Expression of LMP1 in A549 cells has been shown to increase transcription of the endogenous MMP9 gene and MMP9 protein levels in an NFκB dependent manner [³⁷]. Consistent with previous studies, expression of LMP1 increased the levels MMP9 in condition medium (Figure 1A). In contrast, when a reporter construct containing a 700 bp fragment of the MMP 9 promoter region was co-transfected with the LMP1 expression plasmid, LMP1 expression resulted in suppression of reporter activity (Figure 1B). Selective suppression of reporter activity by LMP1 has been previously reported [³⁵], though suppression of reporter activity in that study correlated with suppression of endogenous promoter activity. To further investigate the LMP1 induced disparity between activity on endogenous and exogenous promoters, A549 cells were co-transfected with the LMP1 expression vector and a series of reporter plasmids. Based on previous reports, LMP1 expression was expected to increase AP-1 and NF-κB responsive reporter activity while having no effect on the constitutive reporters FOP Flash, SV40, pGL3-Control and pRL-TK (renilla luciferase driven by the HSV1 thymadine kinase promoter). LMP1 expression suppressed reporter activity in assays utilizing the LEF/TCF responsive TopFlash reporter, the mutated LEF/TCF control reporter (FopFlash), an NF-κB responsive reporter, an AP1 responsive luciferase reporter construct, and a viral promoter driving β-galactosidase (SV40) (Figure 1B).

To test whether the suppression of reporter activity by LMP1 was specific to A549 cells, a similar series of reporter assays were conducted in nasopharyngeal carcinoma cells (CNE-1). LMP1 expression resulted in suppression of reporter activity in each of the constructs utilized (Figure 1C).

To determine that the effect was not an artifact of transfection efficiency, A549 cells were co-transfected with a constant amount of the NFκB responsive reporter along with increasing levels of the LMP1 expression vector (Figure 2A). Very low levels of LMP1 resulted in a modest increase in luciferase activity whereas increasing levels of LMP1 resulted in graded suppression of reporter activity. Reporter activity suppression was also observed when the constitutive firefly construct pGL3 (Figure 2B), the constitutive renilla luciferase reporter containing the HSV1 thymidine kinase promoter (Figure 2C), or the firefly luciferase LEF/TCF responsive TopFlash reporter (Figure 2D) were employed in a dual luciferase assay. Notably, when the LEF/TCF responsive data was normalized to the RL-TK data, an artifactual activation was observed (Figure 2E).

A549 cells were obtained from ATCC. CNE1 cells were obtained from Samuel H. Speck, (Emory University, Atlanta, Georgia, USA) and have been previously described [⁴⁹]. All cells were maintained in Dulbecco's minimal essential medium (Invitrogen, Eugene Oregon) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento CA) and 10 units/ml penicillin and 10 μg/ml streptomycin (Invitrogen). For transient transfection and luciferase experiments, cells were plated in 24-well plates at an initial density of 7.5 × 10⁴ cells per well and transfected with Lipofectamine (Invitrogen) optimized with Plus reagent (Invitrogen) according to the manufacturer's instructions. During chemical treatment, A549 and A549 derived cells were cultured in DMEM + 0.5% FBS.

Generation of the pEhyg*FLAG*LMP1*wt vector and retroviral transduction of cells have previously been described [⁵⁰]. Retrovirally transduced cells were maintained in 250 μg/ml hygromycin until 24 hours prior to plating for experiments. In separate infections, differential LMP1 expression was characterized and the two cell lines designated A549 <LMP1> Hi and A549 <LMP1> Lo; control cells were designated A549 <pEhyg>.

Arsenic trioxide (ATO) (Sigma Aldrich, St Louis Mo, #311383) was used at a concentration of 10 nM for treatment of A549 derived cell lines. Lithium Chloride (Sigma) was used at a concentration of 10 mM. The pcDNA*Flag*LMP1*wt vector was kindly provided by Nancy Rabb-Traub (UNC-Linberger Cancer Institute). Dominant active IκB plasmid has been previously characterized [⁵¹]and was a gift from Dean Ballard (Vanderbilt University Medical School, Nashville, TN, USA). The 700 bp promoter region MMP9 luciferase construct (MMP9-Luc) containing two AP-1 responsive sites and one NF-κB responsive site was a gift from Douglas Boyd (MD Anderson Cancer center, Houston, TX, USA), and has been previously characterized [⁵²]. The LEF/TCF responsive reporter pSuper8TopFlash_Luc containing eight LEF/TCF responsive elements and the control, pSuper8FopFlash_Luc containing mutations in these sites were obtained from AddGene (http://www.addgene.org/) and have been previously described [⁵³]. The NF-κB responsive reporter construct (pNFκB_Luc), control reporter plasmids pGL-3_Control luciferase and pSV40_bGal b-galactosidase were obtained from Promega (Madison, Wisconsin). The pAP1_Luc reporter construct containing multiple AP1 responsive sequences fused to a TATA-like promoter was obtained from Clonetech (Mountain View, CA).

