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Originally published In Press as doi:10.1074/jbc.M407435200 on November 2, 2004

J. Biol. Chem., Vol. 280, Issue 5, 3862-3874, February 4, 2005
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The Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus Modulates Cellular Gene Expression and Protects Lymphoid Cells from p16 INK4A-induced Cell Cycle Arrest*{boxs}

Feng-Qi An{ddagger}, Nicole Compitello{ddagger}, Edward Horwitz{ddagger}, Michael Sramkoski§, Erik S. Knudsen¶, and Rolf Renne{ddagger}||

From the {ddagger}Division of Hematology/Oncology and the Department of Molecular Biology and Microbiology, §Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106 and the Department of Cell Biology, University of Cincinnati, Cincinnati, Ohio 45229

Received for publication, July 2, 2004 , and in revised form, October 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Latently infected Kaposi's sarcoma-associated herpes-virus (KSHV)-associated tumor cells have both endothelial and lymphoid origins and express a limited set of latent viral genes. One such gene, ORF73, encodes the latency-associated nuclear antigen (LANA), a multifunctional protein that plays roles in viral DNA replication, episome maintenance, and transcriptional regulation. LANA interacts with cellular proteins involved in transcriptional regulation such as the tumor suppressors, retinoblastoma (Rb) and p53, and RING3 family members. Although several reports about specific LANA-regulated promoters exist, only limited data are available that address how LANA expression in KSHV-infected cells globally affects cellular gene expression, thereby potentially contributing to KSHV pathogenicity. To investigate this question, we generated an Epstein-Barr virus-negative Burkitts lymphoma line that expresses LANA from a tetracycline-inducible promoter (BJAB/Tet-On/LANA), and we performed microarray-based gene expression profiling. Expression profiling at different time points post-induction revealed that 186 genes were activated or repressed over 2-fold in the presence of LANA. Of these genes, 41 are regulated in the Rb/E2F pathway, whereas 7 are related to p53 signaling. To determine whether these gene expression changes translate into LANA-dependent changes in cell cycle regulation, we overexpressed p16 INK4a, a CDK4/6 inhibitor that efficiently induces cell cycle arrest in Rb-positive cells. Under these conditions, LANA expression protects lymphoid cells from p16 INK4a-induced cell cycle arrest and induces S-phase entry.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kaposi's sarcoma-associated herpesvirus (KSHV)1 is believed to be the causative agent of KS and two lymphoproliferative diseases, primary effusion lymphomas (PEL) and a subset of multicentric Castleman's disease (1-3). In these malignancies, the majority of cells is latently infected and expresses a small set of latent viral genes (4-6), one of which is the latency-associated nuclear antigen (LANA) (7, 8). LANA is a multifunctional protein and shows functional homology to DNA replication/transcription proteins such as SV40 large T antigen of polyomaviruses, EBNA-1 of Epstein-Barr virus (EBV), and E1/E2 of human papillomaviruses. LANA is the only viral protein required for latent viral DNA replication and segregation in latently infected cells (9-12).

In addition to its role in genome maintenance, LANA interacts with a variety of host cellular proteins to regulate viral and cellular gene expression. Among these cellular proteins are mSin3 and RING3, proteins that belong to a large family of chromatin-associated factors involved in transcriptional suppression (13, 14) and transcription factors CREB/ATF and CREB-binding protein (15, 16). LANA also interacts with the tumor suppressors Rb and p53, thereby stimulating transcription from a cyclin E promoter and suppressing a p53-binding site-containing reporter. Furthermore, co-expression of LANA and H-Ras in rat embryonic fibroblasts (but not expression of LANA alone) leads to colony formation and suggests that LANA may contribute to tumorigenesis (17, 18). Additionally, Fujimuro et al. (19, 20) demonstrated that LANA signals through the wnt pathway by stabilizing {beta}-catenin, thereby promoting S-phase entry into endothelial cells. Together, these data suggest that LANA globally modulates cellular gene expression in latently infected cells.

A limited number of LANA-responsive promoters have been identified by transient transfection studies. LANA, like EBNA-1 of EBV, positively regulates its own synthesis. Several promoters such as interleukin-6, telomerase, and the human immunodeficiency virus-long terminal repeat are activated or repressed by LANA (14, 21-25). No cellular genes regulated in either the Rb, p53, or the wnt pathway have been identified as LANA targets.

To identify LANA-responsive genes, we created inducible LANA expression lines in lymphoid cells and performed microarray-based gene expression profiling (26, 27). After identifying cellular genes that were altered in their expression in the presence of LANA, we demonstrate that the observed changes in gene expression translate into cell cycle regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—BJAB and DG75 cells (both EBV-negative Burkitt's lymphoma cell lines) were generously provided by Dr. Elliot Kieff, Harvard University. Primary effusion lymphoma cell lines (BCBL-1, BC-1, and BC-3) have been described previously (2, 28, 29). All lymphoid cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 2 mM L-glutamine.

To generate B cell lines that express LANA from a tetracycline-inducible promoter (30), 5 x 106 BJAB cells were transfected with 12 µg of linearized pTet-On (Clontech) by electroporation (250 V, 950 micro-farads) using a Bio-Rad Gene Pulser. For selection, cultures were grown in the presence of 500 µg/ml G418. Resistant clones were generated 2-3 weeks post-transfection by limited dilution, and single cell clones were expanded. Transient transfection assays utilizing a tetracycline-responsive promoter construct driving luciferase were used to screen more than 100 individual clones. One clone was selected that showed a 22-fold induction of luciferase activity with doxycycline (Dox) treatment and no background expression, compared with commercially available Jurkat/Tet-On and 293/Tet-On cells (Clontech). This clone along with Jurkat/Tet-On and 293/Tet-On cells was transfected with the linearized pTRE2hyg containing the full-length LANA coding region downstream of a TRE-containing promoter. After subcloning 80-100 clones for each cell line and analyzing LANA expression in the absence and presence of Dox by Western blot analysis and immunofluorescence, we selected one clone each for further characterization and gene expression profiling based on the following criteria: (i) no detectable background level of LANA, and (ii) high levels of LANA expression after induction.

