BamHI-A rightward frame 1, an Epstein-Barr virus-encoded oncogene and immune modulator.

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.

Staurosporine increases toxicity of gemcitabine in non-small cell lung cancer cells: role of protein kinase C, deoxycytidine kinase and ribonucleotide reductase.

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.

Irinotecan-induced cytotoxicity to colon cancer cells in vitro is stimulated by pre-incubation with trifluorothymidine.

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.

In vitro optimization of non-small cell lung cancer activity with troxacitabine, L-1,3-dioxolane-cytidine, prodrugs.

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.

Modulation of signaling enhances the efficacy of the combination of satraplatin and erlotinib.

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-encoded BARF1 protein is a decoy receptor for macrophage colony stimulating factor and interferes with macrophage differentiation and activation.

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.

Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma.

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.