The R-enantiomer of ketorolac reduces ovarian cancer tumor burden in vivo.

BACKGROUND: Rho-family GTPases, including Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), are important modulators of cancer-relevant cell functions and are viewed as promising therapeutic targets. Based on high-throughput screening and cheminformatics we identified the R-enantiomer of an FDA-approved drug (ketorolac) as an inhibitor of Rac1 and Cdc42. The corresponding S-enantiomer is a non-steroidal anti-inflammatory drug (NSAID) with selective activity against cyclooxygenases. We reported previously that R-ketorolac, but not the S-enantiomer, inhibited Rac1 and Cdc42-dependent downstream signaling, growth factor stimulated actin cytoskeleton rearrangements, cell adhesion, migration and invasion in ovarian cancer cell lines and patient-derived tumor cells. METHODS: In this study we treated mice with R-ketorolac and measured engraftment of tumor cells to the omentum, tumor burden, and target GTPase activity. In order to gain insights into the actions of R-ketorolac, we also performed global RNA-sequencing (RNA-seq) analysis on tumor samples. RESULTS: Treatment of mice with R-ketorolac decreased omental engraftment of ovarian tumor cells at 18 h post tumor cell injection and tumor burden after 2 weeks of tumor growth. R-ketorolac treatment inhibited tumor Rac1 and Cdc42 activity with little impact on mRNA or protein expression of these GTPase targets. RNA-seq analysis revealed that R-ketorolac decreased expression of genes in the HIF-1 signaling pathway. R-ketorolac treatment also reduced expression of additional genes associated with poor prognosis in ovarian cancer. CONCLUSION: These findings suggest that R-ketorolac may represent a novel therapeutic approach for ovarian cancer based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor. R-ketorolac modulates relevant pathways and genes associated with disease progression and worse outcome.

Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer.

Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. To overcome these hurdles, we developed a new experimental model of clinical dormancy employing patient-isolated Circulating Tumor Cells (de novo CTCs) and their injection in xenografts with subsequent tumor monitoring and CTC characterization (ex vivo CTCs). We hypothesized that significant distinctions exist between signaling pathways of bone marrow-homing vs metastasis-competent CTCs upon transplantation in xenografts. Comparative transcriptomic analyses of ex vivo vs de novo CTCs identified increased mTOR signaling-a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy-with contrasting actions by its two complementary arms (mTORC2/mTORC1). Heightened mTORC2 downstream targets augmented quiescent CTCs (Ki67-/RBL2+ cells) in paired breast cancer tissues, along with high mTORC2 activity in solitary BMRCs and tissue-resident CTCs. Further, shRNA mediated the knockdown of RICTOR, an essential component of mTORC2, and augmented Ki67/PCNA biomarker expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling regulates CTC-associated mitotic and/or dormancy characteristics.

Sublaminar polyester band fixation construct in the treatment of neuromuscular scoliosis.

PURPOSE: Multiple fixation techniques exist for treating progressive neuromuscular scoliosis including pedicle screws, sublaminar bands/wires, hooks or a combination of instruments. Most sublaminar band constructs are supplemented with pedicle screws, hooks and/or sublaminar wires particularly at the top of the construct. There are no studies to date that describe an all/predominant sublaminar band construct. The purpose of this study was to investigate the outcomes of a sublaminar polyester band construct to treat neuromuscular scoliosis. METHODS: A retrospective review was conducted of 32 cases of neuromuscular scoliosis treated with posterior spinal fusion using a sublaminar band construct between 2013 and 2016 by a single surgeon at a single centre. Preoperative, immediate postoperative and two-year follow-up radiographs and clinical records were reviewed. Sagittal, coronal and pelvic obliquity correction was measured. Blood loss, length of surgery and complications were recorded. RESULTS: In all, 29 patients were included. Mean postoperative coronal plane correction was 57% (0% to 92%) and maintained at two-year follow-up. Mean sagittal balance was 2.3 cm (-2.5 to 6.4). Mean lumbar lordosis angle decreased by 7° (44° to 37°). Mean thoracic kyphosis angle increased by 9° (23° to 32°). Mean pelvic obliquity decreased by 50% (from 15° to 7°). There were four major complications (14%) and eight minor complications (21%). Mean blood loss was 1304 cc (250 cc to 2450 cc). CONCLUSION: Sublaminar polyester band fixation constructs provide a viable option in correction of deformity in patients with neuromuscular scoliosis with comparable outcomes with what is reported with other constructs. LEVEL OF EVIDENCE: V.