Gene Expression Profiling and Data Analysis—Total RNA was extracted from 4 x 107 cells using RNAzol (Teltest Inc., Friendswood, TX) as recommended by the manufacturer. Induction protocol and time schedules for RNA extraction post-induction of LANA expression are described in Fig. 2A. Prior to gene expression profiling, each RNA preparation was analyzed by Northern blot using LANA and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)-specific radiolabeled probes (Fig. 2B).



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  		FIG. 2.
Gene expression profiling in BJAB/Tet-On/LANA cells. A, experimental design. A single culture at a density of 2 x 105 cells/ml was divided into 6 flasks (40 ml each). Four flasks were induced with Dox (1.2 µg/ml), and two flasks were left untreated. Total RNA from induced cells was extracted 12, 24, 36, and 48 h post-induction and from untreated control cultures at 12 and 36 h, respectively. B, verification of LANA expression and RNA quality prior to expression profiling. At each time point an aliquot of cells was analyzed for LANA expression by immunofluorescence assay. Cells at 48 h post-infection show nuclear LANA staining. Induction of LANA mRNA and RNA quality was assessed by Northern blot analysis. High quantities of LANA-specific mRNA are detectable at all time points post-induction but not prior to induction. As loading controls, blots were stripped and re-probed for GAPDH mRNA. C, data analysis revealed 199 genes that are changed in their expression in the presence of LANA. 450 changes were observed between 12 and 36 h in the presence of Dox (green). 299 genes changed during this time in the untreated control cells (red). 163 genes overlapped between both groups. These genes were removed except for 28 genes that were altered more than 2-fold in the presence of Dox. A total of 423 expression changes was observed at all time points. Application of selection criteria and removal of ESTs yielded a total of 199 genes (13 genes were present more than once on the array give the final number of 186 genes) (Table I). D, hierarchical clustering of final 186 genes over time shows that most expression changes occurred between 12 and 36 h post-LANA induction.

 
Gene expression profiling was performed by the Case Western Reserve University and Ireland Cancer Center Gene Expression core facility using Affymetrix technology. Oligonucleotide-based HG-U95Av2 arrays containing 12,626 probe sets corresponding to known human genes and ESTs (www.affymetrix.com) were used for all experiments. Selection of poly(A)+ mRNA, reverse transcription, and generation of biotin-11-CTP and biotin-16-UTP (Ensue Diagnostics)-labeled cRNA by T7 in vitro transcription were performed as recommended by Affymetrix. Prior to HG-U95Av2 chip hybridization, a test chip containing 100 probe sets was probed with an aliquot of the cRNA preparation. 75 µg of cRNA were used for each chip. Samples were run on the fully automated Affymetrix GeneChip Fluidics station, which performs all steps from hybridization to washing and staining with streptavidin-phycoerythrin (Molecular Probes). The arrays were scanned using a Hewlett-Packard confocal microscope scanner (Affymetrix) at a wave-length of 560 nm.

Prior to data analysis, we determined the percentage of "present" and "marginal present" genes for each array, which was greater than 40% for each experiment. The signal ratio of 3' to 5' probe sets for GAPDH and actin was less than 2, indicating efficient reverse transcription and high RNA quality. Repeated array hybridizations for identical RNA samples were performed to evaluate the stability of our chip data. For comparisons across experiments, the scale factor was used to normalize the average signal of each array to 1500. Hybridization data were analyzed by Microarray Suite 5.0 (MAS 5.0) software. Gene fold changes were determined based on the signal log ratio parameter. Microsoft access was used for all pairwise comparisons and to determine common changes between multiple data sets.

The following criteria were applied to raw data to determine changes in gene expression levels. Up-regulated genes must be present or marginal present, and expression levels should be greater than 500 in one sample; fold changes should be "+2 or more," and the change call should be "increase" or "marginal increase." Down-regulated genes must be present or marginal present, and expression levels should be greater than 500 in one sample; fold changes should be "-2 or less," and the change call should be "decrease" or "marginal decrease." We used the GeneSpring 4.2 software (Silicon Genetics, Redwood City, CA) to perform hierarchical cluster analysis between data sets and to create Venn diagrams outlining common changes between different cell lines. Raw data are available at NCBI GEO web page accession number GSE1880 [NCBI GEO] .

Confirmation of Selected Genes by Northern Blot Analysis and Real Time RT-PCR—Northern blotting and hybridization have been described previously (23). 5 µg of total RNA was electrophoretically separated on denaturing agarose gels, blotted to Hybond membrane (Amersham Biosciences), and hybridized to specific probes overnight at 65 °C in Church buffer (31). Probes were labeled with [32P]dCTP using the Redivue random priming kit (Amersham Biosciences).

To eliminate contaminating DNA, 10 µg of total RNA was treated with RQ1 RNase-free DNase (Promega) prior to cDNA synthesis. As an additional control, reverse transcription reactions were performed in the presence and absence of RT and amplified by regular PCR using GAPDH primers. For reverse transcription, 500 units of Moloney murine leukemia virus-RT (Invitrogen) was used in a total 50-µl reaction volume containing 10 mM dithiothreitol, 0.5 mM dNTPs, 15 units of RNase inhibitor (Invitrogen), and 2.5 µl of oligo(dT)12-18 primer (Invitrogen). Reactions were incubated at 42 °C for 35 min, followed by 95 °C for 5 min.

