophage colony stimulating factor, as supported by recent functional and structural data. Therefore, BARF1, an intracellular and secreted protein, not only has multiple pathogenic functions but also can function as a target for immune responses. Deciphering the role of BARF1 in EBV biology will contribute to novel diagnostic and treatment options for EBV-driven carcinomas. Herein, we discuss recent insights on the regulation of BARF1 expression and aspects of structure-function relating to its oncogenic and immune suppressive properties.

ABSTRACT 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.

Epstein-Barr virus (EBV), like many other persistent herpes viruses, has acquired numerous mechanisms for subverting or evading immune surveillance. This study investigates the role of secreted EBV-encoded BARF1 protein (sBARF1) in creating an immune evasive microenvironment. Wild-type consensus BARF1 was expressed in the human 293 cell line and purified. This native hexameric sBARF1 had inhibitory capacity on macrophage colony stimulating factor (M-CSF)-stimulated, and not on granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated growth and differentiation of myeloid cells. Antibodies specific to hexameric sBARF1 were able to block this effect. M-CSF was shown to interact with sBARF1 via the protruding N-terminal loops involving Val38 and Ala84. Each BARF1 hexamer was capable of binding three M-CSF dimers. Mutations in the BARF1 loops greatly affected M-CSF interaction, and showed loss of growth inhibition. Analysis of the activation state of the M-CSF receptor c-fms and its downstream kinase pathways showed that sBARF1 prevented M-CSF-induced downstream phosphorylation. Since M-CSF is an important factor in macrophage differentiation, the effect of sBARF1 on the function of monocyte-derived macrophages was evaluated. sBARF1 affected overall survival and morphology and significantly reduced expression of macrophage differentiation surface markers such as CD14, CD11b, CD16, and CD169. Macrophages differentiating in the presence of sBARF1 showed impaired responses to lipopolysaccharide and decreased oxygen radical formation as well as reduced phagocytosis of apoptotic cells. In conclusion, EBV sBARF1 protein is a potent decoy receptor for M-CSF, hampering the function and differentiation of macrophages. These results suggest that sBARF1 contributes to the modulation of immune responses in the microenvironment of EBV-positive carcinomas.

ABSTRACT 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.

Background BamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing. Results Most NPC isolates revealed specific BARF1 nucleotide changes compared to prototype B95-8 virus. At the protein level these mutations resulted in 3 main substitutions (V29A, W72G, H130R), which are not considered to cause gross tertiary structure alterations in the hexameric BARF1 protein. At least one amino acid conversion was detected in 80.3% of NPC samples compared to 33.3% of non-NPC samples (p < 0.001) and 40.0% of healthy LCLs (p = 0.074). NPC isolates also showed more frequent codon mutation than non-NPC samples. EBV strain typing revealed most isolates as EBV type 1. The viral load of either NPC or non-NPC samples was high, but only in non- NPC group it related to a particular BARF1 variant. Serology on NPC sera using IgA/EBNA-1 ELISA, IgA/VCA-p18 ELISA and immunoblot score showed no relation with BARF1 sequence diversity (p = 0.802, 0.382 and 0.058, respectively). NPC patients had variable antibody reactivity against purified hexameric NPC-derived BARF1 irrespective of the endogenous BARF1 sequence. Conclusion The sequence variation of BARF1 observed in Indonesian NPC patients and controls may reflect a natural selection of EBV strains unlikely to be predisposing to carcinogenesis. The conserved nature of BARF1 may reflect an important role in EBV (epithelial) persistence.

Gemcitabine, a deoxycytidine analog, active against non-small cell lung cancer, is phosphorylated by deoxycytidine kinase (dCK) to active nucleotides. Earlier, we found increased sensitivity to gemcitabine in P-glycoprotein (SW-2R160) and multidrug resistance-associated protein (SW-2R120), overexpressing variants of the human SW1573 non-small cell lung cancer cells. This was related to increased dCK activity. As protein kinase C (PKC) is higher in 2R120 and 2R160 cells and may control the dCK activity, we investigated whether gemcitabine sensitivity was affected by the protein kinase C inhibitor, staurosporine, which also modulates the cell cycle. Ten nmol/l staurosporine enhanced the sensitivity of SW1573, 2R120 and 2R160 cells 10-fold, 50-fold and 270-fold, respectively. Staurosporine increased dCK activity about two-fold and the activity of thymidine kinase 2, which may also activate gemcitabine. Staurosporine also directly increased dCK in cell free extracts. Staurosporine decreased expression of the free transcription factor E2F and of ribonucleotide reductase (RNR), a target for gemcitabine inhibition. In conclusion, staurosporine may potentiate gemcitabine by increasing dCK and decreasing E2F and RNR, which will lead to a more pronounced RNR inhibition.