Do fluoroscopic and radiographic images underestimate pin protrusion in paediatric supracondylar humerus and distal radius fractures? A synthetic bone model analysis.

PURPOSE: Fluoroscopy is commonly used to confirm acceptable position of percutaneously placed pins when treating paediatric fractures. There is a paucity of literature investigating the accuracy of fluoroscopic imaging when determining pin position relative to the far cortex of the fixated bone. The purpose of this study was to evaluate the accuracy of fluoroscopic and radiographic imaging in measuring smooth pin protrusion from the far cortex of a bone model. METHODS: Eight bone models were implanted with smooth pins and anteroposterior fluoroscopic and radiographic studies were obtained. All images were evaluated by orthopaedic attending physicians, residents and medical students. The length of pin protrusion from the model surface was estimated on fluoroscopic imaging and measured on radiographs and compared with actual lengths measured on the bone models. RESULTS: 20 evaluators took a total of 320 pin measurements on images of 8 models. There was a significant difference between fluoroscopic measurements compared to radiographic measurements and actual pin lengths. There was no significant difference between radiographic measurements and actual pin lengths. Level of training of examiner was not statistically significant. On average, fluoroscopic estimations of pin protrusion were 1.53 mm shorter than the actual measured length. CONCLUSION: Fluoroscopic images underestimate the length of smooth pins protruding from a bone model surface when compared with radiographs and actual measurements. Orthopaedic surgeons using fluoroscopy should be aware of this discrepancy when assessing intraoperative fluoroscopic images to decide on acceptable implant position. LEVEL OF EVIDENCE: Level V.

Characterization of Seven Outbreaks of Hemorrhagic Hepatopathy Syndrome in Commercial Pullets Following the Administration of a Salmonella Enteritidis Bacterin in California.

Between April 2013 and April 2015, seven flocks belonging to three different major commercial egg producers inCalifornia experienced a mild increase in mortality 2 to 3 wk after administration of Salmonella Enteritidis bacterins. Strains of chickens involved were H&N (flock A1, A2, B2, C1, C2, and C3) and Lohmann white (flock B1). Vaccination was administered individually through injection either in the breast muscles or subcutis in the legs between 11 and 18 wk of age in all flocks. Clinical signs ranged from inapparent to lameness, reluctance to walk, greenish diarrhea, and retching-like symptoms. The mortality ranged from 0.16% to 1.38% per week, with the highest peaks occurring usually 2 to 3 wk postvaccination, and then declined rapidly. Postmortem examinations revealed enlarged livers with disseminated hemorrhages and pale foci of necrosis. Also, severe extensive hemorrhages in the intestine, heart, and proventriculus were observed in a few birds. Various degrees of productive, exudative giant cell granulomatous myositis were observed invading deeply the muscles and subcutis at the site of vaccination. The myositis was always associated with optically empty vacuoles positive for neutral lipids by Oil Red O stain. Droplets of Oil Red O material were also noticed in the affected livers and intestines. Congo red stain highlighted the presence of amyloid in moderate to severe amounts in the breast muscles and moderate amounts in livers, spleens, and intestines. Salmonella antigens were detected in the injection sites and livers by immunohistochemical staining. No viruses or toxic substances were recovered from the liver, spleen, intestine, and pectoral muscles, and the few bacteria isolated were interpreted as secondary postmortem invaders. In addition, livers and bile tested for hepatitis E virus were negative by reverse-transcriptase polymerase chain reaction.

R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis.

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans.

A Novel Pharmacologic Activity of Ketorolac for Therapeutic Benefit in Ovarian Cancer Patients.

PURPOSE: We previously identified the R-enantiomer of ketorolac as an inhibitor of the Rho-family GTPases Rac1 and Cdc42. Rac1 and Cdc42 regulate cancer-relevant functions, including cytoskeleton remodeling necessary for tumor cell adhesion and migration. This study investigated whether administration of racemic (R,S) ketorolac after ovarian cancer surgery leads to peritoneal distribution of R-ketorolac, target GTPase inhibition in cells retrieved from the peritoneal cavity, and measureable impact on patient outcomes. EXPERIMENTAL DESIGN: Eligible patients had suspected advanced-stage ovarian, fallopian tube or primary peritoneal cancer. Secondary eligibility was met when ovarian cancer was confirmed and optimally debulked, an intraperitoneal port was placed, and there were no contraindications for ketorolac administration. R- and S-ketorolac were measured in serum and peritoneal fluid, and GTPase activity was measured in peritoneal cells. A retrospective study correlated perioperative ketorolac and ovarian cancer-specific survival in ovarian cancer cases. RESULTS: Elevated expression and activity of Rac1 and Cdc42 was detected in ovarian cancer patient tissues, confirming target relevance. Ketorolac in peritoneal fluids was enriched in the R-enantiomer and peritoneal cell GTPase activity was inhibited after ketorolac administration when R-ketorolac was at peak levels. After adjusting for age, AJCC stage, completion of chemotherapy, and neoadjuvant therapy, women given perioperative ketorolac had a lower hazard of death (HR, 0.30; 95% confidence interval, 0.11-0.88). CONCLUSIONS: Ketorolac has a novel pharmacologic activity conferred by the R-enantiomer and R-ketorolac achieves sufficient levels in the peritoneal cavity to inhibit Rac1 and Cdc42, potentially contributing to the observed survival benefit in women who received ketorolac.