Real time quantitative PCR was performed on a LightCycler3 (Roche Diagnostics). One µl of cDNA was added to 2 µl of Mastermix (Light-Cycler Faststart DNA master hybridization probes, Roche Diagnostics) in a total volume of 20 µl containing 3 mmol/liter MgCl2, 1.5 µmol/liter of each 3' and 5' primers. Reactions were incubated at 95 °C for 10 min followed by 40 PCR cycles (15 s 95 °C and 60 s 58 °C). SYBR green fluorescence was measured at the end of each cycle. Threshold values were set at the midpoint SYBR green fluorescence and the threshold cycle (CT) was determined as the cycle at which a statistically significant increase in fluorescence was initially detected. Primers were designed based on Affymetrix probe sets for each target gene using primer 3 input software (primer3-www.cgi V 0.2) (supplemental Table 1). These primers and those for GAPDH were included in parallel reactions. Gene expression signals at the point of threshold cycle were normalized to GAPDH as an endogenous reference. A standard curve was generated by amplifying GAPDH from four 10-fold serial dilutions from a cDNA template that resulted in 0.35 cycles/fold change. To allow comparison between array data and real time PCR, the difference of CT values between samples was converted to fold changes.

Protein Analysis by Western Blotting and Immunofluorescence Assays—Immunofluorescence assays to detect LANA were performed as described previously (23). Briefly, cells were spotted onto Teflon-coated slides, fixed with methanol/acetone, and blocked with 3% bovine serum albumin in PBS. To detect LANA (ORF73), a polyclonal rabbit antibody raised against three ORF73 peptides from its central repeat domain was used (66). As secondary antibody, a fluorescent isothiocyanate-conjugated goat anti-rabbit was used (1:100 in blocking buffer). Photographs were taken by using either an inverted fluorescence microscope (Nikon, Inc.) or a confocal microscope (Leica, Inc.).

For Western blot analysis, whole cell lysates were separated on 6% polyacrylamide gels and transferred to nylon membranes. To detect LANA, we used a polyclonal serum as described above; for {beta}-catenin, we used a rabbit antibody (Sigma) followed by a peroxidase-conjugated secondary antibody. As loading control, all membranes were re-probed using tubulin-specific monoclonal antibody (Oncogene Research Products, Boston).

Cell Cycle Analysis after p16 INK4a Treatment—BJAB and BJAB/Tet-On/LANA Dox-treated or untreated were either mock-infected or infected with recombinant adenovirus expressing p16 INK4a (32) and recombinant adenovirus expressing green fluorescent protein (GFP) (kindly provided by Dr. Steven Fisher, Case Western Reserve University) at a ratio of 3:1. 40-45 h post-infection, cells were stained with 5 µg/ml Hoechst 33342 at 37 °C for 30 min, washed with PBS, and resuspended in 2% bovine serum albumin/PBS at a concentration of 107 cells/ml. Cell cycle analysis was performed on a LSR1 flow cytometer (BD Biosciences). The DNA histogram was created by gating on GFP+ cells, and raw data were further analyzed by ModFit LT 3.0 software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of LANA-inducible Cell Lines—The majorities of cells within PELs, MCD, and KS skin lesions are latently infected with KSHV and express a limited number of viral genes. Although all KSHV-associated tumors express LANA, latent gene expression regarding additional viral genes that can influence cellular gene expression (i.e. v-Cyclin, v-FLIP, LANA2 (vIRF-3), Kaposin (K12), and K15 substantially varies between KSHV-associated tumor types (for review see Refs. 33 and 34).

In this study, we focused on how LANA contributes to global cellular gene expression by generating B cell lines that express LANA from a tetracycline-inducible promoter (30). The advantage of this approach is that we can measure the effect of a single viral protein on cellular gene expression in an otherwise isogenic background. To generate LANA-inducible cells, we chose BJAB, a Burkitt's lymphoma-derived (BL) EBV-negative cell line, which has been used previously to stably express LANA (9, 23).

Briefly, cells were transfected with pTet-On expressing the tetracycline-inducible RTA transactivator. Cells were selected, and after single cell cloning, inducibility was compared with highly inducible and commercially available 293/Tet-On cells using transient transfection assays. Next, selected clones were transfected with pTRE2/LANA, and a second round of selection and single cell cloning was performed. After expansion, we tested for LANA expression in the absence and presence of Dox to identify clones of each cell line that exhibited low background and high inducibility. Fig. 1 shows the induction kinetic of BJAB/Tet-On/LANA as well as 293/Tet-On/LANA and Jurkat/Tet-On/LANA cells; LANA was undetectable in the absence of Dox but highly expressed in the presence of Dox. In BJAB/Tet-On/LANA cells, maximum expression levels were reached between 12 and 24 h post-Dox treatment that is comparable with those levels observed in BCBL-1 cells (Fig. 1) and (23).



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  		FIG. 1.
Generation of LANA-inducible cell lines. Tetracycline-inducible cells established from single cell clones were induced for the indicated times and analyzed for LANA expression by Western blot analysis. Whereas no LANA expression is detectable before Dox treatment, high levels of LANA are expressed by 24 h post-induction in all three cell lines. Identical blots were stripped and re-probed with a tubulin antibody as loading control. LANA shows a characteristic doublet at 220 kDa. Bands at lower molecular weights are degradation products that are also observed in KSHV-infected PEL cell lines (23). A, BJAB/Tet-On/LANA. B, 293/Tet-On/LANA. C, Jurkat/Tet-On/LANA. D, LANA expressed in BJAB/Tet-On/LANA cells trans-represses TR-dependent transcription from a luciferase reporter. Uninduced and induced cells were transfected by electroporation with a luciferase reporter containing 7 copies of TR. In the presence of Dox, luciferase was down-regulated 2.2-fold.