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. The main drawback in the use of most nucleoside anticancer agents originates from their hydrophilic nature, which property requires a high and frequent dosage for an intravenous administration. To overcome this problem several troxacitabine prodrugs modified in the aminogroup with a linear aliphatic chain with a higher lipophilicity were developed. To determine whether these prodrugs have an advantage over Troxacitabine pancreatic cancer cell lines were exposed to Troxacitabine and the lipophilic prodrugs. The addition of linear aliphatic chains to troxacitabine increased sensitivity of pancreatic cancer cell lines to the drug > 100-fold, possibly due to a better uptake and retention of the drug.

SN38 is the active metabolite of the anti-cancer agent irinotecan (CPT-11) and is a potent inhibitor of topoisomerase-I (topo-I), leading to DNA strand breaks and eventually cell death. The pyrimidine analog trifluorothymidine (TFT) is part of the anti-cancer drug formulation TAS-102, which was developed to enhance the bioavailability of TFT in vivo, and is currently being evaluated as an oral chemotherapeutic agent in phase I clinical studies. In this study, the combined cytotoxic effects of dual-targeted TFT with SN38 were investigated in a panel of human colon cancer cell lines (WiDr, H630, Colo320, SNU-C4, SW1116). We used different drug combination treatment schedules of SN38 with TFT, and possible synergism was evaluated using median drug effect analysis resulting in combination indexes (CI), in which CI<0.9 indicates synergism, CI=0.9–1.1 indicates additivity and CI>1.1 indicates antagonism. Drug target analysis was performed to investigate the effect of TFT on SN38-induced DNA damage, cell cycle delay and apoptosis. Simultaneous exposure to SN38 in combination with TFT was not more than additive, whereas pre-incubation with TFT resulted in synergism with SN38 (CI=0.3–0.6). Only for Colo320 synergism could be induced for both simultaneous and sequential drug combinations. SN38 and TFT induced most DNA damage in H630 and Colo320 cells, which was increased in combination. TFT pre-incubation further enhanced SN38-induced DNA strand breaks in H630 and Colo320 (>20%), which was most pronounced in H630 cells (p<0.01). Exposure to SN38 alone induced a clear cell cycle G2M-phase arrest and pre-incubation with TFT enhanced this effect in WiDr and H630 (p<0.05). Both drugs induced significant apoptosis; SN38-induced apoptosis increased significantly in the presence of TFT (p<0.01), either when added simultaneously (about 3-fold) or at pre-incubation (about 2-fold). Topo-I protein levels varied among the cell lines and TFT hardly affected these. In conclusion, TFT pre-incubation can enhance SN38-induced cytotoxicity to colon cancer cells resulting in synergism between the drugs, thereby increasing DNA damage and apoptosis induction.

Oxaliplatin (OHP) is an anticancer agent that acts by formation of Platinum-DNA (Pt-DNA) adducts resulting in DNA-strand breaks and is used for the treatment of colorectal cancer. The pyrimidine analog trifluorothymidine (TFT) forms together with a thymidine phosphorylase inhibitor (TPI) the anticancer drug formulation TAS-102, in which TPI enhances the bioavailability of TFT in vivo. In this in vitro study the combined cytotoxic effects of OHP with TFT were investigated in human colorectal cancer cells as a model for TAS-102 combinations. In a panel of five colon cancer cell lines (WiDr, H630, Colo320, SNU-C4 and SW1116) we evaluated the OHP-TFT drug combinations using the multiple drug–effect analysis with CalcuSyn software, in which the combination index (CI) indicates synergism (CI<0.9), additivity (CI=0.9–1.1) or antagonism (CI>1.1). Drug target analysis was used for WiDr, H630 and SW1116 to investigate whether there was an increase in Pt-DNA adduct formation, DNA damage induction, cell cycle delay and apoptosis. Trifluorothymidine combined with OHP resulted in synergism for all cell lines (all CI<0.9). This was irrespective of schedule in which either one of the drugs was kept at a constant concentration (using variable drug ratio) or when the two drugs were added in a 1 : 1 IC50-based molar ratio. Synergism could be increased for WiDr using sequential drug treatment schedules. Trifluorothymidine increased Pt-DNA adduct formation significantly in H630 and SW1116 (14.4 and 99.1%, respectively; P<0.05). Platinum-DNA adducts were retained best in SW1116 in the presence of TFT. More DNA-strand breaks were induced in SW1116 and the combination increased DNA damage induction (>20%) compared with OHP alone. Exposure to the drugs induced a clear cell-cycle S-phase arrest, but was dose schedule and cell line dependent. Trifluorothymidine (TFT) and OHP both induced apoptosis, which increased significantly for WiDr and SW1116 after TFT–OHP exposure (18.8 and 20.6% respectively; P<0.05). The basal protein levels of ERCC1 DNA repair enzyme were not related to the DNA damage that was induced in the cell lines. In conclusion, the combination of TFT with the DNA synthesis inhibitor OHP induces synergism in colorectal cancer cells, but is dependent on the dose and treatment schedule used.

Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis. Simultaneous and sequential exposure (preexposure to 3-AP for 24h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC(25) concentration for 24h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR. Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR. In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.

l-1,3-Dioxolane-cytidine, a potent anticancer agent against leukemia, has limited efficacy against solid tumors, perhaps due to its hydrophilicity. Herein, a library of prodrugs were synthesized to optimize in vitro antitumor activity against non-small cell lung cancer. N4-Substituted fatty acid amide prodrugs of 10-16 carbon chain length demonstrated significantly improved antitumor activity over l-1,3-dioxolane-cytidine. These in vitro results suggest that the in vivo therapeutic efficacy of l-1,3-dioxolane-cytidine against solid tumors may be improved with prodrug strategies.

Proc Amer Assoc Cancer Res, Volume 47, 2006 1117 Troxacitabine (Troxatyl) is the first unnatural L-nucleoside to demonstrate potent antitumor activity against leukaemia as well as various solid tumors in vitro and in vivo .1 Troxacitabine is currently being evaluated in pivotal Phase II/III clinical trials for the third-line treatment of AML and in phase I/II dose-ranging trials in patients with refractory pancreatic cancer. The main drawback in the use of most nucleoside anticancer agents originates from their hydrophilic nature, which requires a high dosage for a continuous infusion. Unlike other nucleoside anti-tumor agents, troxacitabine appears to predominately enter tumor cells by passive diffusion rather than by nucleoside transporters,2 although this may be model dependent. Thus, more lipophilic analogs than the parent compound may provide advantages for cell penetration. In the present studies, in order to evaluate the relationship between their lipophilicity and the antitumor activity, a library of 20 troxacitabine prodrugs has been synthesized using a fast and efficient parallel approach. Synthesized compounds were tested as inhibitors of two non-small cell lung cancer cell lines (A549 and SW1573), which were selected because they had a similar sensitivity to gemcitabine, while SW1573 had a higher activity of deoxycytidine kinase, required for activation of both gemcitabine and troxacitabine. The results indicate that lipophilic analogs exhibit better antitumor activity profiles than that of troxacitabine ([Figure 1][1]). Prodrugs with long linear aliphatic chains (>8 CH2) are clearly more potent than that of troxacitabine with IC50 values in the nanomolar range for the best derivatives. Prodrugs, which contain a benzoyl group at the N4-position were less active than troxacitabine. [Figure 1][1] 1 Grove, K.L.; Guo, X.; Liu, S.-H.; Chu, C.K.; Cheng, Y.-C. AntiCancer Activity of b- L -Dioxolane-cytidine, a Novel Nucleoside Analogue With the Unnatural L Configuration. Cancer Res. 1995, 55, 3008-3011. 2 Gourdeau, H.; Clarke, M. L.: Ouellet, F.; Mowles, D.; Selner, M.; Richard, A.; Lee, N.; Mackey, J. R.; Young, J. D.; Jolivet, J.; Lafreniere, R. G.; Cass, C. E. Mechanism of Uptake and Resistance to Troxacitabine, a Novel Deoxycytidine Nucleoside Analogue, in Human Leukemic and Solid Tumor Cell Lines. Cancer Res. 2001, 61, 7217-7224. ![Figure][2] [1]: #F1 [2]: pending:yes

A series of β′-hydroxy-α,β-unsaturated ketones were prepared by means of an iron(III) catalyzed domino process. The in vitro antiproliferative activities were examined in the human solid tumor cell lines A2780, SW1573, and WiDr. The results showed that β′-hydroxy-α,β-unsaturated ketones were more potent than α,β-unsaturated ketones. The best activity profiles were obtained for the derivatives bearing cyclic or branched substituents on the side chains.