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

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.

Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral load and IgG antibody level in pigs.

Porcine Torque teno virus (TTV) has a single-stranded circular DNA genome and is currently classified into a new genus Iotatorquevirus with two species in a newly established family Anelloviridae. Viral DNA of both porcine TTV species (TTSuV1 and TTSuV2) has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States and in Europe. However, the prevalence of specific TTSuV antibodies in pigs remains unknown. In this study, the putative ORF1 capsid protein from TTSuV2 isolate PTTV2c-VA was expressed in Escherichia coli. The purified recombinant ORF1 protein was used as the antigen for the development of Western blot and indirect ELISA to detect TTSuV2-specific IgG antibodies in pig sera. The results revealed a relatively high rate of seropositivity to TTSuV2 in conventional pigs from different sources but not in gnotobiotic pigs. Overall, pigs with undetectable TTSuV2 viral load were more likely to have a lower anti-TTSuV2 antibody level. An analysis of 10 conventional pigs during a 2-month period showed that decreased viral loads or presumed virus clearance were associated with elevated anti-ORF1 IgG antibody levels. Interestingly, porcine circovirus associated disease (PCVAD)-affected pigs had a significantly lower level of TTSuV2 antibody than PCVAD-unaffected pigs (p<0.01). This is the first study to establish essential serodiagnostic tools for investigation of TTSuV seroprevalence and infection dynamics, which will help elucidate the potential pathogenicity of TTSuV infection in pigs.

Molecular phylogenetics of the gomphoid-phalloid fungi with an establishment of the new subclass Phallomycetidae and two new orders.

Molecular phylogenetic analyses for the gomphoid-phalloid fungi were conducted based on the five gene dataset with extensive taxon sampling. The monophyly of the gomphoid-phalloid clade was strongly supported, and four well supported major subclades were recognized. Three of the four subclades were represented entirely by gastroid taxa, and only Gomphales contained both gastroid and non-gastroid taxa. While the gastroid morphology is derived from epigeous, nongastroid taxa in Gomphales, the topology of Phallales indicated that truffle-like form is an ancestral morphology of the stinkhorn fruiting bodies. Although basidiospore maturation occurs within the enclosed fruiting bodies of the stinkhorn, the elevation of the mature spore-producing tissue represents an independent origin of the stipe among Basidiomycota. Comparisons are made between previous and new classification schemes, which are based on the results of phylogenetic analyses. Based on the results of these analyses, a new subclass Phallomycetidae, and two new orders, Hysterangiales and Geastrales, are proposed.

Sensitivity of renal cell carcinoma to aminoflavone: role of CYP1A1.

PURPOSE: The aminoflavone analogue (AF) exhibits antitumor activity in vitro, particularly against neoplastic cells of renal origin. We identified cellular correlates of responsiveness to AF in continuous human tumor renal cell carcinoma lines and in tumor cell isolates, termed renal carcinoma cell strains, from patients with clear cell and papillary renal neoplasms. MATERIALS AND METHODS: In vitro antiproliferative activity of AF was evaluated using the sulforhodamine B protein dye assay. In vivo antitumor activity of the drug was determined in mice bearing xenografts. Covalent binding of AF/metabolite(s) was assessed following exposure of cells to AF for 16 hours. CYP1A1 and CYP1B1 mRNA and apoptosis were quantitated by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: AF produced total growth inhibition in vitro in 3 of 6 human tumor renal cell lines at concentrations of 90 to 400 nM. In vivo treatment of mice bearing xenografts of the Caki-1 renal cell carcinoma, sensitive to AF in vitro, resulted in significant antitumor activity, including tumor-free survivors. Studies in 13 renal cell strains isolated from patients with clear cell (9) or papillary (4) renal cell carcinoma indicated that 3 of 4 papillary strains were sensitive to AF compared with 2 of 9 clear cell strains. AF sensitive renal cell lines and strains exhibited induction of CYP1A1 and CYP1B1 gene expression, increased covalent binding of AF metabolite(s) and apoptosis. CONCLUSIONS: AF has noteworthy antitumor activity against certain human tumor renal cell lines in vitro and in vivo, which correlates with drug metabolism to covalently binding metabolites after CYP1A1 and CYP1B1 gene expression. We hypothesize that it leads to apoptosis induction. AF sensitive renal cell strains are predominantly of the papillary histological type. These results are limited by the small numbers of cell lines and cell strains but they are suggestive of the need for further testing in larger collections of cell strains.