 
To ensure that LANA is functional in these cell lines, two assays were performed. Previously, we have shown that LANA trans-represses the enhancer activity of the viral terminal repeat (TR) which is dependent on the ability of LANA to bind to TR (35). Transient transfection assays in BJAB/Tet-On cells showed that a TR-containing reporter was repressed in the presence of LANA (Dox+) at 48 h post-induction (Fig. 1D); similar results were obtained for 293/Tet-On/LANA cells (data not shown). LANA is the only viral protein required for long term maintenance of TR-containing plasmids (10, 12). Transfection of a plasmid containing two copies of TR and a GFP reporter leads to long term maintenance in induced BJAB/Tet-On/LANA cells.2 These experiments illustrate the functionality of LANA in BJAB/Tet-On/LANA cells because it binds to DNA and supports replication and maintenance of TR-containing plasmids.

In BJAB Cells, Expression Levels of 186 Genes Are Significantly Altered in the Presence of LANA—In the first set of experiments, we analyzed the effect of LANA on cellular gene expression by comparing Dox-induced cells to noninduced cells over time. A single cell culture at a density of 2 x 105/ml was divided into mock- and Dox-treated cells, and total RNA was extracted at 0, 12, 24, 36, and 48 h. Prior to gene expression profiling, RNA quality and LANA protein expression were analyzed by immunofluorescence assay and Northern blot analysis (Fig. 2B). Synthesis of cRNA, hybridization, washing, and raw data analysis were performed as described under "Materials and Methods." We used the oligonucleotide array HG-U95Av2 (Affymetrix, Inc.), which represents more than 12,000 known human genes and ESTs.

Fig. 2 outlines the experimental design and provides the number of observed gene expression changes at each time point post-induction in BJAB/Tet-On/LANA cells. At 12 h, expression levels in induced compared with mock-induced cells revealed only 17 changes. In contrast, 450 changes were observed when comparing profiles from Dox-treated LANA-expressing BJAB/Tet-On/LANA cells at 36 to 12 h post-induction. Notably, the control comparison between 36 and 12 h in the absence of Dox also revealed 299 changes, of which 163 overlapped with the above 450 changes (Dox+), suggesting that these changes are the result of the growth conditions (increasing cell density over time) and occurred independent of LANA expression. We sub-tracted these 163 genes from the original 450. Following further analysis of these 163 genes, we identified 28 that were induced or repressed more than 2-fold when LANA was present; therefore, these gene changes were re-entered in the analysis. Comparison of induced and control cells at 24 h post-induction revealed an additional 79 genes. Comparison of cells at 48 to 36 h post-induction yielded 12 additional gene changes. Subsequently, we observed differential gene expression with significant p values as determined by the Wilcoxon's signed rank test (MAS 5.0 software) for a total of 423 probe sets in the presence of LANA.

To define further the group of differentially expressed cellular genes, we applied two additional exclusion criteria. First, probe sets expressed at very low levels (whose mean fluorescent signal intensities did not reach 500 at any time point) were disregarded; second, all ESTs were excluded. Application of these criteria revealed 199 genes of which 13 where represented by duplicate probe sets. Hence, a total of 186 genes were significantly altered in response to LANA expression (Fig. 2C). We confirmed the Affymetrix profile for 8 randomly selected genes by real time RT-PCR. Although the extent of up- or down-regulation for each sample differed between the assays, overall trends of gene expression patterns were confirmed (Tables I and II). As additional quality control, array-based profiling on two samples was performed twice. Using 2-fold change as single criteria revealed less than 0.06% gene changes between replicates (80 of 12,625); applying the above-mentioned criteria reduced the observed changes to 0.006%. Hierarchical cluster analysis of these 186 genes showed that the majority of expression changes occurred in congruence with the expression kinetics of LANA between 12 and 36 h post-induction (Fig. 2D).


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  		TABLE I
186 genes modulated by LANA in BJAB cell

 


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  		TABLE II
BJAB/Tet-On/LANA cell line Affx Chip data confirmed by real time PCR Light cycler RT-real time PCR using cyber green incorporation from Roche Applied Science.

 
In BJAB Cells, LANA Targets Many Rb/E2F-regulated Genes—By utilizing GenBankTM annotations in combination with Affymetrix data mining tools and GeneSpring software, all 186 genes were categorized into 13 functional groups (Table I). Based on data from Radkov et al. (18), who showed that LANA interacts with Rb, we analyzed our data for the presence of genes that are regulated in the Rb/E2F pathway. In a comparison of our data to three recent reports on identifying genes that are Rb/E2F-regulated (36-38), we identified 41 genes in this pathway (Table I). Some genes are related to cell cycle control (cyclin E1, CDC2, and CDC25A) and to DNA replication (RPA1, dihydrofolate reductase, primase, thymidylate synthetase, and polymerase-{epsilon}), whereas other genes are involved in chromatin remodeling, transcriptional control, and signal transduction (SWI/SNF-related, Pou domain class 2 transcription factor, and inhibitor of DNA binding 1). Furthermore, we found 7 genes that were related to p53 signaling. These include anti-apoptotic gene Bcl2-like 1, GADD45B (involved in growth arrest following DNA damage), and human 14-3-3{epsilon}, a protein involved in many signal transduction pathways including the wnt pathway (Table I, marked by *).