For luciferase assays, 7.5 × 10⁴ cells were plated in each well of a 24-well plate, transiently transfected as stated above with 100 ng of the specified reporter plasmid per well, and harvested at 24 hours. For co-transfection and plasmid dose response assays, the total amount of DNA was held constant between wells. Treatment with 10 mM LiCl for four hours was used as a positive control for Top-Flash luciferase activity. Luciferase activity was quantified using the Luciferase Assay System (Promega # E1500) according to manufacturer's protocol, using a single tube luminometer (EG&G Berthold Lumat LM9507) programmed for a two second delay followed by a ten second read.

Nuclear and membrane fractions of indicated cell lysates were separated using the Qproteome Cell Compartment Kit (Qiagen, Valencia, CA, #37502) according to the manufacturer's directions. Briefly, 5 × 10⁶ cells were harvested in proprietary extraction buffer, separated by centrifugation, and the cytosolic fraction (supernatant) was removed. The remaining pellet was resuspended in proprietary extraction buffer, separated by centrifugation, and the membrane fraction (supernatant) was removed. After nuclease treatment, the pellet was resuspended in 500 ul of proprietary extraction buffer, separated by centrifugation, and the nuclear fraction (supernatant) was removed. Nuclear (PML and membrane (LMP1) fractions were combined with 4X Laemmli Buffer ( 240 mM Tris, 8% SDS, 40% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue) and 30 micrograms of protein per well were loaded in a 10 well NuPage Mops 4-12% gradient minigel (Invitrogen). Proteins were separated by electrophoresis at 130 V for 1.5 hours. Separated proteins were transferred for 1.5 hours at 30V to PDVF membrane (Invitrogen). Protein transfer was verified by Ponsou-S staining. Membranes were blocked in 5% BSA in PBST for 1hr prior to application primary antibody overnight at 4°C while shaking at 55 rpm. Rabbit polyclonal primary antibody to PML (Santa Cruz Biotechnologies, Santa Cruz, CA, #sc-5621) was used at a dilution of 1:200. Rabbit polyclonal primary antibody to β-Actin (Cell Signaling, #4967) was used at a dilution of 1:1000. Mouse monoclonal primary antibody to LMP1 was (B D Bioscience, Bedford, MA, #559898) used at a dilution of 1:1000. The secondary antibodies were fluorophore conjugated IRDye 680 goat anti-mouse IgG (LiCor Lincoln, NE, #926-32220) or IRDye 800CW goat anti-rabbit IgG (LiCor, #926-32211), and were used at a dilution of 1:15,000. Membranes were imaged using an Odyssey Infrared Imaging System (LiCor).

A549 cells were plated on 8-chambered Lab-Tek slides at 2 × 10⁴ cells per well; chemical treatment began 24 hours after plating. At specified time points, cells were rinsed twice with PBS and fixed in 4% Paraformaldehyde (freshly diluted from 16%, Electron Microscopy Science, Hatfield, PA) for 10 min at room temperature. The cells were then rinsed twice with PBS and permeabilized with 0.5% Triton X100 (Sigma) in PBS for 15 min at room temperature. Primary antibodies were applied overnight in a humidified chamber at 4° C. Following 3 rinses in PBS, secondary antibodies (1:500 dilution) were applied for 1 hour at room temperature. The chambers were removed and Vectashield with DAPI (Vector Laboratories, Burlingame, CA # H1200) was applied with a cover slip mount. The slides were imaged using a Nikon Eclipse 80i microscope and a SensiCam QE camera (Cooke Corporation) and IPLab V3.65a software (Scanalytics). Cells were grown on the same chamber slide and were exposed to the same antibody concentration and wash times. Exposure conditions were optimized for the brightest field of the specified conditions and held constant for subsequent exposures. Primary mouse monoclonal antibody (sc-966) and rabbit polyclonal antibody (sc5621) to PML were purchased from SantaCruz and used at a 1:500 dilution. Primary antibody to LMP1 was purchased from BD Bioscience (#559898) and used at a dilution of 1:500. Secondary antibodies that were employed were Alexa Fluor 594 goat anti-mouse (Invitrogen #A11005), AlexaFluor 594 goat anti-rabbit (Invitrogen#A21207) and AlexaFluor 488 goat anti-mouse (Invitrogen #A11008) and were as used at a dilution of 1:500.

Individual comparisons were analyzed by two-tailed unpaired t tests; multiple comparisons were analyzed by ANOVA with Modified Bonferroni post hoc test. A p-value < 0.05 was considered significant. For figures, (*) denotes p < 0.05 compared to control, (**) denotes p < 0.01 compared to control, (***) denotes p < 0.001 compared to control, (#) denotes p < 0.05 ATO compared to ATO/GCV co-treatment, (###) denotes p < 0.001 ATO compared to ATO/GCV co-treatment. The presented data is representative of multiple experiments performed in triplicate. Data is represented as the mean (+/-) SEM.