Cardiorespiratory events in preterm infants referred for apnea monitoring studies.

BACKGROUND: Episodes of apnea, desaturation, and bradycardia are common in preterm infants. Such infants who have persistent cardiorespiratory events detected by clinical bedside monitoring often are referred for overnight apnea monitoring studies. OBJECTIVE: To characterize apnea, bradycardia, and desaturation events in infants referred for an overnight apnea monitoring study and compare them with corresponding events in control infants of similar age and weight with no bedside monitor alarms. METHODS: Twelve-hour bedside apnea monitoring studies were performed on 68 preterm infants before hospital discharge. This population included 35 infants who were referred by their attending physicians because of persistent bedside monitor alarms (referral group) and 33 infants who had no documented cardiorespiratory events for at least 2 days before the study (control group). Each study monitored respiration via respiratory inductance plethysmography, oxygen saturation (Sao2), and heart rate. Events were defined as meeting 1 of the following criteria: apnea > or =20 seconds, bradycardia < or =80 beats per minute, or Sao2 < or =80%. RESULTS: The incidence of apnea > or =20 seconds was low, with no significant difference between infant groups. Referral infants exhibited a higher occurrence of desaturation episodes (20 +/- 6 vs 6 +/- 3 episodes/12-hour study) and a higher occurrence of bradycardia episodes (4.3 +/- 0.8 vs 1.1 +/- 0.3 episodes/12-hour study) than controls. These episodes of desaturation and bradycardia were always preceded by a respiratory pause, which was shorter in the referral infants (10.0 +/- 0.4 seconds vs 12.0 +/- 1.0 seconds). Baseline Sao2 was lower in referrals than controls (95 +/- 1% vs 98 +/- 1%), and the incidence of periodic breathing was significantly higher. CONCLUSIONS: Infants referred for apnea monitoring studies because of persistent bedside monitor alarms have very infrequent prolonged apnea but a higher frequency of desaturation and bradycardia in response to short respiratory pauses than infants without persistent bedside monitor alarms. Referral infants also exhibit a lower baseline Sao2. These abnormalities in oxygenation and cardiorespiratory control may be markers for subtle residual lung disease or functional central nervous system abnormalities.

Inhibition of IFN-gamma signaling by an Epstein-Barr virus immediate-early protein.

Viruses have evolved elaborate mechanisms to target many aspects of the host's immune response. The cytokine IFN-gamma plays a central role in resistance of the host to infection via direct antiviral effects as well as modulation of the immune response. In this study, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, BZLF1, inhibits the IFN-gamma signaling pathway. BZLF1 decreases the ability of IFN-gamma to activate a variety of important downstream target genes, such as IRF-1, p48, and CIITA, and prevents IFN-gamma-induced class II MHC surface expression. Additionally, BZLF1 inhibits IFN-gamma-induced STAT1 tyrosine phosphorylation and nuclear translocation. Finally, we demonstrate that BZLF1 decreases expression of the IFN-gamma receptor, suggesting a mechanism by which EBV may escape antiviral immune responses during primary infection.

Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing.

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 microm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

Epstein-Barr virus immediate-early protein BRLF1 induces the lytic form of viral replication through a mechanism involving phosphatidylinositol-3 kinase activation.

Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.

Epstein-Barr virus immediate-early protein BRLF1 interacts with CBP, promoting enhanced BRLF1 transactivation.

The Epstein-Barr virus (EBV) immediate-early protein BRLF1 is a transcriptional activator that mediates the switch from latent to lytic viral replication. Many transcriptional activators function, in part, due to an interaction with histone acetylases, such as CREB-binding protein (CBP). Here we demonstrate that BRLF1 interacts with the amino and carboxy termini of CBP and that multiple domains of the BRLF1 protein are necessary for this interaction. Furthermore, we show that the interaction between BRLF1 and CBP is important for BRLF1-induced activation of the early lytic EBV gene SM in Raji cells.