These data suggest that, in BJAB cells, LANA expression broadly targets cellular gene expression through its interaction with Rb. Thus, in LANA-expressing cells that do not express Rb, these genes should not be changed. As described above, we also generated 293/Tet-On/LANA cells and subsequently performed gene expression profiling. 293 cells are E1A-transformed, which efficiently causes Rb degradation (39). After LANA induction for up to 42 h in 293/Tet-On/LANA cells, we observed significantly less gene changes than in BJAB cells (81 genes, see supplemental Table 2); only one was related to the Rb/E2F signaling pathway (calreticulin). To rule out that LANA expression might stabilize Rb, we performed Western blot analysis and could not detect Rb in 293/Tet-On/LANA cells in either the presence or absence of LANA (data not shown). This suggests that in the absence of Rb, LANA modulates the expression of a completely different set of genes. Indeed, only one gene (TAX1BP1), a protein with anti-apoptotic activity that binds to the HTLV-1 tax protein, was found to be similarly down-regulated in BJAB and 293 cells. Notably, in 293 cells the majority of genes were moderately down-regulated in response to LANA expression (67 of 81), whereas in BJAB cells, 83 of 186 genes were up-regulated.

Modulation of LANA of the Rb/E2F-dependent Genes Contributes to Cell Cycle Regulation and Activation of {beta}-Catenin in BJAB Cells—Many of the observed changes within the 41 genes involved in Rb/E2F signaling were moderate (between 2- and 2.5-fold) (Table I). To confirm that these changes translate into cell cycle regulation, we expressed p16 INK4a, a potent negative regulator of CDK4 and CDK6, in BJAB/Tet-On/LANA cells utilizing a recombinant adenovirus (32). When overexpressed, p16 INK4a drives cells into G0/G1 cell cycle arrest (41). BJAB/Tet-On/LANA cells (Dox+ or Dox-) were co-infected with two adenoviruses expressing either p16 INK4a or GFP to mark infected cells. Dox treatment did not alter the number of adeno/p16-infected cells (Fig. 3). Following a 42-45-h incubation period, cells were stained with Hoechst 33342 dye, and GFP+ cells were gated and analyzed for cell cycle phases by flow cytometry. LANA induction and p16 INK4a expression was confirmed by Western blot analysis (Fig. 4A). In control BJAB cells, 47.9% of cells were in G0/G1 with 40.3% of cells in S-phase. p16 INK4a expression in noninduced BJAB/Tet-On/LANA cells caused 65.6% of cells to arrest in G0/G1, with only 23.8% of cells remaining in S-phase. In contrast, p16 INK4a expression in LANA-expressing BJAB/Tet-On/LANA cells (Dox added 24 h prior to infection with Ad/p16 INK4a) caused only 55.2% of cells to arrest in G0/G1 and 32.8% of cells in S-phase. Fig. 4B shows average percentages from three independent experiments. Results were statistically significant because the p value was <0.05 (Student's t test). These data show that LANA expression protects a significant number of cells from p16 INK4a-induced cell cycle arrest and supports S-phase entry.



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  		FIG. 3.
The presence of LANA has no effect on the number of adenovirus-infected cells. BJAB/Tet-On/LANA cells in the absence or presence of Dox were infected with Ad/p16 INK4a and Ad/GFP. 45 h post-infection, cells were stained with Hoechst 3332 dye and were analyzed for GFP expression by fluorescence microscopy and by flow cytometry analysis. As can be seen in both cases, the number of GFP-expressing cells is about 8.7% in the presence and absence of LANA.

 



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  		FIG. 4.
LANA expression protects BJAB/Tet-on/LANA cells from p16 INK4a-induced cell cycle arrest. To overexpress p16 INK4a, cells were infected with Adeno/p16 INK4a and Adeno/GFP at a ratio of 3 to 1. 42-45 h post-infection, cells were Hoechst-stained and the GFP-expressing population was analyzed for cell cycle distribution using ModFit LT 3.0 software. A, Western blot analysis of Ad/p16 INK4a Ad/GFP and mock-infected BJAB/Tet-On/LANA cultures in the presence and absence of Dox. LANA expression is only detectable after Dox treatment (3rd and 6th lanes), and p16 INK4a expression was only detectable in infected cells (4th to 6th lanes). {alpha}-Tubulin was used as loading control. B, cell cycle analysis of p16 INK4a-expressing cells in the presence and absence of LANA. Shown are the average percentiles of three independent experiments. C, LANA induces {beta}-catenin expression in BJAB cells but not in 293 cells. LANA-inducible lines were tested for {beta}-catenin expression in the presence and absence of Dox. In addition parental cell lines BJAB and 293 were analyzed. Western blot analysis shows that, in BJAB cells, {beta}-catenin is only detectable in LANA-expressing cells (3rd lane). In contrast, 293 cells constitutively express {beta}-catenin. As a loading control, blots were reprobed for {alpha}-tubulin expression.

 
It is important to note that the extent of p16-induced cell cycle arrest in lymphoid cells is lower than in fibroblast cultures; our results are in agreement with observations published previously (13) for p16-induced cell cycle arrest in PEL cell lines. Our data further confirm data from Fujimuro et al. (19, 20) who showed that LANA can induce S-phase entry in primary dermal microvascular endothelial cells by stabilization of {beta}-catenin, which is also enriched in PEL cell lines.

Subsequently, we examined whether LANA expression causes accumulation of {beta}-catenin in BJAB/Tet-On/LANA and 293/Tet-On/LANA cells. {beta}-Catenin was undetectable by Western blot analysis in control BJAB and noninduced BJAB/Tet-On/LANA cells. Following induction of LANA, the accumulation of {beta}-catenin was readily detectable (Fig 4C). In 293/Tet-On/LANA cells, which exhibited no Rb/E2F-related gene expression changes, {beta}-catenin is constitutively activated, and LANA had no influence (Fig. 4C). These data suggest that the extent to which LANA modulates cellular gene expression in lymphoid cells correlates with its ability to induce the accumulation of {beta}-catenin.

Does LANA Expression Contribute to Gene Expression Profiles in PEL-derived Cell Lines?—The majority of PELs are dually infected with EBV and KSHV (2, 29). We wanted to define a subset of genes that exhibited differential expression specific to PEL cell lines, compared with BL lines that are associated with EBV; thus we examined the gene expression profiles of three PEL and three BL cell lines. Infection status of these cell lines is outlined in Fig. 5A, left lower corner. All cell lines were grown under identical conditions with respect to growth media and cell density. Expression profiling, data analysis and comparison were performed as described under "Materials and Methods."



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  		FIG. 5.
Comparative gene expression profiling of PEL- and BL-derived cell lines. A, Venn diagram showing all pairwise comparisons and the number of common genes between each pair. 180 genes were commonly expressed in all KSHV-infected PEL cell lines (BCBL-1, BC-3, and BC-1). KSHV and EBV infection status of all cell lines are also outlined. B, hierarchical clustering using GeneSpring 4.2 software. Genes that are most related in their expression pattern are placed adjacent. Red indicates high expression, and blue indicates low expression levels. C, comparison of gene changes between BCBL-1, BC-3, and BJAB cells to LANA-induced gene changes in BJAB/Tet-On/LANA cells. This Venn diagram was produced by using GeneSpring 4.2 software focused on the differences between PEL (BCBL-1 and BC-3) to BJAB cells and BJAB/Tet-On/LANA cells, with the same genetic background. As can be seen, a total of 66 (22 + 31 + 13) genes overlap to the 186 genes that were altered in the presence of LANA.

 
To identify genes with expression patterns specific to KSHV-infected PEL-derived lines, we flagged genes that differed in expression levels 2-fold or more up or down in both BCBL-1 and BC-3 cells when compared with two EBV-negative Burkitt's lymphoma cell lines, BJAB and DG75. Our analysis, represented by a Venn diagram in Fig. 5A, lead to a list of 298 (118 plus 180) probe sets. By including the comparison between BC-1 (KSHV and EBV) and Raji (EBV) in this analysis and by removing 29 duplicate probe sets, we further defined our list to be 151 genes commonly expressed in all three PEL-derived cell lines.

Hierarchical cluster analysis confirmed that these genes are indeed differentially expressed between PEL and BL-derived cell lines. As shown in Fig. 5B, within these 151 genes, roughly 50% exhibit high expression in PEL cells but low expression in BL cells and vice versa. Thus, the expression levels of each of these 151 genes can predict PEL versus BL origin (supplemental Table 3). To validate the array data, five genes were analyzed by Northern blot analysis and two genes by real time PCR (Fig. 6 and Table III).



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  		FIG. 6.
Confirmation of gene expression profiling results by Northern blot analysis. Each lane contains 10 µg of total RNA from the same pool used for profiling. Probes for each gene were generated by PCR amplification from genomic DNA using primers shown in supplemental Table 1. PCR fragments were radiolabeled by random priming. As loading control, one membrane was hybridized with a probe specific for GAPDH. Numerical values below each lane give the signal intensity observed by gene expression profiling, and capital letters indicate the expression call (i.e. P indicates presence and A indicates absence).

 


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  		TABLE III
PEL cell lines Affx Chip Data confirmed by real time PCR Light cycler RT-real time PCR using cyber green incorporation from Roche Applied Science.

 
Two recent studies analyzed large cohorts of PEL-derived cell lines and primary tumor biopsies. Klein et al. (42) analyzed a total of 62 samples and identified a group of 125 genes that had a PEL-specific expression pattern when compared with various B cell tumors. Jenner et al. (43) compared 36 lymphoid tumor lines and 2 primary lesions and identified a group of 270 genes that were differentially expressed in PEL cells. Based on our comparison of 6 cell lines, we found that 60 genes within the 151 genes were also reported in these two studies as PEL-specific. In congruence with Klein et al. (42), we observed a lack of mature B cell markers (CD19, CD22, CD 79a/b, CD52, and BLNK) in PEL cells. Additionally, Bcl6 and integrin-{beta}2 were down-regulated. Also in congruence with the previous two studies, the vitamin D receptor, IRF4, interleukin 10, and the interleukin 2 receptor {beta} genes were highly expressed in PEL cells. Additionally, we found ENPP2, an autotaxin, GPR37, a G protein-coupled receptor, and HFL1, a complement-like factor, highly expressed in PEL compared with BL (supplemental Table 3).

To ask whether LANA contributes to the overall observed gene expression pattern in PEL cells, we compared the 151 PE-specific genes to those affected by LANA in BJAB/Tet-On/LANA cells. Six genes were identified (CD19, intersectin 2 (ITNS2), DEK, DCK, NCOA3, and BCL6); from the 81 genes found changed in 293/Tet-On/LANA cells, we gained three additional genes (G protein-coupled receptor 37 (GPR37), annexin 2 (ANXA2), and calreticulin). From these 9 genes, 8 were also listed in two previous studies on PEL-specific gene expression (42, 43). Furthermore, by comparing all genes (186 in BJAB plus 81 in 293 cells) affected by LANA to reported studies revealed an additional 12 genes. In total, we identified 21 genes that are modulated by LANA and whose expression is similar in PEL cells (see Table I and supplemental Table 2, marked by #). Within this group of 21 genes, 3 are Rb/E2F regulated, namely DCK, PTPRC, and NCOA3.