Enhancement of adenovirus vector entry into CD70-positive B-cell Lines by using a bispecific CD70-adenovirus fiber antibody.

Although many recombinant adenovirus vectors (rAd) have been developed, especially by using group C adenoviruses, to transfer and express genes, such rAd do not readily infect B-cell lines due to the lack of the coxsackievirus-adenovirus receptor. Bispecific antibodies have been used in different cell systems to facilitate entry of rAd into otherwise nonpermissive cells. Bispecific antibody is synthesized by covalently linking two monoclonal antibodies with distinct specificities. It has been shown that lymphoproliferative tumors commonly express the cell surface protein CD70, while this receptor is normally expressed on only a small subset of highly activated B cells and T cells. We therefore investigated whether a bispecific antibody with specificities for the adenovirus fiber protein and CD70 can facilitate rAd entry and subsequent expression of rAd-encoded genes in CD70-positive B cells. We found high CD70 expression on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), as well as some, but not all, Burkitt lymphoma (BL) lines. We show here that rAd encoding green fluorescent protein (Ad-GFP) infects EBV-transformed LCLs and a CD70-positive BL line 10- to 20-fold more efficiently in the presence of the CD70-fiber bispecific antibody. In contrast, the bispecific antibody does not enhance Ad-GFP infection in CD70-deficient BL cells. Using the CD70-fiber bispecific antibody, we increased the ability of rAd vectors encoding the EBV immediate-early proteins BZLF1 and BRLF1 to induce the lytic form of EBV infection in LCLs. These results indicate that the CD70-fiber bispecific antibody can enhance rAd infection of CD70-positive B cells and suggest the use of this vector to explore EBV-positive LCLs.

Epstein-barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies.

Although the immediate-early proteins of both herpes simplex virus (HSV) and cytomegalovirus (CMV) are known to modify promyelocytic leukemia (PML) (ND10) bodies in the nucleus of the host cell, it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect. The PML protein is induced by interferon, involved in major histocompatibility complex class I presentation, and necessary for certain types of apoptosis. Therefore, it is likely that PML bodies function in an antiviral capacity. SUMO-1 modification of PML is known to be required for the formation of PML bodies. To examine whether Epstein-Barr virus (EBV) lytic replication interferes with PML bodies, we expressed the EBV immediate-early genes BZLF1 (Z) and BRLF1 (R) in EBV-positive cell lines and examined PML localization. Both Z and R expression resulted in PML dispersion in EBV-positive cells. Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines. We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function. As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins, Z reduces the amount of SUMO-1-modified PML. We also found that Z itself is SUMO-1 modified (through amino acid 12) and that Z competes with PML for limiting amounts of SUMO-1. These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV. Furthermore, Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification.

Activation of lytic Epstein-Barr virus (EBV) infection by radiation and sodium butyrate in vitro and in vivo: a potential method for treating EBV-positive malignancies.

The consistent presence of the EBV genome in certain tumors offers the potential for novel EBV-directed therapies. Switching the latent form of EBV infection present in most EBV-positive tumor cells into the cytolytic form may be clinically useful because lytic EBV infection leads to host cell destruction, and very few normal cells contain the EBV genome. It would also be therapeutically advantageous to induce expression of EBV-encoded lytic proteins that convert the nucleoside analogues ganciclovir (GCV) and 3'-azido-3'deoxythymidine (AZT) into their active, cytotoxic forms. In this report, we have explored two different approaches for activating the lytic form of EBV infection in tumors. We show that gamma-irradiation at clinically relevant doses induces lytic EBV infection in lymphoblastoid cell lines in vitro as well as in EBV-positive B-cell tumors in SCID mice. In addition, sodium butyrate (given as a single i.p. dose) is effective for activating lytic viral infection in some EBV tumor types in SCID mice. We also examined whether low-dose gamma-irradiation treatment of EBV-positive lymphoblastoid cells in vitro promotes GCV or AZT susceptibility. The combination of radiation with either GCV or AZT induced significantly more cell killing in vitro than either radiation or prodrug treatment alone. Most importantly, we found that the combination of gamma-irradiation and GCV was much more effective in treating EBV-positive lymphoblastoid tumors in SCID mice than either agent alone. Thus, GCV or AZT treatment could potentially enhance the therapeutic efficacy of radiation therapy for EBV-positive lymphomas in patients.