Additional analyses were performed with BCBL-1 and BC-3 cells compared with BJAB and 186 LANA-modulated genes in induced BJAB/Tet-On/LANA cells. This analysis, which more closely accounts for the genetic background, revealed that 66 (22, 31, and 13) of the original 186 LANA-induced gene expression changes overlapped between groups (Fig. 5C). This group contained 17 of the 41 Rb-related genes described previously, including those related to DNA replication (i.e. RPA, TTK, DCK, PRIM2A, and TOP2A), and contained the 8 genes that overlapped with the list of 151 PEL-specific genes from this study and previously reported PEL-specific genes (42, 43). In summary, these results suggest that a significant proportion of virally induced gene expression changes can be attributed to LANA expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During latency, KSHV expresses viral genes such as LANA, v-Cyclin, and v-FLIP that target cell cycle control and proliferation (for review Refs. 44 and 45). Our molecular screening approach implementing inducible cell lines allowed us to study the impact of LANA on cellular transcription without the influence of additional viral genes. Our profiling data from lymphoid cells that express LANA in an inducible manner confirm that one mechanism by which LANA modulates cellular gene expression is targeting the Rb/E2F pathway. Our data further expand on data from Radkov et al. (18), who showed that LANA can interact with the pocket domain of Rb, and activate the cyclin E promoter in reporter assays presumably by releasing E2F (18). Promoters of genes in this pathway contain binding sites for different E2F family members that can be divided into activators (E2F1, E2F2, and E2F3) and repressors (E2F4 and E2F5). Activator complexes are negatively regulated by binding to the pocket domain of Rb. Upon phosphorylation by CDK4 and/or CDK6, Rb releases E2F, which activates genes expressed during the transition from G1 to S and are involved in DNA replication. The suppressor complexes E2F4 and E2F5 are active when bound to the pocket proteins p107 and p130, also called Rb2. Dissociation of these complexes leads to de-repression of genes that are involved in DNA damage checkpoints during G1 and G2, chromatin assembly, DNA repair, and mitosis (for review see Refs. 46 and 47).

We found that of the 186 genes differentially expressed in BJAB/Tet-On/LANA cells, 41 (22%) were related to Rb/E2F signaling. This group contained genes with established roles in cell cycle control, such as cyclin E, CDC25A, and CDC2, or markers of proliferation (human 14-3-3{epsilon} and MKI67) (Table I). Additional genes were involved in DNA metabolism, replication, and repair functions: RPA1, AK3, dihydrofolate reductase, primase, thymidylate synthetase, polymerase {epsilon}, TOP2A, DCK, and MCM4. Furthermore, LANA affected the gene expression levels of mitotic checkpoint control genes and mitotic genes such as TTK, MAD2, and HEC. We verified the relevance of this LANA-induced gene expression pattern by providing functional data demonstrating that LANA protects a significant number of BJAB/Tet-On/LANA cells from p16 INK4a-induced cell cycle arrest (Fig. 4). Hence, these data show that LANA expression alone is sufficient to induce cell cycle progression in B lymphoid cells. Fujimuro et al. (19) recently demonstrated that LANA manipulates the wnt signaling pathway and induces S-phase entry in endothelial cells. Two domains of LANA specifically interact with and inhibit GSK-3{beta}, the main negative regulator of {beta}-catenin (20). This inhibition of GSK-3{beta} leads to a stabilization of {beta}-catenin and, as a result, activates transcription together with Tcf-4 (for review see Refs. 48 and 49). This pathway also activates cyclin D, which through CDK4/6 and the subsequent phosphorylation of RB induces S-phase entry (19, 20). Together, these data suggest that LANA targets the major checkpoint regulator Rb by two different mechanisms as follows: direct binding of Rb and/or stabilization of {beta}-catenin. In addition to this unique redundancy, KSHV encodes a v-Cyclin that phosphorylates CDK6, thereby further contributing to cell cycle progression (50, 51).

Given the role of LANA in viral DNA replication during latency (10-12), it is conceivable that direct binding to Rb provides a mechanism by which a specific subset of E2F-regulated genes are targeted. Evidence for some specificity was reported by Radkov et al. (18) who showed that, in vitro, LANA only interacts with Rb but not with the pocket proteins p107 or Rb2 (18). We wanted to know whether those 41 Rb/E2F-related genes affected by LANA were preferentially known to be regulated by E2F1-3 versus E2F4,5. Ren et al. (38) has analyzed E2F-binding sites in over 200 genes by CHIP using E2F1-(activator complexes) or E2F4 (repressor complexes)-specific antibodies. E2F1- or E2F4-binding sites alone or in combination were found in many promoters of known genes involved in DNA replication and cell cycle control but also in checkpoint genes for DNA damage repair and apoptosis, chromosome segregation, and mitosis (38). Examination of these 41 genes revealed that most genes contained binding sites for both activator and repressor complexes. We found a small subset of genes, however, which harbor only E2F4-binding sites and whose expression is de-repressed in p107 and Rb2 knock-out cells (38). These included the mitogen-activated kinase (MAP3K7) and the mitotic checkpoint proteins MAD2L1, TTK, and HEC. The fact that these E2F4-regulated genes were affected by LANA suggests that, in BJAB cells, LANA modulates transcription of Rb/E2F target genes upstream of Rb by stabilizing {beta}-catenin. In agreement with studies from Fujimuro et al. (19, 20), Western blot analysis showed that LANA stabilized {beta}-catenin expression in BJAB cells (Fig. 4C). In 293 cells, {beta}-catenin is constitutively activated and is not changed by the presence of LANA (Fig. 4C). This observation, together with the lack of Rb in 293 cells (39), explains why LANA expression did not impact targets related to Rb or p107 and Rb2 in these cells.

We additionally analyzed our data for cellular genes involved in the wnt signaling pathway (52-54), and we found 9 genes that are regulated by Tcf-4, downstream of {beta}-catenin. These genes include GADD45B, CTNND2, CNN3, ALDH1B1, SLC25A1, SLC4A2, ANXA2, TOB1, and TIEG. Although this group is relatively small, it is important to point out that in 293 cells {beta}-catenin is highly expressed; in BJAB cells, however, many target genes within this pathway are already deregulated by high levels of c-Myc, which in nontransformed B cells is regulated downstream of {beta}-catenin (48, 49, 55).

Predominantly, cellular genes in 293 cells are moderately down-regulated between 2- and 2.5-fold under the influence of LANA and may be explained by the association of LANA with mSin3 and RING3 (transcriptional repressors) (14, 40). In addition to this indirect mechanism of repression, it has been shown that the C terminus of LANA, when bound to DNA, can modulate transcription. LANA binds to GC-rich motifs within the viral terminal repeats and its own promoter (35, 56). Most interestingly, three genes (dual specificity phosphatase 8 (DUSP8), FKBP8, and a leucine-zipper containing transcription factor (LZTR1)) were strongly down-regulated between -9.8- and -17-fold in 293/Tet-On/LANA cells. One of these genes, FKBP8, contains a GC-rich element in its promoter, and it is tempting to speculate that LANA binds directly to cellular promoters (57), a topic currently under investigation.

The observed profiling data for BJAB and 293 cells suggests that LANA-dependent regulation of E2F target genes involves both Rb, and the pocket proteins p107 and Rb2, and is likely a result of wnt signaling upstream of Rb binding. To examine whether wnt signaling is needed for LANA-dependent regulation of cellular gene expression, we performed expression profiling of cells containing aberrations in the wnt signaling pathway. Staal et al. (58) reported that neither transfection of dominant negative GSK-3{beta} nor chemical inhibition by lithium treatment induced the accumulation of {beta}-catenin in Jurkat cells; hence {beta}-catenin regulation is independent of GSK-3{beta} in these cells. In Jurkat/Tet-On/LANA cells, LANA expression resulted in only 3 gene changes between 18 and 42 h post-induction, none of which were related to the Rb/E2F pathway. Whereas these results further suggest that the ability of LANA to inactivate GSK-3{beta} and stabilize {beta}-catenin is fundamental to the regulation of cellular gene expression, we are aware that other explanations including cell type-specific differences are possible.

Our array analysis of LANA-induced gene expression changes in three different cell lines containing aberrations in either Rb or wnt signaling suggests that the major pathway by which LANA modulates cellular gene expression is upstream of Rb and is dependent on a functional wnt pathway. In congruence with these data, Fujimuro et al. (19, 20) also reported high levels of {beta}-catenin in PEL-derived cell lines.

We further investigated the impact of LANA on gene expression levels in PEL cell lines. By comparing LANA-responsive genes from BJAB/Tet-On/LANA and 293/Tet-On/LANA cells to expression profiles from PEL cell lines from this study and to previous reports (42, 43), we identified a set of 21 genes similarly expressed in PEL lines and or primary tumor samples (Table I and supplemental Table 2, marked by #). Not surprisingly, this list contains genes regulated by E2F (DCK, NCOA3, and PTPRC) and/or the wnt signaling pathway (ANNXA2, ALDH1B1, and calreticulin). Our data are consistent with the hypothesis that LANA modulates cellular as well as viral transcription in KSHV-associated tumors.

KSHV also encodes v-Cyclin, which activates E2F signaling through direct phosphorylation of CDK6 (51, 59, 60). Most intriguingly, KSHV has somehow evolved to encode two proteins that target Rb. In endothelial cells (the main entity of KS lesions), LANA expression can be detected very early after infection (61, 62); however, LANA expression alone is not sufficient to transform such cells (63). It is tempting to speculate that the effect of LANA on the Rb pathway helps to create a cellular environment supportive of DNA replication and susceptible to the establishment of viral latency. LANA is the only viral protein required for latent DNA replication and episomal maintenance in dividing cells. In vivo, CD19+ B cells are believed to be the main reservoir for latently infected cells (64, 65). Our results show that LANA expression in B lymphocytes modulates cell cycle control. The further establishment of a direct relationship between the ability of LANA to modify cellular transcription and its role in DNA replication and genome maintenance requires in vitro primary cell infection studies. The elucidation of the mechanism(s) by which LANA modulates both viral and cellular gene expression and contributes to viral DNA replication and genome maintenance during latency may provide new targets for the development of therapeutic approaches for KS treatment.


   FOOTNOTES
 
* This work was supported by grants from the American Cancer Society and National Institutes of Health Grants CA88763 and CA097939 (to R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1-3. Back

|| To whom correspondence should be addressed: Dept. of Molecular Genetics and Microbiology and Shands Cancer Center, University of Florida, Gainesville, FL 32610. Tel.: 352-392-9848; Fax: 352-392-5802; E-mail: rrenne{at}ufscc.ufl.edu.

1 The abbreviations used are: KSHV, Kaposi's sarcoma-associated herpesvirus; LANA, latency-associated nuclear antigen; EBV, Epstein-Barr virus; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; Rb, retinoblastoma tumor suppressor; PEL, primary effusion lymphomas; KS, Kaposi's sarcoma; CREB, cAMP-response element-binding protein; Dox, doxycycline; PBS, phosphate-buffered saline; GFP, green fluorescent protein; TR, terminal repeat; BL, Burkitt's lymphoma; EST, expressed sequence tag. Back

2 R. Johnson and R. Renne, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Steven Fisher, Don Ganem, and Andy Polson for providing reagents. In addition, we thank Marty Veigl and Patrick Leahy for their help with gene expression profiling and Rebecca Johnson for critical reading and discussion of the manuscript.



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